An investigation into some antibiotics produced by ... University Institutional Repository An...

Loughborough UniversityInstitutional Repository

An investigation into someantibiotics produced by

Pseudomonas antimicrobica

This item was submitted to Loughborough University's Institutional Repositoryby the/an author.

Additional Information:

• A Doctoral Thesis. Submitted in partial ful�lment of the requirements forthe award of Doctor of Philosophy of Loughborough University.

Metadata Record: https://dspace.lboro.ac.uk/2134/10381

Publisher: c© Ernest Attafuah

Please cite the published version.

https://dspace.lboro.ac.uk/2134/10381

This item was submitted to Loughborough University as a PhD thesis by the author and is made available in the Institutional Repository

(https://dspace.lboro.ac.uk/) under the following Creative Commons Licence conditions.

For the full text of this licence, please go to: http://creativecommons.org/licenses/by-nc-nd/2.5/

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"I LOUGHBOROUGH UNIVERSITY OF TECHNOLOGY .

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AN INVESTIGATION INTO SOME ANTIBIOTICS PRODUCED'

Supervisors:

BY PSEUDOMONAS AN11MICROBICA

by

Ernes.t Attafuah, BSc (Hons), MSc.

A Doctoral Thesis Submitted in Partial Fulfilment of the

Requirements for the Award of the Degree of Doctor of Philosophy

of

. Loughborough University of Technology

September1991 .

W. G. Salt R. J. Stretton

... t. ",

o by Ernest Attafuah, 1991

Loughborough University of Tecnnoi8gy libr~ry

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DEDICATION

To

Almighty God, my mother and father

i

ACKNO~DGEMENTS

I am grateful to Drs. W. G. Salt and R. J. Stretton for their supervision of this· work, and for their advice, helpful comments and constructive criticisms of this manuscript. I am also grateful to Dr. D .. Brown for excellent work done and for his supervision during the X-ray crystallography work. My thanks are also due to my research colleagues in the organic/medicinal chemistry section, for the occasional 'brain storming' sessions we had.

I would like to thank all the technical staff I have been involved with, including Elizabeth AlIen, Jill Thorley, Jane Owens and John Brennan.

I am grateful to my father, without whose discovery, financial assistance, constant encouragement and patience, this project would never have been initiated nor concluded.

Finally, I would like to thank Mrs. M. Brooks for her patience during the excellent . typing of this thesis.

i i

ABSTRACf

Two strains (NCm 9897 and 9898; strains A and B respectively) of a Pseudomonas·

species have been shown to display antifungal and antibacterial activity on solid

media. Biochemical tests indicate that the organisms may be two distinct strains of

a new species. Cell lllorphology was studied using scanning electron microscopy.

Chemically defined media, established for the organisms, indicate non-fastidous

characteristics.

Four liquid media, able to elicit antibiotic production from Strain A have been

developed: a chemically defined medium (antibacterial), a chemically defined

medium and a complex medium (antifungal) and a chemically defined medium

(antibacterial and antifungal).

Nitrogen and magnesium limitation significantly increased yields. Magnesium content

in a medium (without a magnesium salt component) and in whole cell samples grown

in the said medium were assessed using atomic absorption spectroscopy and

elemental analysis respectively.

Optimization experiments for antibacterial and antifungal activity, assessed by a disc

diffusion assay, increased yields, in 250 ml conical flasks by a factor of X9 and 109%

respectively. A 6 litre laboratory-scale fermentor was used for larger batch

cultivations .

. Procedures for extraction of the active compounds from the biological matrices were .

developed leading to the isolation of one antibacterial compound, ABl (yellow

crystalline) and three antifungal compounds, AFl, AF2 and AF3 (pale yellow and

. amorphous).

Structure determination of ABl, involving mass spectrometry, IR/UV spectroscopy,

lH-NMR and x-ray diffraction, indicated it to be 1.6 dimethyl pyrimido[5,4-e ]-1,2,4-

triazine-5,7(IH,6H)-dione (Xanthothricin; Toxoflavin), a toxic metabolite previously

i i i

detected in foods contaminated with Ps. cocovenenans. Selective media, developed '

for Strain Aand Strain B, did not support growth of Ps. cocovenenans. Preliminary

structural analysis suggests that AFl may possess a mono-substituted ring system with

CHz chain and a terminal hydroxyl group; that AF2 may belong to the polyene group

of antifungal antibiotics and that AF3 may be an aliphatic ketone with hydroxyl

group ..

Agar diffusion, minimum inhibitory concentration, assays for the compounds, indicate

activity to be in the ~g!ml range for sensitive microorganisms. Antibiotic challenge

against test microorganisms suggest bacteriostatic activity for ABl, fungistatic activity

for AFl and AF3 and fungicidal activity for AF2.

iv

TABLE OF CONTENTS

Page No.

Dedication i

Acknowledgements ii

Abstract iii

Table of Contents v

Index of Tables xvi

Index of Figures xix

Abbreviations xxix

CHAPTER 1 INTRODUcrION

1.1 The Pseudomonads 1

1.2 Antibiotics: a general/historical approach 2

1.3 Sources and production of antibiotics 8

1.4 The fermentation process 19

1.5 Downstream processing and identification· of antibiotics . 23

1.6 Some Pseudomonas antibiotics 25

1.7 A novel Pseudomonas 28

1.8 Aims 29

CHAPTER 2 MATERIALS AND METHODS 32

2.1 MATERIALS 32

2.1.1 Microorganisms 32

2.1.2 Routine media 33

2.1.3 Growth curve media 34

2.1.4 Media tested for antibiotic production 35

v

2.2 GENERAL METHODS

2.2.1 Culture maintenance

2.2.2 Working stocks

2.2.3 Inoculum development

2.2.4 Biochemical/tolerance tests

2.2.5 Transmission electron microcopy (TEM) - Negative straining .

2.2.6 Selective medium development

2.2.7 Growth curves

2.2.8 Freeze drying

2.2.9 Some comparison/acidophily tests (strain A and Ps. cocovenenans)

2.2~1O Preliminary detection of antifungal activity

2.2.11 Preliminary extraction of water-soluble active . substances

2.3 ANTIBACfERIAL ACfIVITY STUDIES

2.3.1 Elicitation of antibacterial activity in liquid media

a) Assay procedure for antibacterial activity

2.3.2 Pigmented inoculum experiments

a) Orbital incubation

b) Static incubation

2.3.3 Preliminary testing of the active broth

a) Heat stability

b) A test for pyrrols

vi

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38

38

38

38

38

39

39

39

40

40

41

41

42

42

42

43

43

43

44

44

44

2.3.4 Optimization experiments

a) Medium component single-deletion of the basal medium (MCSD) [SLR]

b) MCSD of the basal medium (AR)

c) Variation of nutrient concentration

d) Variation of physical parameters

23.5 Cultivation in a laboratory-scale fermentor

2.3.6 Statistical error calculation

2.3.7 Thin-layer chromatography (TLC) plate preparation

a) Analytical TLC

b) Preparative TLC

2.3.8 Detection of active compound

a) Ultravioletlvisible (direct observation) .

b) Charring

c) Bioautography

.2.3.9 Isolation and punfication of the antibacterial principal

a) Determination of appropriate solvent system for extraction

i). Solvent for broth extraction

ii) Solvent for liquid/liquid extraction

iii) Solvent for TLC extraction

iv) Solvent for trituation

(b) Liquid/liquid extraction procedure

(c) Liquid/liquid/TLC extraction procedure

vi i

Page No.

44

44

45

45

45

47

47

47

47

48

48

48

48

49

49

49

49

51

51

51

51

52

----------------------------------------------,

2.3.10 High pressure liquid chromatography (HPLC) of isolate

2.3.11 Dose-response calibration curve·

2.3.12 Calculation of percentage efficiency of extraction

2.3.13 Physical properties

a) Melting point

b) pH/thermal stability test

. c) Ultraviolet light stability test

d) Retention factor determination

2.3.14 Antimicrobial properties

a) Minimum inhibitory concentration (MIC) determinations .

i) Tube dilution method

ii) Agar diffusion method

b) Assessment of microbistatic/cidal activity

2.3.15 Chemieal properties

2.3.16 Spectral data for antibacterial substance

a) Ultraviolet/visible scan··

b) Infra-red scan

c) Mass spectroscopy

2.3.17 X-ray crystallography.

a) Crystal formation

b) X-ray diffraction·

viii

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53

54

54

54

54

54

55

55

55

55

55

55

56

56

56

56

57

57

57

57 ..

58

Page No.

2.4 THE EFFECT OF MAGNESIUM SALT OMISSION 58 FROM THE MEDIUM RECIPE

2.4.1 Cell propagation experiments 58

a) Strain A in Med A2 (SLR) 58

b) Strain A in Med A2 (AR) 59

c) Ps. aeruginosa in Med A2 (SLR) 59

2.4.2 Atomic absorption measurements of Mg2+ in Med A2 SLR· 59 and AR .

a) Calibration curve 59

b) Mg2+ measurements (samples) 59

2.4.3 Analysis of Mgz+ in cells of strain A grown in basal medium 60 and Med A2

2.4.4 Viable counts of strain A grown in basal medium and Med 60 A2

2.5 ANTIFUNGAL ACTIVITY STUDIES 61

2.5.1 Elidtation of antifungal activity in liquid media 61

2.5.2 Assay of antifungill activity 61

2.5.3 Construction of PDLM for antifungal production 61

a) Oxoid PDA batch No. 15621497 61

b) Oxoid PDA Lot No. 07040305 61

c) Oxoid Potato Extract 62

d) Potato Liquid Extract 62

2.5.4 Optimization of antifungal activity 62

a) Medium component single-detection of medium q 62

ix

b) Variation of nutrient concentration

c) Variation of physical parameters

2.5.5 Cultivation in a laboratory-scale fermenter


2.5.7 Isolation and purification of the antifungal substances

. a) Determination of appropriate solvent system for broth extraction

b) Development of a solvent system!fLC strategy for extraction

2.5.8 A check for purity

a) HPLC

b) Gas chromatography


a) MIC determination of an antifungal compound

b) Zone stability comparisons of the 3 antifungal compounds

2.5.10 Spectral data for the antifungal substances

a) Ultraviolet/visible scans

b) Infra-red scans

c) Nuclear magnetic resonance scan

CHAPTER 3: RESULTS

3.1.0 GENERAL

3.1.1 Transmission electron microscopy

3.1.2 Selective media development

x

Page No.

62

63

63

63

63

63

64

64

64

65

65

65

66

66

66

66

67

68

68

68

68

3.1.3 Biochemical/tolerance test

3.1.4 Some comparison studies between strain A and Ps. ocovenenans

Page No.

68

69

3.1.5 Growth curve studies 70

3.1.6 Invasive growth inhibition of A. niger by some Pseudomonas 71 species

3.1.7 Preliminary extraction of water-soluble active substances

3.2.0 ANTffiACfERIAL ACfIVITY STUDIES

3.2.1 Elicitation of antibiotic production in liquid media

3.2.2 Preliminary testing of antibacterial substance/s

a) Heat stability


3.2.3 Pigmented inoculum experiment


. b) Static incubation


a) Medium component single-deletion (SLR)

b) Medium component single-deletion (AR)

c) Glucose variation

d) Ammonium chloride variation

e) pH variation

t) Agitation rate (RPM) variation

g) Temperature variation

h) Initial optical density (O.D.) variation

xi

71

71

71

72

72

72

72

72

72

73

72

74

74

76

77

79·

80

81

Page No.

i) Summary 82

k) Statistical error calculation 82

k) Scale-up fermentations 82

3.3.4 Isolation, purification and quantification of the antibacterial 83 principal

a) Determination of appropriate solvent system for extraction 83

i) Solvent for broth extraction 83

ii) Solvent/solvent extraction 83

iii) SolventtTLC extraction 84

b) Calculation of percentage efficiency of extraction 86

c) A check for purity 86 .

d) Assessment of antibiotic concentration in fermentation broth 86

3.3.5 Physical properties 86

a) Melting point 86

b) Retention factor determinations 87 ---_. -,

c) pH/thermal stability test 87 I

d) Ultraviolet light stability test 87

3.3.6 Microbiological properties 87

a) Minimum inhibitory concentrations 87

b) Effect of the antibacterial substance on growth of E. coli 88

3.3.7 Chemical properties 88

3.3.8 Spectral data (antibacterial substance) 88

a) Ultra violet/visible scans 88

b) Infra-red scan 89

xi i

Page No.

c) Mass spectrometry 89

3.3.9 X-ray crystallography 89

a) Solvent crystallization 89

b) X-ray data 89

3.4.0 THE EFFEcr OF MAGNESIUM SALT OMISSION 90 FROM THE MEDIUM RECIPE GROWTH

3.4.1 Cell propagation experiments 90

3.4.2 Atomic absorbtion measurements of Mg2+ in basal media' 90

3.4.3 Analysis of Mg2+ in cells of strain A grown in basal medium 91 and in Med A2

3.4.4 Viable growth CUIVes . 91

3.5.0 ANTIFUNGAL AcrIVITY STUDIES 92'

3.5.1 Elicitation of antibiotic production in liquid media 92

3.5.2 Preliminary testing of antifungal substance/s (Heat stability) 92

3.5.3 Pigmented inoculum experiment 92

a) Orbital incubation 92

b) Static incubation 93

3.5.4 Optimization experiments 93

a) Medium component single-deletion of medium q .93· "

b) Sodium nitrate variation 95.

c) Sucrose variation 98

d) Glycerol variation 99

e) pH variation 102

xiii

----~---.- - .

-------------------------------------------------

1) Agitation rate variation.


h) Summary

i) Statistical error calculations

j) Scale-up fermentations

3.5.5 Isolation and purification of the antifungal principal

a) Determination of appropriate solvent system

i) Solvent for broth extraction .

ii) Solvent for TLC extraction

b) A check for purity

i) HPLC

ii) Gas chromatography

3.5.6 Microbiological properties

3.5.7 Spectral data

a) Ultravioletlvisible spectra

b) Infra-red spectra

c) Nuclear magnetic resonance spectra·

d) Mass spectrometry (antibacterial substance only)

CHAPTER 4: DISCUSSION/CONCLUSION

4.1 The microorganism

4.2 Antibacterial activity studies

4.3 The effect of magnesium salt omission from the medium recipe

xiv

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103

105

106

107

107

107

107

107

108

108

108

108

109

109

109

110

110

110

191

191

194

209

4.4 Antifungal activity studies

4.5 Conclusion and future trends

APPENDIX: TRACE ELEMENT COMPOSITION OF MEDIA CONSTITUENTS

a) Medium for antibacterial substance production

b) Medium for antifungal substance/s production

PUBLICATION:

BIBLIOGRAPHY:

xv

.. Page No.

211

223

226

.226

·226

227

229

--- -- - -- ------------------------------,

INDEX OF TABLES

TABLE 1.1 Some chemical modifications of PeniciIIins (P) and . Cephalosporins (C)

TABLE 2.1 Construction of media tested for antibiotic production

TABLE 3.1 Zone of inhibition results of muItidisk code V4 tested against strain A, strain B and a range of microorganisms

TABLE 3.2 Biochemical/tolerance test results of strain A and B, with 3 pseudomonads as control

TABLE 3.3 Mean generation times (min) of strain A, Band Ps. aeruginosa (control), growth in various constructed media to obtain respective chemically defined media

TABLE 3.4 Invasive growth inhibition of A. niger by strain A, B and other pseudomonads grown on PDA and on CDA

TABLE 3.5 Result of various media assayed for antibiotic activity

TABLE 3.6 Sequence of optimization experiments with corresponding factorial increases in production

TABLE 3.7(a) Summary of a simple scale-up optimization trial in the laboratory-scale fermentor (variation of agitation and aeration)

TABLE 3.7(b) Summary of a further optimization trial on optimal conditions achieved at table 3.7(a) (variation of time (day))

Page No.

6

37

111

112

116

116

118

137

137

137

TABLE 3.8 Retention factors of spots from active broth filtrates 141 . and fresh, uninoculated medium/control, obtained from analytical TLC elutions, viewed under V.V. light and charred

TABLE 3.9 Retention factor determinations of the antibacterial 142 substance, using a selection of solvent which cover a cross-section of solvent polarities

TABLE 3.10 The effect of pH/temperature on the activity/stability of the antibacterial substance, with increasing time (mins)

xvi

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TABLE 3.11 The effect of V.V. light on the activity/stability of the 143 antibacterial substance, with increasing time (days)

TABLE 3.12 Minimum inhibitory concentration determinations of 143 the antibacterial substance, against a range of microorganisms, assessed by the agar diffusion assay

TABLE 3.13 Probable elemental ratios (empirical formulae) of the 147 antibacterial substance, as generated by the mass spectrometer data system

TABLE 3.14 Positional parameter and equivalent isotropic, 148 temperature factors arising from the X-ray diffraction' of the antibacterial crystal

TABLE 3.15 Bond lengths arising from the X-ray diffraction of the 148 . antibacterial crystal

TABLE 3.16 Bond angles arising from the X-ray diffraction of the 148 antibacterial crystal

TABLE 3.17 Atomic absorbtion measurements of Mg2+ 152 concentration in

a) SLR and AR basal media

b) SLR and AR basal media, previously inoculated with strain A, incubated for 20 mins, then membrane-filtered

TABLE 3.18 Sequence of optimization experiments with corresponding percentage increase in antifungal zone of inhibition

TABLE 3.19(a)

TABLE 3.19(b)

Summary of simple scale-up optimization trials in the laboratory-scale fermentor. (Variation of agitation and aeration)

Summary of further optimization trials . (variation of time (days» on optimal conditions achieved at table 3.19(a)

TABLE 3.20 Retention factor results for the active broth showing progress through a) first tier, b) second tier of the Azzalos and Issaq solvent system strategy, done on analytical TLC plates, viewed under U.V. light and charred.

xvii

178

178

178

179

- - - - -- -------------------~--------------------------------------

TABLE 3.21 Minimum inhibitory concentration de terminations of the antifungal antibiotic, AFl, against a range of microorganisms, assessed by the agar diffusion assay.

xviii

Page No.

183

INDEX OF FIGURES

Page No.

FIGURE 1.1: Structure of some representative antibiotics 8 showing quite a remarkable diversity in . molecular structures

FIGURE 1.2: A pie chart showing percentage distribution of 9 naturally occurring antibiotics and their sources

FIGURE 1.3: Schematic representation of the interplay 10 between the organism and its environment .

FIGURE 1.4: Secondary metabolite· (antibiotic) production 12 during the growth phase of microorganisms

FIGURE 1.5: Use of mutation and selection in the 18 development of improved penicillin producing strains of f. chtysogenum .

FIGURE 1.6: Schematic representation of a stirred tank 22 reactor

FIGURE 1.7: Column chromatography. Successive steps in 24 formation of chromatogram of green leaf pigments

FIGURE 1.8: Schematic diagram of a modern 24 chromatographic instrument

FIGURE 1.9: Structures of some Pseudomonas antibiotics 27

FIGURE 1.10: A photomicrograph of the mealy bug 29 Planococcoides njalensis from which Ps. antimicrohica was first isolated

FIGURE 1.11: ... A flowchart of the project 31

FIGURE 2.1: A Biolafette 6-litre laboratory-scale fermenter 47 in operation

FIGURE 2.2: A modified Aszalos and Issaq solvent system 50 flowchart, used to determine the appropriate solvent system for the active broth filtrate

FIGURE 3.1: An electron-micrograph of an individual cell of 113 strain A. negatively stained with PTA and photographed under TEM

xix

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FIGURE 3.2: Control for the selective medium experiment 113 showing growth of strain A and growth of Ps. cocovenenans on NA after 24 hrs at 37°C

FIGURE 3.3: Test of a selective medium showing growth of 113 strain A but no growth of Ps. cocovenenans on NA impregnated with sulphafurazole

FIGURE 3.4: Growth profiles of strain A, obtained from 114 comparison studies done, showing growth in basal medium(SLR) at pH 4.0; growth in basal medium (SLR) at pH 7.2 and growth in Med A2 (AR) at pH 7.2

FIGURE 3.5: Growth profiles of Ps. cocovenenans, obtained 114 from the comparison studies done, showing growth in basal medium (SLR) at pH 4.0, growth in basal medium (SLR) at pH 7.2 and growth in Med A2 at pH 7.2

FIGURE 3.6: Control for the comparison studies (acidophyly) 11S done, showing growth of strain A and of Ps. cocovenenans on NA at pH 7.2 after 24 hrs, at 37°C

--- _. FIGURE 3.7: Test for the comparison studies (acidophyly) 11S done, shoWing profuse growth of strain A and no growth of Ps. cocovenenans, on NA, at pH 4.0 after 24 hrs, at 37°C

FIGURE 3.8: Control for the invasive growth of A. niger after 117 7 days incubation at 30°C

FIGURE 3.9: Inhibition of A. niger invasive growth by strain 117 A after 7 days incubation at 30°C

FIGURE 3.10: Inhibition of A. niger invasive growth by Ps.cocovenenans after 7 days incubation at 30°C

117

FIGURE 3.11: Summation of growth profiles of strain A, 119 grown in the basal medium (SLR) MCSD experiment

FIGURE 3.12: Summation of antibacterial antibiotic 119 concentration/time profiles, produced by strain A in the basal medium (SLR) MCSD experiment

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FIGURE 3.13: Summation of growth. profiles of strain A grown 120 in the basal medium (AR) MCSD experiment

FIGURE 3.14: Summation of antibacterial> antibiotic 120 concentration/time profiles, produced by strain A in the basal medium (AR) MCSD experiment

FIGURE 3.15: Summation of growth profiles of strain A, 121 grown in Med A2 of various percentage glucose concentrations (0.3 - 1.0%)

FIGURE 3.16: Summation of growth profiles of strain A grown 121 in Med A2 of various percentage glucose concentrations (0.0 - 0.3%)

FIGURE 3.17: Summation of antibacterial antibiotic 122 concentration/time profiles, produced by strain A in Med A2 of various percentage glucose concentrations (0.3- 1.0%)

FIGURE 3.18: Summation of antibacterial antibiotic 122 concentration/time profiles, produced by strain A in Med A2 of various percentage glucose concentrations (0.0 - 0.3%)

------ FIGURE 3.19: Summation of antibacterial antibiotic 123 concentration/glucose concentration profiles, produced by strain A in Med A2 at various times

FIGURE 3.20: Growth profile, antibacterial antibiotic 124 concentration profile and pH profile of strain A, grown in Med A2

FIGURE 3.21: Summation of growth profiles of strain A, 125 grown in Med A2 of various percentage NH.CI concentrations (0.06 - 0.3%)

FIGURE 3.22: Summation of growth profiles of strain A, 125 grown medium A2 of various percentage NH.CI concentrations (0.0 - 0.06%)

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Page No.

FIGURE 3.23: Summation of antibacterial antibiotic 126 concentration/time profiles, produced by strain A in Med A2 of various percentage NH4CI concentrations (0.06 - 0.3%)

FIGURE 3.24: Summation of antibacterial antibiotic 126 concentration/time profiles, produced by strain A in Med A2 of various percentage NH4CI concentrations (0.0 - 0.06%)

FIGURE 3.25: Summation of antibacterial antibiotic 127 concentration/percentage NH4CI concentration profiles, produced by strain A in Med A2 at various times

FIGURE 3.26: Growth profile, antibacterial antibiotic 128 concentration profile and pH profile of strain A, grown in medium 1

FIGURE 3.27: Summation of growth profiles of strain A, 129 grown in medium 1 at various pHs

FIGURE 3.28: Summation of antibacterial antibiotic 129 concentration/time profiles, produced by strain A in medium 1 at various pHs

FIGURE 3.29: Summation of antibacterial antibiotic 130 concentration/pH profiles, produced by strain A in medium 1 at various times

FIGURE 3.30: Growth profile, antibacterial antibiotic 131 concentration profile and pH profile of strain A, grown in medium 2

FIGURE 3.31: Summation of growth profiles of strain A, 132 grown in medium 2 at various r.p.ms.

FIGURE 3.32: Summation of antibacterial antibiotic 132 concentration/time profiles, produced by strain A in medium 2 at various r.p.ms.

FIGURE 3.33: Growth profile, antibacterial antibiotic 133 concentration profile and pH profile of strain A, grown in medium 2 at 120 r.p.m.

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.

FIGURE 3.34: . Summation of growth profiles of strain A, 134 grown in medium 2 (120 r.p.m.) at various temperatures

FIGURE 3.35: Summation of antibacterial antibiotic 134 concentration/time profiles, produced by strain A in medium 2 (120 r.p.m.) at various temperatures

FIGURE 3.36: Summation of antibacterial antibiotic 135 concentration/temperature profiles, produced by strain A in medium 2 (120 r.p.m.) at various times

FIGURE 3.37: Summation of growth profiles of strain A, 136 grown in medium 2 (120 r.p.m.) at various initial O.Ds.

FIGURE 3.38: Summation of antibacterial antibiotic 136 concentration profiles, produced by strain A in medium 2 (120 r.p.m.) at various initial O.Ds.

FIGURE 3.39a: Determination of an appropriate solvent system 138 for broth extraction using the Aszalos and Issaq solvent system strategy

--~----- -.. -- FIGURE 3.39b: Determination of an appropriate solvent system 138 for the liquidJIiquid extraction

FIGURE 3.40: Determination of appropriate solvent systems 138 for the liquidffLC extraction

FIGURE 3.41: Constructed flow chart for the isolation and 139 purification of the antibacterial substance, using a liquidJIiquid extraction route

FIGURE 3.42: Constructed flow chart for the isolation and 140 . purification of the antibacterial substance using

a liquidffLC extraction route

FIGURE 3.43: A chromatogram of an active concentrate of 141 solvent 1, derived from active broth, eluted with solvent 3 and as viewed under UV light

FIGURE 3.44: Antibiotic concentration/zone of inhibition log- 142 dose response calibration curve, used for quantitative assessment of the· antibacterial substance present in active broth filtrates

xxiii

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FIGURE 3.45: . The effect of mid-log phase antibacterial 144 . antibiotic MIC challenge against E. coli grown

in basal medium: microbistatic/cidal assessment

FIGURE 3.46: A uv/vis spectrum of the antibacterial, basal 145 medium broth filtrate'

FIGURE 3.47: A uv/vis spectrum of the purified antibacterial 145 substance, dissolved in UV ethanol

FIGURE 3.48: A KBr. disc infra-red scan of the antibacterial 146 substance

FIGURE 3.49: A high resolution mass spectrum of the 147 antibacterial substance

FIGURE 3.50: Molecular structure of the antibacterial crystal. 149 as generated by the X-ray diffractometer, from bond lengths and angles and from an atomic numbering scheme

FIGURE 3.51: Unit cell contents of the antibacterial substance 150 as generated by the X-ray diffractometer

FIGURE 3.52: (A) The 'effect of cell propagation on growth and 151

(B)

antibiotic production of strain A, from basal . medium, along:

i) a series of Med A2 (SLR) ii) a series of Med A2 (AR)

The effect of cell propagation on growth of Ps. aeruginosa from basal medium, along a series of Med A2 . (SLR)

FIGURE 3.53: Atomic absorbtion calibration curve used for Mg2+ assessment

FIGURE 3.54:

FIGURE 3.55: .

Viability of strain A grown in basal medium and in Med A2

Growth profile, zone of inhibition profile and pH profiel of strain A, grown in pDLM-l

xxiv

151

152

153

154.

- -------------------------------------------------------------------------,

FIGURE 3.56:

FIGURE 3.57:

FIGURE 3.58:

FIGURE 3.59:

FIGURE 3.60:

FIGURE 3.61:

FIGURE 3.62:

FIGURE 3.63:

FIGURE 3.64:

FIGURE 3.65:

FIGURE 3.66:

Growth profile, zone of inhibition profile and . pH profile of strain A, grown in PDLM-1 under static conditions

Summation of growth profiles of strain A, grown in the Med B1 MCSD experiment

Summation of antibacterial zone of inhibition/time profiles, produced by strain A in the Med Bl MCSD experiment

Summation of antifungal zone of inhibition/time profiles, produced by strain A in the Med B1 MCSD experiment

Summation of growth profiles of strain A, grown in medium 3 of various NaND! percentage concentrations

Summation of antibacterial zone of inhibition/time profiles, produced by strain A in medium 3 of various percentage concentrations

.. Summation of antifungal zone of inhibition/time profiles, produced by strain A in medium 3 of various percentage concentrations

Summation of antibacterial zone of . inhibition/percentage NaND! concentration profiles, produced by strain A in medium 3, at various times .

Summation of antifungal zone of inhibition/percentage NaND! concentration profiles, produced by strain A in medium 3, at various times

Growth profile, antibacterial zone of inhibition profile, antifungal zone of inhibition profile and pH profile of strain A, grown in medium 4

Summation of growth profiles of strain A, grown in medium 4 of various sucrose percentage concentrations

xxv

. Page No.

154

155

156

156

157

158

158

159

159

160

161

-- -----------------------------------------------------------

Page No.

FIGURE 3.67: . Summation of antibacterial zone of 162 inhibition/time profiles, produced by strain A in medium 4 of various sucrose percentage concentrations

FIGURE 3.68: Summation of antifungal zone of inhibition/time 162 profiles, produced by strain A in medium 4 of various sucrose percentage concentrations

FIGURE 3.69: Growth profile, antibacterial zone of inhibition 163 profile, antifungal zone of inhibition profile and pH profile of strain A, grown in medium 4

FIGURE 3.70: Summation of growth profiles of strain A, 164 grown in medium 4 of various glycerol percentage concentrations

FIGURE 3.71: Summation of antibacterial zone of 165 inhibition/time profiles, produced by strain A in medium 4 of various glycerol percentage concentrations

FIGURE 3.72: Summation of antifungal zone of inhibition/time 165 profiles, produced by strain A in medium 4 of various glycerol percentage concentrations

----_ .. ,. FIGURE 3.73: Summation of antibacterial zone of 166 inhibition/percentage glycerol concentration· -profiles, produced by strain A in medium 4 at various times

FIGURE 3.74: Summation of antifungal zone of 166 inhibition/percentage glycerol concentration profiles, produced by strain A in medium 4, at various times

FIGURE 3.75: Growth profile, antibacterial zone of inhibition ~ 167 profile, antifungal zone of inhibition profile and pH profile of strain A, grown in medium 5

FIGURE 3.76: Summation of growth profiles of strain A, 168 grown in medium 5 at various pHs

FIGURE 3.77: Summation of antibacterial zone of 169 inhibition/time profiles, produced by strain A, in medium 5 at various pHs

xxvi

FIGURE 3.78:

FIGURE 3.79:

FIGURE 3.80:

FIGURE 3.81: .

FIGURE 3.82:

FIGURE 3.83:

FIGURE 3.84:

FIGURE 3.85:

FIGURE 3.86:

FIGURE 3.87:

FIGURE 3.88:

FIGURE 3.89:

FIGURE 3.90:

Summation of antifungal zone of inhibition/time profiles, produced by strain A in medium 5 at . various pHs

Growth profile, antibacterial zone of inhibition profile and pH profile of strain A, grown in medium 5 at pH 3

Growth profile, antifungal zone of inhibition profile and pH profile of strain A, grown in medium 6

Summation of growth profiles of strain A, grown in medium 6 at various r.p.ms.

Summation of antifungal zone of inhibition/time profiles, produced by strain A in medium 6 at various r.p.ms,

Summation of antifungal zone of inhibition/r.p.m. profiles, produced by strain A in medium 6, at various times

Growth profile, antifungal zone of inhibition profile and pH profile of strain A. grown in medium 6 at 120 r.p.m.

Summation of growth profiles of strain A. grown in medium 6 at various temperatures

Summation of antifungal zone of inhibition/time . profiles, produced by strain A in medium 6 at various temperatures

Growth profile, antifungal zone of inhibition profile and pH profile of strain A. grown in medium 6 (120 r.p.m.) at 32°C

Growth profile, antifungal zone of inhibition profile and pH profile of strain A. grown in medium 6 (120 r.p.m.) at 37"C

. Determination of an appropriate solvent system for broth extraction, using the Aszalos and Issaq solvent system stragety

Determination of an appropriate solvent system for broth extraction using an array of solvent systems of high polarity

xxvi i

Page No.

169

170

171

172

172

173

174

175

175

176

177

180

180

Page No.

FIGURE 3.91: Flow chart arising from determination of 181 appropriate solvent systems for liquidfTLC extraction

FIGURE 3.92: Constructed flow chart for the isolation of the 3 182 . antifungal antibiotics using a liquid(fLC

extraction route

FIGURE 3.93: Stable zones of inhibition created by antifungal 184 antibiotic, AF1, against T. mentagrophytes

FIGURE 3.94: Stable zones of inhibition created by antifungal 184 antibiotic, AF1, against A. niger

FIGURE 3.95: Stable zones of inhibition created by antifungal 184 antibiotic, AF1, against f. chrysogenum

FIGURE 3.96: Stable zones of inhibition created by antifungal 185 antibiotic, AF1, against ~. aureus

FIGURE 3.97: . Stable zones of inhibition exhibited by 185' antifungal antibiotics AF2, but not AF1 and AF3, against C. albicans, after' 5. days incubation at 30°C

FIGURE 3.98: A uv/vis spectrum of antifungal antibiotic, AF1 186

FIGURE 3.99: A uv/vis spectrum of antifungal antibiotic, AF2 186

FIGURE 3.100: A uv/vis spectrum of antifungal antibiotic,AF3 186

FIGURE 3.101: An infra-red spectrum of antifungal antibiotic, 187 AF1

FIGURE 3.102: A 60MHz INMR spectrum of antifungal 188 antibiotic, AF1



xxviii

ABBREVIATIONS

General

AA Atomic absorbtion

Ab Antibacterial substance

ABl Antibacterial substance 1 "

AcOH Ethanol

ADP Adenosinediphosphate

Af Antifungal substance/s

AFl Antifungal substance 1

AF2 Antifungal substance 2

AF3 Antifungal substance 3

AMP Adenosinemonophosphate

AP Activator protein

AR Analar reagent

--~-.

kg Arginine·

ATCC American type culture collection

ATP Adenosinetriphosphate

cAMP Cyclic adenosinemonophosphate

CCR Carbon catabolite repression

CDA Czapek Dox Agar

CDLM . Czapek Dox Liquid Medium

CDM Chemically defined medium

CV Coefficient of variation

D.N.A. Deoxyribonucleic acid

xxix

-- -----------------------------------------------------

E.C. Energy charge

elm Electron/mass ratio

E.T.G. Electron transfer chain

EtOAc Ethylacetate .

FAD Flavin adenine dinucleotide

G.C. Gas chromatography

GDH Glutamate dehydrogenase

Gr Greek

GS-GOGAT Glutamine synthetase-glutamate aminotransferase

His

HPLC

IMI

IR

IJSB

IV

LDso

LVCC

Lys

MCSD

MeOH

Met

MIC

MR/VP

MS

NA

Histidine

High pressure liquid chromatography

International Mycological Institute

Infra red

. International Journal of Systematic Bacteriology

Intra venous

Median lethal dose

Log viable cell count

Lysine

Medium component single deletion

Methanol

Methionine

Minimum inhibitory concentration

Methyl redNoges-Proskauer

Mass spectrometer

Nutrient agar

xxx

NAD Nicotinamide adenine dinucleotide

NADH Reduced NAD

NADPH Reduced nicotinamide adenine dinucleotide phosphate

NB Nutrient broth

NCIB National collection of industrial bacteria

NCIB 9897 Strain 'A

NCIB 9898 Strain B

NCfC . National collection of type cultures

NCYC

IHNMR

NspA

NspB

OD

ONPG

Om

PABA

PDA

PDLM

PDLM-1

Phe

Pi

PrOH

PTA

QSAR

Rf

National collection of yeast cultures

Proton nuclear magnetic resonance

Neurospora Medium Agar

Neurospora Medium Broth

Optical density

O-nitrophenol beta D-galactopyranoside

Ornithine

Para amino benzoic acid

Potato Dextrose Agar

Potato Dextrose Liquid Medium

Potato Dextrose Liquid Medium of batch No. 15621497

Phenylalanine

Inorganic phosphate

Propanol

Phosphotungstic acid

Quantitative structure activity relationship

Retention factor

xxxi

R.P.M.

SLR

TEM

TLC

TIC

uv/vis

-- ._---------------------

Revolutions per minute

Standard laboratory reagent

Transmission electron microscopy

Thin layer chromatography

TriphenyltetracoIium chloride

Ultra-violet/visible _

xxxi i

------ ------------------------

Media used for optimization (Abbreviated nomenclature)

a) Basal medium

Glucose

omitted from the basal medium recipe





b) Czape.k Dox Liquid Medium - modified (Med q)

Magnesium glycerol phosphate

KC!

. Sucrose

Glycerol

Carbon source

MedB2

Optimization sequence

Medium I

. Medium 2

Medium 3

Medium 4

Medium 5

omitted from Med q recipe





. omitted from Med q recipe

omitted from Med q recipe .

Med A2·of 0.06% NH4C! concentration

Medium I at pH 6

:MedB8

Medium 3 of 0.05% NaN03 concentration

Medium 4 of 2% glycerol concentration

xxxiii

-MedAl·

.- MedA2

- Med A3

- Med A4

- Med AS

-Med BI·

- Med B2

- Med B3

- Med B4

- Med B5

- Med B6

~ Med B7

- Med B8

-- -_._._---------------,

CHAPTER 1

INmODUCflON

1. INTRODUCfION

1.1 The Pseudomonads

Organisms in the genus Pseudomonas (false unit; Gr) are mostly free-living

bacteria, widely distributed in soil and water and, in general, are so versatile that , . .

they have been known to multiply in almost any moist environment containing

trace amounts of organic compounds. While some are plant pathogens, a smaller

number is associated with specific diseases in man. The most important of this

latter group include Ps. aeruginosa (urinary tract infection), Ps. pseudomallei

(melioidosis) and Ps. mallei (glanders; MacFaddin, 1983). Ps. aeruginosa in

particular, is now becoming increasingly important as a source of general infection

and has also become an important pathogen among debilitated, burned and

immuno-compromised individuals. (Bennett, 1974; Bodey, 1983).

The Pseudomonads are Gram negative microorganisms, usually occurring as single

cells which are straight or curved rods but not helical. . Their dimensions are

generally 0.5-1 I'M by 1.5-4 I'M. They are, with rare exceptions, motile by polar

flagella and may be monotrichous or multitrichous. They do not produce sheaths

or prosthecae and no resting stages are known.

The metabolism of the genus is respiratory and never fermentative. Some are

facultative chemolithotrophs, able to use H2 or CO as an energy source.

Molecular oxygen is the universal electron acceptor though some can denitrify

using nitrate as an alternative acceptor in the presence of specific substrates.

Except for the latter, they are strict aerobes. They are catalase positive and with

the exception of Ps. maltiphilia are classically oxidase positive. The G & C

content of the· DNA of those species so far examined ranges from 58 - 70 moles

%. The type species is Pseudomonas aeruginosa (Palleroni, 1984).

Most pseudomanads morphologically resemble the enteric bacilli, the aeromonads

and the vibrios and tend to grow well in differential enteric media. Also, some

Pseudomonas spp. resemble and are often confused with members of the genus

Xanthomonas (MacFaddin, 1983).

There are strains which produce water-soluble, yellow green, fluorescent pigments

while others synthesize in addition, various type-specific phenazine pigments.

Many others however, are non-pigmented (Davis et ai, 1980). The Pseudomonads

are now. becoming increasingly important as producers of 'clinically useful'

antibiotics (Hunter and Baumberg, 1989).

1.2 Antibiotics: a general/historical approach

Antibiotics may be broadly defined as substances produced by living organisms

which kill or inhibit the growth of microorganisms. Most are secondary

. metabolites and are produced towards the end of the growth phase, in situations

where the cells have more or less stopped dividing. The role of the antibiotic in

the life-cycle of the producing organism may be manifold although not entirely

clear. They may be secreted during stressful competition with other organisms for

scarce/ depleting nutrients and this may give the producing organism a survival

advantage. It has also been postulated that antibiotics may inhibit those sites

involved in primary metabolism, in order to conserve cellular energy during the

stationary phase of the growth cycle (Khokhlov et ai, 1973). This to some extent

is supported by the observations that with some bacteria which are unable to

. produce secondary metabolites, the viability of the cells during the stationary

phase decreases relatively rapidly, suggesting an antibiotic function which may

result in maintained viability during the stationary phase (Edwards, 1980; Hara,

and Beppu, 1982; Nisbet and Porter, 1989). A further suggestion centres on the

fact that in sporulating antibiotic producing organisms, antibiotics appear at the

time of sporulation. Sporulation requires the de-novo synthesis of the spore coat

and is associated with the break down of the existing cell wall and with the

subsequent release of cell wall components (D-amino acids, novel sugars) which

. are similar in structure to many antibiotics. Hence it could well be that the

altered cell wall metabolism of sporulating organisms might provide the precursors

that results in the biosynthesis of many antibiotics (Davis et ai, 1980).

Although it was known for a long time that some microorganisms are capable of

producing antibiotics, this natural phenomenon was, in the past, merely viewed as

an interesting artefact with a hint of a potential therapeutic application. Thus

2

. i

when Alexander Flemirig in 1929, returning from holiday to his laboratory in St.

Mary's Hospital, made his famous observation on an old, contaminated culture

plate of Staphyloccoci, he was merely one in a long line of workers who had

noticed similar phenomena. It must be noted, however, that it was principally . .

Flemirtg's observationthat initiated events leading to the development of penicillin

as the first non-toxic antibiotic in the strict sense of the term. It must also be

noted, as has now. become clear from subsequent attempts to reproduce the

phenomenon, that the lysis of Staphylococci in the area surrounding a

contaminant Penicillium colony, as on Fleming's original plate, could only have

arisen by an extraordinary coincidence (Greenwood, 1983; MacFarlane, 1984). An

early glimpse was thus caught of the erratic nature of the antibiotic triggering

mechanism in microorganisms.

Although Fleming was hopeful about the possible therapeutic value of his

discovery, he was unable to purify and concentrate the substance. It was left to

the German biochemist, Ernst C.hain, working with Howard Florey, at the Sir

William Dunn School of Pathology in Oxford,. to obtain pure and stable extracts

of the penicillin. The extracts eventually obtained, which were thought to be pure

. but· later shown to contain less than' 1 % penicillin, were used in controlled

.' therapeutic experiments, first on mice then on men, with remarkably encouraging' . . . . . ... _-."

results (Chain et aI, 1940; Abraham et aI, 1941). From all this, it was indeed

fortunate that problems of serious toxicity arising from the. impurities were not

encountered at these early trials, as this might have resulted in the unfortunate

termination of the whole enterprise.

. Further development of penicillin in Britain was difficult because of the 2nd

World War, so Florey visited U.SA in 1941 to enlist the support of the American

authorities and drug firms. Once they were convinced of its potential, all research

on it became classified until after the war. During the war, progress was rapid

and by 1944 - 5, bulk production was in progress and the drug was beginning to

. become readily available (Hoover and Dunn, 1979; Greenwood, 1983). Although

.its chemotherapeutic properties were well known by 1940, it was five years later,

after intensive research effort by workers. at Sir William Dunn School of

Pathology, Oxford, and at both Merck and Pfizer pharmaceutical companies, .

3

U.S.A., directed towards purification and structure elucidation, using chemical

degraqation and x-ray crystallography, that its structure was finally established

(Florey et aI, 1949) .. Its spectrum of activity revealed it to be an antibacterial

agent, particularly effective against Gram positive bacteria ..

Antibiotics derived from soil miCroorganisms now became important and, in 1940,

Selman Waksman initiated a systematic search for non-toxic antibiotics, principally

from the Actinomycetes. This group includes Streptomyces spp. which was later

to yield many therapeutically useful compounds ... It has been postulated that

Waksman may have been influenced in his decision to undertake this study by the

then recent discovery by Rem~ Dubos (an ex-pupil of Waksman) of the antibiotic

complex tyrothricin, in culture filtrates of Bacillus brevis (Dubos and Hotchkiss,

1941). Like Dubos's tyrothricin, Waksman's first discoveries were far too toxic for.

systemic use although they included actinomycin (discovered in 1940 from

Actinomyces antibioticus; Waksman and Woodruff, 1940),a compound later used

in cancer chemotherapy. The first real breakthrough came in 1943 with the

discovery of Streptomycin, the first of the aminoglycoside antibiotics from a ..

fermentation broth of Streptomyces griseus (Schatz et aI, 1944). It was found to

have a spectrum of activity that neatly complemented that of penicillin by

inhibiting many Gram negative bacteria but more importantly at that time, it also

inhibited the growth of Mycobacterium tuberculosis, the. causative organism of

tuberculosis (Greenwood, 1983).

Between 1945 and 1948, Giuzeppe Brotzu of Sardinia, whilst investigating the

. microflora of sewage outflow in the hope of discovering naturally occurring

antibiotic substances, isolated a species of cephalosporium (Brotzu, 1948). This

displayed striking inhibitory activity against several bacterial species including

Salmonella ttohimurium which was at that time considered beyond the reach of .

penicillin. Lacking the means to proceed further, samples were dispatched to the

. Sir William Dunn School of Pathology at Oxford where two antibiotics were

reported to be isolated (Greenwood, 1983). They were called cephalosporin P

and cephalosporin N because the former inhibited Gram positive organisms such

as Staphyloccoci and Streptococci whilst the latter was active against Gram

negative organisms such as Escherichia coli and SalmoneIla ttPhimurium. As it

4

. turned out, neither of these substances was a cepholosporin in the sense that term

is used today: the cephalosporin P fraction proved to be a mixture of 5 antibiotics

(PePs) with steroid like structures (Crawford, et aI, 1952; Burton and Abraham,

1951; Burton et aI, 1956) and cephalosporin N turned out to be a penicillin

(adicillin) (Abraham et aI, 1953; Abraham and Newton, 1954; Abraham et aI,

1954). The forerunner of the cephalosporin now in use, cephalosporin C, was

detected later by Abraham and Newton as a minor component on fractionation

of cephalosporin N. (Newton and Abraham 1955; Newton and Abraham 1956).

It was also apparently present, in minor quantities, in the original fermentation

. mixture.

Overall, it was the appearance of Streptomycin which really triggered the general . hunt for naturally occurring antibiotics and when the pharmaceutical companies

joined the chase, soil samples by the thousand, from all over the world, were

screened for antibiotic producing microorganisms. Subsequently, hundreds of

antibiotic substances were discovered and rediscovered. Although most failed

preliminary toxicity tests, by the mid 1950's representatives of most of the major

families of antibiotics, including the aminoglycosides, chloramphenicol, tetracycline

and the macrolides had been discovered. Indeed it has been argued that by 1960,

practically all the antibacterial agents required by modern medicine were known

(Greenwood, 1983).

New antibiotics, however, keep cropping up in unusual places and it is now

apparent that discoveries of important novel bioactive compounds often depend

upon the development of strategies for the isolation and characterization of novel

and rare microorganisms. (Nolan and Cross 1988). It has to be emphasised

nevertheless, that since 1960, only a very few truly novel antibiotic substances have

been discovered and the rate of discovery has been falling. Furthermore, with

regard to discoveries made over the last 20 years, clinical success rate has dropped

from 5% to less than 1% (Primrose, 1987).

In contrast to the above situation, a surprising number of naturally occurring

antibiotic substances, displaying molecular variations on established antibiotic

structures, have recently emerged and these include a total number of 20

5

Position

I . 2·P

Z·C

2·". i3 3-C 3'

3P."C

5P.6C

penicillins, 20 actinomycins, 10 polymyxins, 10. bacitracins and 3 neomycins

(Primrose, 1987).

An important alternative approach to obtain new antimicrobial agents has been

to . modify the chemical structures of existing antibiotics and thus obtain

compounds with enhanced active properties. Here, a rationale based on

quantitative structural activity relationships (QSAR) is used (Nogrady, 1988).

Previously active compounds which have now been made obsolete by resistant

strains may also be given a new lease of life with this approach and on this point,

QSAR has proved most successful with the penicillin and cephalosporins where

numerous semi-synthetic derivatives exist. (Flynn, 1972). Table 1.1.

Table 1.1:

From"

5 ICH,): H,

H

Some Chemical Modifications of Penicillins (P) and'

Cephalosporins (C); after Hoover and Dunn, (1979).

Si CH,Q

'.-¥ -.v p- L~-j;CH,jl ~. ~

To

so. SO,. SCH, H, =CHSAr: H. OAc: H. CH,: CH,. H: =CH,: Br. CH,Br:

H. CH,SR: CH,SR. H: =CHSR: =0. OAc. Br. OCH.

CH,OAc CHO. CO,H.-.=CH,I. OH. OR. Cl. F. :->H,. :'<HCOOR OAc

CO,H

H

H. OH. OCOR. OR. SR. :--IC,H,X. S,O,Na. :'oi" :'oIH,. C:--I. SO., CH,. S,C=NHII<'R,. SiC=S·,:'oIR,. SiCS,OR. SCOR. SO,Ar. Br. OCONH,. OCONHR. S·hetorocycle. ",CS. Ar. heteroc::cle

H. CO,R. CO,CH,OCOR. CONH,. CO,OCOR. CONHCHI RICO,H. CO,SiR •. CO:'i,. CH:OH. CH,CO,H. ·::OCH:'oI:. COCH,CI. C:"l. retr:J.zole

CH,.OCH, 6·/3·P.7·13·C RCONH H. "'H,. R'CO:'iH. R':--I-CH=:-I .. "'rC=:-I. R,:'i. R.'1H. RSO,:-IH.

R.PONH. R:--<HCONH. R:--IHCS:--iH, KOCO!'H. RCOO. RCOCH,. RCONI"'H .. '

6·",·P.7·",·C H CH,. OCH;. RCONH. :'-IH,. Cl. OH. OCOR. CH:OH. CH,CI. CH:F. CH,NH,. CH,CH,C:'oI. SCH,. (H,COOCH,. CH,M. CO,H. CH,COAr. CHIOHlle..".,. NHCOOC,H! =s 7·?8·C =0

Medicinal chemists and chemotherapists have also been active in exploiting the

antimicrobial potential of fully synthetic substances, although as yet only a very

small number have been devised by premeditated attack on known biochemical

pathways. Of the antimicrobial drugs presently used therapeutically, only the

6

diaminopyrimidines, trimethoprims and pyrimethamines really fall into this

category (Greenwood, 1983). An example of others under development and study

is a family of amino acid derivatives (at present under investigation by Hoffman

La Roch), one of which is alaphosphin (L-alanyl-L-amino-ethylphosphonic acid),

a synthetic cell wall attack drug (AlIen et ai, 1978). Other established synthetic

antimicrobial agents intended for chemotherapy include the nitrofurans, the

quinolones, the imidazoles, the naphthyridines (and related compounds of the

nalidix acid type) (Greenwood, .1983). The underlining motive of the researchers

has always been selective toxicity by which the drug specifically targets the .

invading pathogen whilst leaving the host cells unaffected.

Without question, the appearance in the late 1930's and early 1940's, of potent,

non-toxic, antimicrobial agents, selectively active against bacteria, revolutionised

the treatment of infection. Indeed the discovery of the. first 'miracle drugs' the

sulphonamides, penicillin and streptomycin was declared by some to herald the

disappearance of bacterial infection as a disease entity of any importance

(Greenwood, 1983). At present, with over 50 years hindsight and hundreds of

chemotherapeutic agents available for use, a more modest and dispassionate view

of the benefits and limitations of antimicrobial drugs is possible:

1. . bacterial pathogens have not been eradicated by chemotherapeutic agents

and many now show a remarkable resistance to them;

2. the pattern of bacterial disease, particularly hospital-acquired infection has

altered considerably, mainly due to new operation procedures,

instrumentation techniques and treatment regimens which tend to severely

compromise the patients·own capacity to withstand infection;

3) the use of antibiotics often disrupts the delicately balanced bacterial flora

of the body, allowing for the proliferation of resistant species and

sometimes initiating potentially more serious new infections;

4) no antibacterial drug is entirely free from deleterious side-effects and as

such, the use of these agents has its own attendant risks.

Finally, it should be borne in mind that most successes in the battle against

diseases have tended to be limited to the treatment of those caused by bacteria

and that those other numerous infections caused by viruses, protozoa, helminths

7

--.. ---c----~------------------------

and fungi are, \Vith some notable exceptions (imidazoles with candidal infections,

nitroimidazole with protozoan infections), less amenable to chemotherapy.

1.3 Sources and Production of Antibiotics

. To date, the total figure of antibiotics so far· discovered is well over 2,500 and the

diversity of molecular structures is immense (Fig. 1.1). Many are produced as

mixtures of related compounds (Berdy, 1974; Primrose, 1987; Berdy,1988). The

majority are produced by the Actinomycetales, accounting for over 58%. This

group contains ten classes of which· three are of importance· as antibiotic

. OH CH,OH 0 . ~II 11

NO,~CH-CH-NH-C-CHCI, HO

CH, CH,

01H H'~ OH. OH

Chloramphenicol COOH o OH OH OH OH 0 OH

Amphotericin 8

H,C, ..... CH,

~'" H,C CH, ..

o ~... H H"yC 05CH,

CH, ... ~_~OH . . 0 CH,

CH, I CH,

H OH

NHCNH, Erythromydn

OH H OH " H. NH

Streptomycin

OH . 0 . CONH,

OH 0 0- " . ,,5, .... CH, . ~ A CH,-C-NH:- TH-TH T ...... CH,.

. O=C-N--CH-COOH

Fig. 1.1:

" Tetracycline Penicillin G

Structures of some representative antibiotics showing quite· a

remarkable diversity in molecular structures (After Primrose, 1987).

8

------- -- --------------------------,

producers and ofthese three, the Streptomycetes is clearly the most significant;

Fungi also produce a large number of antibiotics, contributing approximately 18%

of the total and here, the sole group responsible is the· Aspergillales. True

bacteria are also significant producers, yielding almost 9% of all antibiotics and

of these,· the two families Bacillaceae and Pseudomonads predominate. The

remaining bacteria produce only a very small number of antibiotics. Other

antibiotic producers include the higher plants, with 12% and algae, lichens and

animals, all together producing 3% (Edward, 1980; Hunter and Baumberg, 1989;

Fig. 1.2).

ACTINOMYCETALES ___

(Streptomyces)

58%

_ FUNGI --'-1-:8%~ (Aspergi llales)

__ - BACTERIA 9% (Bac i 11 aceae and

Pseudomona )

----OTHER SOURCES

Fig. 1.2: A pie chart showing percentage distribution of naturally occurring antibiotics and their sources.

The elicitation and consistent production of secondary metabolites such as

antibiotics and pigments has been revealed by many workers to be one of the

most erratic of all phenotypic properties of microorganisms (Holliman, 1961;

Hellinger, 1951). Antibiotic and pigment production are often linked and may in

many cases be acutely sensitive to anyone of, or a combination of, variations in

pH, time, temperature, aeration (oxygenation) nutrient/trace element

concentration and inoculum level (Goodhue et aI, 1986; Fig. 1.3).

9

Carbon and energy Oxygen sources ._-_ .. ----.---. -~ ___ , ____ . ___ _ other nutrients ' /" + _ ~

A9ito~ Aerotio~ )lSure

1'~,uPpIY I , C02 -remoyol- -----,1 Lt fEl~.;;;;;;;;jjt =Rheology'..; ---.=.'=-_____ Temperoture

~" rJ ~--------l conce,ntration .~ ~ ?hOI0

9y

Fig. 1.3:

L, __ -'-____ .. Production _----------1 Schematic representation of the interplay between the organism and

its environment. (After Hutter et al. 1978)

For example, Jamieson (1942) using tap water of lake origin in the preparation

of his media,ended up after culturing,with blue green pigments from organisms

'he had previously considered to be Achromobacterspp. Even cultivation, in '

, media containing proteose-peptone which is known to enhance pigmentation

(McCombie and Scarborough, 1923; Wrede, and Strack, 1924; Elema and

Sanders, 1931; Swan et aI, 1957), when prepan~dwith distilled water, had failed

toyield pigment and this therefore suggested that it was the trace elements in the

lake water that may have initiated pigment production. He consequently

recommended a modification of the classification of the microorganisms by

suggesting they be placed under Pseudomonas spp. (SW"" ~,1~5})

Schoental (1941) and Young (1947) also found that no pigmentation was

produced from the pigment producer, Ps. reptilovora,. in 'media containing over

1 % glucose: the'inhibition being attributed to the production of excess acid.

del-Rio et al (1972), whilst investigating the optimal conditions for the production

of antimicrobial substances from Ps. reptilovora, found that not all the commercial

peptones employed in the optimal medium gave the same antibiotic yield. They

further found that when different batches of thesame kind of peptone was used, ,

a marked difference in antibiotic activity was observed. Atomic absorption

spectroscopy of the iron, manganese, zinc and copper content (trace metals) of

10

the commercial peptone and peptone-like products showed a remarkable

difference in the copper content between peptones which gave good antibiotic

yields and those which inhibited production. Copper was subsequently shown,

during the same investigation, to be directly associated with the antibiotic, YC-73,

also known as fluopsin (Fig. 1.9).

It has been noted that though antiboitic production may readily occur when a

producer organism is grown on solid medium, when grown in an equivalent liquid

medium (minus the agar) very little or no antibiotic production occurs (Holliman,

1961). The explanation for this may be that on solid media, colonial growth, in·

most cases, creates extreme conditions where availability of nutrient in the agar

in the immediate area is soon exhausted. This is especially so for cells near and

around the centre of the colony where diffusion of nutrients from the agar to the

uppermost cells in the colony is limited and where waste products do not readily

diffuse away and therefore accumulate in the colony and in the agar beneath

(Frobisher, 1959). Such adverse conditions, (coupled with availability of the

required nutrients/trace elements) may stress the organism, triggering certain

metabolic pathways associated with antibiotic production. In thewild this would

be a useful survival advantage in face of keen competition. The stress

factor/triggering hypothesis may go some way to explain why antibiotic/pigment

production is more difficult to elicit in . liquid cultures in which such extreme

conditions are not readily found. It may also help to explain why in those liquid

. media capable of supporting antibiotic production, production usually occurs late

in the growth cycle (idiophase) where conditions of stress become prevalent (Fig.

1.4). The terms 'trophophase' (growth phase) and 'idiophase' (production phase)

were first coined by Bulock (1961, 1967) during batch mode studies of antibiotic

production in submerged cultures.

11

-- - - -- - --- < --I

I

-- -- -- --- --------------:----------------

Fig. 1.4:

No, of cells per ml culture

A

c o

I I I I

Time

Secondary metaboIite (antibiotic) production during the growth

phase of micoorganisms. The shaded area represents the zone of

secondary metabolite production. A indicates the lag phase; B

indicates the logarithmic (log) or exponential phase (trophophase);

C indicates the stationary phase (idiophase); D indicates the decline

or death phase (After Edwards, 1980).

A school of thought exists (Davis, et aI, 1980) which disagrees with the notion that

stress factors necessarily trigger antibiotic production thereby challenging the

subsequent inference of a survival advantage for antiboitic producers. The main

reasons cited are as follows:

1) antibiotic producing organisms constitute only a tiny fraction of the

microbial population in soil samples and thus do not appear to have a

striking advantage;

2) The strains found in nature excrete only small amount of an antibiotic;

heavy excretion is an artefact dependent on selection of regulatory

mutants;

3) Antibiotics appear only after growth has ceased rather than during

competition for growth. _

At first glance these 3 points may appear quite valid but dealing with each point

in numerical order, a deeper analysis may suggest alternative explanations:

12

-- .----------------------------~------------------------------------

1) in the wild, normal soil environments may contain such an over-abundance

of essential nutrients (PeppIer, and Perlman, 1979; Reed, 1981; Goodhue

et aI, .1986) that true conditions ~of stress needed to trigger antibiotic

production may in fact be rare,

2) . as already discussed above, antibiotic. production is markedly enhanced

when a microorganism is grown on solid medium as opposed to when

grown in an equivalent liquid medium and it therefore seems that there

may be a need to stress the wild-state organism in order to enhance

production (HoIIiman, 1961);

It should also be noted here tliat having developed an optimized medium

and conditions for antibiotic production for a wild-type strain, growth of its

mutant under the same conditions might well result in Iow to zero yields

(Calam, 1986). AIl this suggests that in the wild, conditions must be just

right before any significant excretion can take place;

3) although point 3) above, subscribes to conventional ideas of antibiotic

production, this may not necessarily be true for all cases as pf9aue~iBR

production can also occur during the trophophase where antibiotic

p~oduction can be triggered quite early on (Demain, 1986; Vining et aI, .

1986; Doull and Vining, 1988). Furthermore, it can be argued that

triggering, occurring just before entry into stationary phase (idiophase) , ".

supports the contention of stress factor elicitation as adverse conditions

becQme more prevalent here due to depleting nutrients/trace elements and

increasing toxic end-products.

To date the stress-factor associated, survival-advantage role of the antibiotic in

nature, remains controversial and some contemporary writers, though clearly in

support, tend to handle it hypothetically (GottIieb, 1976; Katz et aI, 1977; Martin

and Demain, 1980). It is important to note that a survival advantage has already

been clearly demonstrated in Cephalosporium gramineum (Bruehl, et al 1969).

13

A study of how nutritional factors affect antibiotics production is important in any·

yield· improvement study. It is well known that a carbon source is essential for

growth and can provide a source of energy required to drive the cell's metabolic

processes. It has also been noted that at the earlier growth stages of some

antibiotic producing microorganisms, a direct relationship exists between cell mass

and lowlevel antibiotic production (Shehata et aI, 1971). However, as the carbon

source concentration is further increased, antibiotic production and growth may

became depressed via the processes of carbon catabolite repression and substrate

inhibition (Young, 1947; del-Rio et aI, 1972; Bushell, 1989).

Much work has been published on carbon catabolite repression (CCR) as it .

affects antibiotic production (Gallo, et al. 1972; Aharonowitz, et aI, 1978; Revilia,

et ai, 1984; Lebrihi, et ai, 1988) and it has been suggested that CCR in some

microorganisms involves Cyclic adenosine monophosphate (cAMP) as a positive

effector (Pastan et aI, 1976). In antibiotic production, cyclic AMP is thought to

interact with a cAMP receptor protein (activator protein) to form a complex

which binds to the promoter sites of operons coding for inducible enzymes. These

enzymes then go on to activate gene transcription leading to the production of

antibiotic biosynthetic enzymes. High levels of cAMP in many organisms tends

to increase antibiotic production. For example, the addition of cAMP to

Streptomyces kanamyceticus cultures has been reported to relieve the glucose

repression of kanamycin production: apparently acting on kanamycin synthetase

repression (Satah, et ai, 1976). Increasing tylosis production by mutational means

in Streptomyces fradiae resulted in a 20 - 50% increase in intracellular cAMP

levels. (Colombo, et ai, 1982). However, evidence against a lac-operon-like

. system comes from observations of non-reversible glucose repression by cAMP in

actinomycin producers (Brown, et ai, 1983). Furthermore, the rise and fall of

cAMP levels during the course of batch culture is also inconsistent with a lac

operon-type model and it has therefore been suggested by Demain (1986) that

carbon source repression in Streptomyces species may in fact be operated by a

completely different mechanism, possibly involving glucokinase or its catabolic

products (Demain, 1986; Lebrihi et ai, 1988). It should be noted that no

molecular level evidence has yet been presented to confirm repression of the

14

-- ------------------------:----------------

transcription of genes coding for inducible enzymes by glucose mediated cAMP _

reduction.

Another nutritional factor of importance in antibiotic production is nitrogen, also

essential for cell growth as it is used to build up amino acids, nucJeotides -and -

hence essential proteins. It has been observed that low nitrogen concentrations

in a medium may significantly increase the antibiotic production whereas high

concentrations decreases production. To explain this, Aharonowitz (1980) has

suggested a form of ammonium repression of secondary metabolic productioD<

which occurs in many cultures where the ion is present in excess. He also

observed two mechanisms under ammonium assimilation in Streptomyces

clavuligerus cultures (Aharonowitz, 1979): a high affinity glutamine synthetase

glutamate aminotransferase (GS-GOGT) enzyme system which operated under

ammonium limitation, and glutamate dehydrogenase (GDH) which was very active

during ammonium excess. He revealed that high cephalosporin rates coincided

with peak GS-GOGAT activity whereas high rates of ammonium assimilation

decreased the production rate. Similar effects have _ been observed in

Streptomyces cattleya (Wax et aI, 1982)) and in Streptomyces venezulae (Vining

. and Chatterjee, 1982). Finally, Shapiro and Vining (1983) also suggested that

GDH and or GS-GOGAT may indeed be linked to a system for repressing

secondary metabolism. -How this system actually works at the molecular/genetic

level remains unresolved. -(Bushell, 1989).

Phosphorus has a role in the cell's energy metabolism and is present in ATP,

NAD and FAD. It is also a constituent of nucleic acids, phospholipids and

nucJeotides, (van Demark, 1986). -Phosphates are also known to regulate_

antibiotic production in many organisms and. an excess can often depress

production. -Two mechanisms have . been proposed to account for this:

repression/inhibition of biosynthetic phosphatases and indirect regulation via

adenosine phosphates which may act as intracellular effectors (Janglova, et aI,

1969; Madry, et al 1979).

-There has been for some time, controversy about which parameter is, in fact; the

intracdlular effector that governs antibiotic biosynthesis .. For example, Atkinson

15

I

I

~-- -~-----------:----------

et al in 1969, proposed an energy charge (E.C.) definition which they maintain

is the regulatory parameter governing all energy utilization/generating pathways

E. c. = ATP + V, ADP ATP+ADP+AMP

.This view however has not been universally accepted (Purich et ai, 1973; Curdova

et ai, (1976). According to Rickenberg (1974) and Bu'Lock (1974), synthesis of

secondary metabolites may well be regulated by cAMP which may be the

fundamental intracellular effector. Again, this view is not well established as

cAMP has not been reported in the Actinomycetales nor the bacilli (Rickenberg,

1974; Hanson, 1975). Finally, both Behal et al (1969) and Martin (1977) have

proposed that intracellular levels of NADPH is the limiting factor in antibiotic

biosynthesis, especially during idiophase.

To date, existing models to explain the regulatory mechanisms of antibiotic

production remain ambiguous in parts (Aharonowitz, 1980; Martin and Demain,

1980; Hunter and Baumberg 1989).

Unlike primary metabolites, such as amino acids, secondary metabolites became

important as products, at a time when little was known about their biosynthetic

pathways and this ~ greatly hamp~red efforts to increase yields by specific

addition/deletion techniques (Primrose, 1987). Even ~oday, details of antibiotic

biosynthesis in many organisms still remain unresolved (Nisbet and Porter, 1989).

One of the techniques (nutritional approach) involves the screening of hundreds

of nutrient additives as possible precursors of the desired product. Occasionally

a precursor is identified that increases production of the secondary metabolites:

an example being the addition of a-aminoadipate which stimulates penicillin

production (Baldwin et ai, 1987). Alternatively, the precursor may direct the

formation of one specific product (directed biosynthesis): an example being the·

addition of phenylacetic acid which promotes the formation of benzylpenicillin

over other penicillins (Primrose et ai, 1987).

Another technique involves a mutational approach and this is based upon the fact

that the. primary determinant of antibiotic type and of product yield, is the

16

organism itself. Consequently a major consideration is strain development in

, which random and induced mutation, fol1owedby selection procedures, are used

to obtain a superior antibiotic production strain. Mutants may be obtained via a

variety of means including X-ray and U.V. light bombardment, application of

mutagenic drugs (Fig. 1.5), continuous subjection to normal drugs at sub-MIC • • -f.

(minimum inhibitory concentration) levelsand the reaping of cells found growing

as secondary colonies on top of senescent colonies (Frobisher, 1959; Greenwood, c,

1984; Primrose, 1987).

Altogether, both nutritional and mutational approaches have been quite successful

but investigations into these can be quite labour and time intensive. Occasionally

they can be combined: for example, where addition of an amino acid has been

shown to be stimulatory, mutational efforts are focused on removing regulatory

controls on the biosynthesis of that amino acid.

Although, in general, research into mutational and selection procedures has been

hampered by lack of information on both the biosynthetic pathways for antibiotic ,

production and on pathway regulation, selective recombinant DNA technology has

recently been successfully exploited. Here, genes controlling entire biosynthetic

pathways can be cloned and the DNA sequenced. This will permit identification

of the number of gen~s i~~olved and would aiso be a good indicator' of the

, number of biosynthetic steps in the pathway. By deleting one or more of the

genes in the pathway, intermediates would accumulate and these can be identified

by conventional analysis. In this way, a clearer picture of the biosynthetic pathway

can be built up which reveals the normal regulatory circuits of the biosynthesis.

To enhance productivity, detrimental regulatory circuits can be eliminated and

additionally, some of the cloned genes can be placed under the control of a known

promoter (Primrose, 1987).

If genes involved in antibiotic biosynthesis are to be cloned, then methods for

their selection are required and here, a number of ways have been developed.

One method is illustrated by candicin biosynthesis in which a key step is the

conversion of chorismate by the enzyme P ABA synthetase. Cloning of the gene

for this synthetase was facilitated by the availability of a, direct selection method

17. I

------

Fig. 1.5:

Penicillium i chrysogenum Northern. ! 60 mgll NRRL-1951 Regional

i I Spontaneous Research

I "I' Laboratory 1 150me/l NRRL-1951,825 I t X-rays Carneo;ie

X-"'2 Institute

300 me/l

T Ultraviolet light

550 mgll WIS 0-176

~ , Ultraviolet light

wise 13-0 la

+ Spontaneous

WIS47-638 University

.t Spontaneous of Wisconsin

WIS47-1564

~ ·Spont~neous WIS48-701

~ Nitrogen mustard

WIS49-133

~ Spontaneous

WIS51-20

... Ultraviolet light

E-l

t Nitrogen mustard

E-3 I Nitrogen mustard "I'

E-4

~ Nitrogen mustard

E-6

+ Nitrogen mustard EIiLilly •

E-8 and Co.

+ Nitrogen mustard

E-9

+ Nitrogen mustard

E-l0

+ Nitrogen mustard

E-12

+ Nitrogen mustard

E-13

+ Nitrogen mustard

E-14

+ Nitrogen mustard

E-1S

{- Spontaneous

7911 E-1S_l

Use of mutation and selection in the development of improved

penicillin-producing strains of f. chrysogenum., (After Primrose,

1987).

18

using the restoration of P ABA Independence to a strain requiring P ABA for ..•

· growth (Gil and Hopwood, 1983). A second method involves the isolation of

mutants, which no longer produce the desired antibiotic and the identification of .

DNA fragments which then restore antibiotic synthesis (McAlpine et aI, 1987) ..

The third method is to determine whether the genes for antibiotic synthesis are

plasmid borne, as is the case of methylenomycin (Chater and Bruton, 1983;

Kinashi et aI, 1987) and if so, this would facilitate their subsequent manipulation.

Finally, another method relies on the fact that an antibiotic producing organism .

· has to be resistant to its own antibiotic otherwise it would be self limiting. Here,

in some cases, the genes specifying antibiotic resistance are linked to those

specifying antibiotic production. For example, Streptomyces IiVidans is not known

to produce any antibiotic related to erythromycin but when the Streptomyces·

erythreus gene for erythromycin resistance was cloned and transferred to it, s.. Iividans became an erythromycin producer (Stonzak, 1986).

· Just as novel proteins can be produced by recombinant DNA techniques, so can

novel antibiotics. The Streptomyces coelicolor gene cluster which is responsible

for the biosynthesis of the isochromanequinone antibiotic, actinorhodin, has been

cloned. When the cloned genes were introduced into a variety of other

Streptomyces spp., producing different isochromanequinones, at least three new .' ,.. -- .

antibiotics were detected. Actinorhodin or one of its precursors, thus appears to·

be a novel metabolite in these other Streptomyces spp, and is subject to further

or different enzymatic modifications (Malpartida and Hopwood, 1986).

A similar approach is used in the synthesis of (j-Iactam antibiotics in which a key ..

step is thecyclization of a tripeptide precursor. When the purified enzyme is fed

novel tripeptides, novel (j-Iactams are produced (Pratt, 1989).

1.4 The fermentation process

Shake culture is an aerated process which represents an important microbiological

technique and provides . a convenient· method of growing microorganisms in

submerged culture' (Calam, 1986). Generally, such aerated processes are

19

I

colloquially known as fermentations though sensu strictu, fermentation is a

biological process occurring in the absence of air (Primrose, 1987).

The shake culture method began to emerge in the 1930's and developed with the

rise of the' antibiotic industry as a small-scale testi~g method for ~edia

optimization in laboratory cultures (Calam, 1986). In industry, shaken cult~res are'

often used for the initial stages of inoculum production (Drew, 1981) and in cases

where it is aimed at screening or yield improvements, rather than at theoretical

studies, complex media are, used. With complex media, growth is at first rapid,

becoming visually apparent after approximately 24 hours for fungi and after

approximately 12 hours for bacteria. After this, the growth rate reduces at cell

concentrations of 10 to 30 gm/litre and this occurs after 5 - 7 days for mycelial

microorganisms and after 24-48 hours for bacteria. Product formation may also

begin 1 - 3 days after inoculation and continue for 3 - 4 days or more. The

duration of production may depend upon the number of viable cells present, the

pH, toxic end-products and product stability ..

The composition of the complex medium used (which could later be translated

to larger scale fermentations) is based on carbohydrates, various proteinaceous .

materials, vegetable oils and a few inorganic salts. During the fermentation, the - ,-.-'

medium components are metabolised at different rates and this provides a long

period during which conditions become. suitable for optimal growth and

metabolism (Calam, 1986). A simple chemically defined medium is however

preferred for studies on the physiology, biochemistry and regulatory mechanisms

of the producer organism, since it minimizes the probability of complicated

interactions between components of more complex medium fermentations.· The

composition of such a medium would, typically, have a specific basis of

carbohydrates, ammonium ions, phosphates, metal salts and vitamins (where

applicable), all of which are deemed essential for antibiotic production (Williams

and Katz, 1977; Greasham and Inamine, 1986;).

The shake and stirred fermentor culture techniques are two important examples

of the generalised technique of submerged culture. Both are much used and

involve aeration and agitation. They are extensively used with filamentous

20

microorganisms. The growth of the cells and the production of metabolites

involve a number of cellular processes such as the dissimilation of substrate

carbohydrates, the formation of intermediates and the generation of energy.

Results can be affected by the pattern of aeration and agitation, the characteristics

of the cells themselves, their efficiency and productivity and their response to

conditions in the fermentor. In the stirred fermentor culture where air is bubbled

through, the consequent agitation stirs and mixes the medium thus enabling

uptake of oxygen by the medium, mainly via diffusion from the air bubbles.· In the

shaken culture on the other hand, the medium obtains oxygen largely by direct

absorption from the air as the culture swirls around the walls of the flask in a thin

layer of large surface area (Calam, 1986). When 100 ml of medium in a 500 ml

flask is incubated in a rotary shaker, oxygen uptake rates of 0.3 to 9.5

mmol/litre/min can be achieved depending on the type of flask used (Miller,

1986). Overall, shaken flasks give much better growth and production than could

be expected from the rate of oxygen uptake (Cooper et aI, 1944) and this may be

due to the fact that there is less. shear stress than in the stirred fermentor

situation.

Differences in agitation and aeration between shakers and stirred cultures tend

to create different metabolic patterns which in turn can give rise to different yields

of the desired product. Mutants can be selected which respond well in one or the

other system and, as is often the case, conditions optimized in shaken flasks will

. not· be transferable to larger-scale vessels. In industry, a highly trained

interdisciplinary team has to be used to solve these problems associated with

scaling-up as experimental runs at this level would need to be, for reasons of cost,

understandably limited in number (Trilli, 1986).

In industry, tanks of 10-500,000 litre capacities are. normally used. The basic

fermentor design consists of a closed vessel fitted with an air inlet and an agitator.

Many other features however, are required and some of these are as represented

at Fig. 1.6. Addition of baffles to the vessel walls can improve the efficiency of

O2 transfer by increasing the turbulence of the agitated culture medium (Primrose,

1987).

21

Inoculum or nutrients

Temperature record and control

~ Steam

Antifoam probe

Anti· foam Alkali

Pumps

.. .. :.:~,~.: . .':t:~ ~;:', . .' " .' '. ,·;··:'~··;"".t:-tr;:~~-::-:· ~ ~.;.~:

Pressure

pH record and control

Filter

9l!2~!E ~ Cooling g; water

~Air

Cooling --"t~;m!ll!llillll& water

A1r-+-==I . Filter

Fig. 1.6:

Harvest line

Schematic representation of a stirred tank reactor. For clarity no

seal is shown between the agitator shaft and the fermenter body and

baffles have been omitted. (Mter Primose, 1987)

22

---- ------c--------;------------

1.5 Downstream processing and identification of antibiotics

Downstream processing is the term commonly used to describe the isolation and

purification of secondary metabolites from fermentation broths and other

biological matrices (Primrose, 1987). The difficulties often encountered at this

stage are legendary and may require several steps in order to effect separation,

(Fig. 1.7). This is because both contents of differentiated plant cells and of

microbial cells and the medium in which the latter has been grown, often presents

an extraordinarily complex mixture of primary and secondary metabolites (some

quite closely related). These metabolites often occur at concentrations which

range from nanograms to milligrams per millilitre where usually, only one of the

compounds may be of interest (White et aI, 1986). Common tools for separation

include thin layer chromatography, liquid phase/liquid phase chromatography,

column chromatography, ion exchange chromatography, high pressure liquid

chromatography and. gas chromatography. When· investigating unknown

antibiotics, especially from novel sources, researchers in this field prefer the first

three techniques as different mobile/solid phases can be more easily manipulated

in the ·search for the appropriate liquid and solid phases for isolation (Calton,

1986). A generalised diagram of a modern chromatographic instrument is shown

at Fig. 1.8. Of late, a new analytical/separation tool, known as super critical fluid

chromatography, has appeared on the market and this constitutes a significant

advance on existing instruments (Ndiomu and Simpson, 1989).

After isolation, certain properties of the antibiotic are investigated and this may

include accurate melting point determination which may help with identification;

pH and thermal stability. tests; efficacy against a range of microorganisms which

constitutes a test of its potential usefulness, and toxicity tests all intended to aid

in ascertaining its use as a chemotherapeutic agent (Arima, 1959 and del Rio et

l!!,1972).

To determine the structure of an unknown antibiotic, spectral analysis is necessary

and this may include ultravioletMsible spectroscopy: able to identify

chromaphores; infrared spectroscopy; not only able to identify functional groups

but valued for the unique "finger-print" it provides; mass spectrometry (MS): able

23

Fig. 1.7:

-- ------------------------------------

j •• ,. 1 .,' I .1 • ~ •••• ' ,/G

i ... ~'"",-i-j_y "j i j-.:.-l ......... " j •• • - y ! '. ~_'( t::::::::l·-Yneoxanthin I r.'!~·"dg"""'YG I I ." f..... I

~ Y . ~""""G

(b)

t::::=J.-Yvio1axanthii1

~a--YG chlorophyll b

lutein + zeaxanthin

chlorophyll a

carotenes

Colum chromatography. Successive. steps in formation of

chromatogram of green leaf pigments. G signifies green: Y, yellow;'

YG, yellow green. (a) Sorption of mixture on powdered sugar from

petroleum ether. (b) Partial separation after washing with petroleum

ether plus n-propanoI. (c) Further separation produced by continued

, washing. (After McGraw and Hill, 1987).

pressure or flow regulation

, s~urc' of . '---Q\o--i mobile phase r

chromatographic column

.. ~.. ;. . "~' . ~.:-":~'.;::::;;., . fraction .~0.L

Fig. 1.8:

sampling port

,~;:.~ignal-j.,-" ,::processing .... --~ =ac:cessories .

recorder

Schematic diagram of a modern chromatographic instrument. The

source of the mobile phase in gas chromatography is a gas cylinder;

in liquid chromatography, a high-pressure pump. Interconnecting

broken lines indicate the parts often used, but not essential to the

basic function. (After McGraw and Hill, 1987).

24

to determine with good confidence, the molecular weight; nuclear magnetic

resonance spectroscopy' (NMR): able '. to . give additional information on the

structural environment around atoms such as hydrogen, fluorine and phosphorous

(Aszalos, 1986 and Dudley et aI, 1987). However, where practical,by far the best

tool for, complete structure elucidation remains X-ray crystallography where

confidence limits of well over 90% are regularly achieved (Glusker and Trueblood,

1972).

1.6 . Some Pseudomonas antibiotics

Pseudomonas aeruginosa has been of interest since the early days of microbial

in.vestigation because it provided one of the first reported examples of microbial

antagonism when Bouchard showed in 1889, that it was antagonistic to some .

species of bacteria. Emmerich and Louw (1899) claimed it 'produced a substance

called pyocyanase in the belief that it was an enzyme which could cure

experimental anthrax. It is also well known from host-parasite inter-relationship

. studies that, as part of the normal flora on man, it can produce substances which

inhibit the growth of Candida species (Wistreich et aI, 1984).

Common strains of Ps. aeruginosa are known to produce a phenazine pigment,

pyocyanine· (Fig. 1.9), the blue colour of which, superimposed upon yellow

pigments also present, produce the typical blue-green colour of pus (Stokes et aI,

1946; Holliman, 1961). . Other examples of phenazine pigments include

hemipy ocyanine, also from Ps. aeruginosa (Schoental, 1945; Hays, et aI, 1945)

iodinin, from Ps. iodina (Gerber and L€:chavalier, 1964) and oxychlororaphine

from Ps. chlororaphis (Miller, 1961). Although at one time the known naturally.

occurring phenazines were all from the Pseudomonads or closely related.

organisms, at least two examples are now known from mould metabolism: These

include Calonuclria erubesai which produces phenazine-1-carboxylic acid, Coriolus .

sanguineus which produces cinnaborin, Streptomyces thioluteus which produces .' .

1,6-dihydroxyphenazine and Streptomyces grisofulvus which produces the

griseofulvins a and b (Cavill, et aI, 1953; Kluyver, 1956; Akabori and Nakamura,

1959; Holliman, 1961). Phenazines are generally known to exhibit a wide

spectrum of antibiotic activity; inhibiting the growth of bacteria, actinomycetes and

25

--- ---~~~~~~~~~---:~~~~~~~---c--

fungi with near to equal effectiveness (Gerber and Lechevalier, 1964). However,

in general, they are highly toxic and are intercalators by mode of action

(Albaraller et aI, 1985; Schafer et aI, 1985).

The genus Pseudomonas also produces other interesting heterocyc1ic compounds

which have antibiotic activity. The 'Pyo' compounds (Fig. 1.9) studied by Hays et

ill. (1945) and by Wells, (1952) are closely related 2-alkyl or alkenyl~4-quinolenes,

the N-oxides of which have been shown to be responsible for the streptomycin-like

activity of Ps. aeruginosa culture filtrates (Holliman, 1961). Others include

comirin (Forsyth, 1959), glyco-lipide (Jarvis and Johnson, 1949; Hawer and

Karnovsky, 1954) and viscosin (Groupe et aI, 1951; Ohno et aI, 1955).

In 1958, Takeda et al isolated a new antibacterial substance, pyoluteorin, from a

strain of Ps. aeruginosa. It turned out to be one of the first known antibiotics to

contain chlorine (Fig. 1.9). Then Arima et al (1964) isolated a new antifungal

antibiotic, pyrrolnitrin (Fig. 1.9), from Ps. pyrrocinia. This also contained chlorine

and is similar in structure to pyoluteorin. It is now commercially available under

the trade name of Pyro-Ace (Japan) and is administered topically against

dermatophytic fungi (Gorman and Lively, 1967) .

. Eg~wa (1970) isolated an antibiotic, referred to as YC-73 (later to be mown as

fluopsin; Fig. 1.9), from a Pseudomonas species. This same antibiotic with its

wide spectrum of activity was also found among three antibiotic substances

produced by Ps. reptilivora.

Fuller et al (1971) isolated an antibacterial substance, referred to as Pseudomonic

acid (Fig. 1.9), from cultures of Ps. fluorescens. It is now marketed under the ~

trade name of Mupirocin and is effective against bacterial skin infections.

Pseudomonic acid is also used in nasal ointment preparations (British National

Formulary, 1991).

In 1981, Kintaka et aI, isolated a novel p-lactam antibiotic, isosulfazecin (Fig. 1.9),

from an acidophilic pseudomonad, Ps. mesoacidophilia. The antibiotic is not as

powerful as existing p-lactam antibiotics.

26

Cl

PYOCYANINE

.r-r.--:T'-CI

PYRROLNITRIN

Cu++

FLUOPSIN

. OH Cl .

&:. c n-I 8 --(N)-CI

~ OH ~

PYOLUTEORIN

OH

~ UN~R

PYO COMPOUNDS [1b) R= (CH2)SCH3 [1c)R= (~aOil

[Ill) R= CH,:,CH(CH2lsO-b

HOOC H . HN . OCH3 ·

HN~CO"X HN~ 2 CO" N

. . 0 . 'S03H

ISOSULFAZECIN

~H OH OH

OH

CH3 H H

.' H R o

R = H

PSEUDOMONIC ACID A

Fig. 1.9: Structures of some Pseudomonas antibiotics.

27

More recently, at least four new antibiotics have been isolated from Pseudomonas

species, three in Japan and one in the UK. They include antibiotic CB-104, an

antibacterial substance (Takeda, 1983); antibiotic Tan 447, a novel macrocyclic

lactone effective against both Gram positive and Gram negative bacteria (Takeda,

1984); antibiotic Tan 456, a polyene antifungal substance (Takeda, 1984) and

MM42842, a novel monocyclic ,8-lactam antibiotic (Gwynn, 1988).

Pyocin, a bacteriocin produced by Pseudomonas, was first reported by Jacob in

1952. It is regarded as a protein with a structure which resembles the lower part

of a bacteriophage (Ishii, 1965; Shinomiya, 1983). It is thought to attack the cell

wall of members of the same or allied species forming pits and thereby causing

cellular leakage. This phenomenon has been exploited in the taxonomy of the

pseudomonads and is known as "pyocin-typing" (Gillies, 1984). Pyocin is stable

. at 55°C but exposure at 70°C for ten minutes completely inactivates it (Kageyama

and Egami, 1962).

1.7 .A novel Pseudomonas

In the early 1960's, in Ghana, during research studies into the control of the

'Black Pod' disease of cocoa, caused by the fungus Phvtophthora palmivora, a

baCterhim was isolated from the gut of the mealy bug Planococcoides njalensis

(Fig. LlO}. It was first identified as a local strain of Ps. aeruginosa by Dr. Y.

Tanada, of the University of California (Auafuah, 1965) but later, during the

course of the present study, reclassified as a new species of Pseudomonas, to be

known as Ps. antimicrobica (Attafuah and Bradbury, 1989; I.J.S.B., 1990). Work

on the organism showed that it was capable of producing substances with a wide

spectrum of antibiotic activity, over and above that displayed by Ps. aeruginosa

itself. Its first application (mid-1960's) was as the antagonist in a carefully

monitored, pilot biological-control programme against Phytophthora. Here,

compared to the controls, it proved quite successful as the development of 'black

pod' disease was not detected on Phytophthora inoculated pods (Attafuah and

Bradbury, 1989).

28

I

Fig. 1.10: A photomicrograph (Mag. X4) of the mealy bug Planococcoides

njalensis from which Ps. antimicrobica was first isolated.

(Reproduced with kind permission from Attafuah, A.).

1.8

The aims (Fig. 1.11) of this study was to:

i) examine the culturallbiochemical characteristics of the

microorganism;

ii) develop a liquid medium able to elicit production of active - -- -- ------ ---- - --._- - ---- ._-.- ----- .. --- .

substance/s from strain A;

Hi) . optimize production of the active substance/s;

29

---- ------~--------------------~--------------

iv) . develop a procedure/s for the separation and isolation of the active

substance/s;

v) investigate some physical, chemical and biological properties of the

active. substance/s;

vi) . attempt an· elucidation of the structure/s of the active substance/so

~.' ..

30

w -

Fig. 1.11

ClASSIFICATION OF BACTERIUM (BIOCHEM TESTS)

..

-... ,- - -

X·RAY CRYSTALLOGRAPHY

STRUCTURAL FORMUlA

r-

r-

I-

A FLOWCHART OF THE PROJECT

MEDIA FOR CONSISTENT . ANTIBIOTIC PROD.

SPECTRAL ANALYSIS MASS SPEC.

.

CHEMICAL TESTS .

EMPIRICAL FORMULA

MEDIUMB (Antibacterial) AB -

MEDIUMC AF/AB

EFFICACY AGAINST MICROORGANISMS

OPTIMIZATION . APPROPRIATE OF ANTIBIOTIC SOLVENT PRODUCTION - EXTRACTION .-

SYSTEM FOR ANTIBIOTIC IS

PREP.TLC . r ISOlATION

OF ANTIBIOTIO-

STABILITY 1- -TESTS

.

SOLVENT EXTRAC-TION

"- STRATEGY-ISOlATION OF ANTI-BIOTIC

CHAPTER 2

MATERIALS.AND METHODS

MATERIALS AND METHODS

2.1 MATERIALS

2.1.1 Microorgariisms

a) Bacteria

Pseudomonas spp; NCIB 9897 - strain A

Pseudomonas spp. NCIB 9898 - strain B

Pseudomonas aeruginosa NCIB 6749

Pseudomonas antimycetica NCIB 8641

Pseudomonas maltiphilia NCIB 9201

Pseudomonas cocovenenans NBIC 9450

Escherichia coli NBIC 9001

Salmonella abony NCfC6017

Serratia marcescens NCfC 11879

Bacillus cereus NCIB 9373

Staphylococcus aureus NCIB 8625

---~ -,---Staphylococcus epidermidis NCIB 7944

Clostridium sporogenes NCIB 532

b) Fungi

Saccharorrwces cerevisiae NCYC 345

Candida albicans NCYC 597

Aspergillus niger IMI 31821

Sporothrix schenckii IMI77984

Penicillium chtysogenum IMI26211

32

I

I

2.1.2 Routine Media.

a) Nutrient Broth (NB)

Substance

Lab lemco

Peptone

NaCI

Amount (g!!itre of deionised H20)

10.0

10.0

5.0

b) . Neurospora Broth (Nsp B)

Substance

Maltose

Yeast extract

Amount (g!!itre of deionised H2Q)

38.0

2.5

Mycological peptone 3.0

Malt extract 2.0

c) Ringer's Solution

. Substance Amount· (g!!itre of deionised H~

NaO 2.25

KO 0.105.

CaCI2 0.12

NaHC03 0.05 ,

FeS04 ·0.01

~S04 0.35

33

d) . Potato dextrose liquid medium (PDLM)·

Substance Amount (gIIitre of deionised H20)

Potato extract.

Glucose

4.0

20.4

Where appropriate, solidified versions of the above media were made by the

addition of Technical Agar (No. I) at 1.5-2.0% (w/v).

Ail media (unless otherwise stated in text) were sterilised by autoclaving at·

121°C, 15 lb/sq in for 15 minutes.

The Lab Lemco was obtained from London Analytical and

Bacteriological Media Ltd., Salford U.K.. The NaCl, KOH and glycerol were

obtained from East Anglia Chemicals, Hadleigh, Ipswich, u.K. Magnesium

glycerolphosphate was from Hopkin and William Ltd., Essex, U.K. The Yeast

Extract, Technical Agar (No. 1), Potato Dextrose Agar, Potato Extract, and.

Bacteriological Peptone were obtained from Oxoid Ltd., Basingstoke,U.K.

. The remaining substances. came from.. Fisons Scientific Apparatus,

Loughborough, U.K.

Ail chemicals (unless otherwise stated in text) were of standard laboratory'

reagent (SLR) grade.

, .. 2.1.3 Growth Curve Media

a) Basal medium (Cruickshank, 1972).

Substance Amount (gIIitre of deionised H20)

NH.Q 3.0

MgCI2.6H20 0.2

34

I

- ------------------------------------------

Na:zSO.

KH2PO.

K2HPO.

Glucose

b) Strain A's Medium

0.2

13.6

17.4

3.0

Basal medium plus histidine and phenylalanine each at 0.1 !iJIitre.

c) Strain B's Medium

Basal medium plus methionine, arginine, lysine and ornithine each at

0.1 g/Iitre. '

d) Ps. aeruginosa's Medium (Guirard, 1970)

Basal medium plus alanine, leucine, lysine, ornithine, histidine each at

0.1 g/Iitre.

e) Yeast Extract Medium

Basal medium plus Yeast Extract at 5 g/Iitre.

2.1.4 Media tested for antibiotic production (See Table 2.1)

a) Nutrient Broth (NB)

b) Neurospora medium broth (NspB)

c) Basal medium minus glucose (BM1)

35

-----------------------------------------------

d) Medium MSl

Substance Amount (glIitre of deionised HJU

KN03 ·5.00

~HPO. 0.40

MgSO •. 7H20 2.00

FeSO. 0.Q1

e) Medium MS2

Substance Amount (gIlitre of deionised H20)

NH.CI 0.40

MgSO •. 7H20 0.25

~HP04 0.25

f) Czapek Dox Liquid Medium minus sucrose (CD1)

Substance Amount (gIlitre of deionised H20)

NaN03 2.00

KO 0.50

Magnesium glycerolphosphate 0.50

FeSO. 0.01

g) Potato Extract (PE)

Variations of the above media were also made by making various

additions/adjustments to their basic formulae (See Table 2.1).

36

';><:":·',:."~"·.·'r~ -i" ,: .. -i . .. ~ I ~:~ .. :,:~t.:..

w "

Table 2.1

MEDIA TESTED FOR ANTIBIOTIC PRODUCTION

.., L- D-:E LW leucine glycine phenyl- rrethio- omi':' => - alanine Cl WJ :s

2 g/L 2 g/L 8g/L 4 g/L 0.1 g/L 1 g/L 0.1 g/L 0.1 g/L 0.1 0.1 g/L

NB

Nspa

fJ~1 d d e e e e

t-lS1 i i i i

M3.2

0 0 8)1 s s

Variations on media listed at Section 2.1.4(a-g) tlere tes;;ed for antibiotic production. (Each modified medium is defined in the table by a 10vler case letter. (See Table 3.5)

Example: ~ledium 9 consists of ~Iedium BMl plus (13.6 gill" glycerol (50 ml/L), FeS04 (0.01 gill at pH 7.2

glucose

g/L 5 g/L

j

u

~'V' _

-. ..,.'." ,;,. .-.

,~ ,

3 g/L I

~sucrose

5g/L 30 --- ~-

k

;·t· .. ~. ::', . . . .]. ;-

.

I ,

Fe9J4 Agar CaC03

15 g/L 3 g/L 1 g/L 5 g/L 7.4

9 f

I m

q p r

y ·V W x

g/L

i

o s

glycine

4g/L

o s

d

0.1 g/L 0.1 g/L 0.1

d e e e e

i

I isted at Section 2.1.4{a-g) \'Iere tes'~ed for antibiotic production. um is defined in the table by a IO~ler case letter. {See Table 3.5}

consists of ~Iedium BMl plus {13.6 giLl" (50 mIlL). FeS04 (0.01 g/L) at pH 7.2

u

glucose .sucrose

g/L, 5 g/L: 3 g/L • ,5 g/L 30

~':\ ". " ... ' . . ,-.-, : ~~ .. ;

,~ -,

j

f h

k

. i- .. ~' ". "--.. -......--, ' -~ ------------~-------- ..

9

q

I

Agar

15 g/L 3 g/L 1 g/L

p

y

I m

r

1562149A 7040305 1 >rlw'rYk

'v w x

Yeast

Extract

5 g/L

f

7.4 7.2

i

6.8 5.6

b

m

t

2.2 GENERAL METHODS

2.2.1 Culture maintenance

Cultures were obtai~ed as freeze-dried powders and were resuscitated according to

the supplying culture collection instructions. All bacteria used were grown on nutrient

agar (NA) slants at 31lC and all fungi used were grown on neurospora medium agar.

(NspA) slants at 30"C. After static incubation to sustainable growth they were all

stored in the dark at 4°C and subcultured every four weeks~ Culture purity was

routinely checked by subculture on selective media and/or by microscopic (X100 to

. XIOOO oil immersion) examination of the morphology and Gram staining attributes

(Gillies and Dodds, 1984).

2.2.2 Working Stocks

The organisms to be used were first inoculated from NA master slants onto fresh NA . 11., A- . .

slants and incubated overnight at 31lC for bacteria, or ontolslants and incubated·

ovemight at 30"C for fungi .. These were then subcultured at regular intervals to serve

as working stocks. Inoculi (colonies for bacteria and spores for fungi) from these

were then transferred into NB for bacteria, NspB for fungi and grown ovemight.

under static conditions at 37°C and 30"C respectively, prior to being used.

2.2.3 Inoculum development

Strain A, obtained from the working stock, was streaked onto a NA plate which was.

incubated ovemight at 37°C. Colonies· from these were then inoculated into basal, .

medium and grown at 37°C in a rotary incubator to an optical density (OD; 650 nm;

1 cm path) of 0.3 - 0.5. Samples of this culture were used as standard inocula .

. 2.2.4 Biochemical(folerance tests

Altogether, 31 biochemical/tolerance tests we~e performed on strain A. With the

exception of urease, haemolysis, nitrate reduction and ONPG tests, which were after

38

--------------------,-------------------

Cowan and Steel (1974), all the rest were after MacFaddin (1983): Strain B (P9898),

· Ps. aeruginosa, Ps. antimycetia ~nd Ps. maltiphilia were also tested at the same time,

and acted as controls.

2.2.5 Transmission Electron Microscopy (TEM) Negative Staining

The specimen was thoroughly washed with deionised water by centrifuging and

· resuspending (X5) at 4000 revolutions/minute (r.p.m.) for 15 minutes. Next, the

negative stain, phosphotungstic acid was added. A droplet of this suspension was

· then put onto a carbon-stabilised formvar coated grid and allowed to dry. Finally, the

grid was viewed and photographed using the AEl EM6B transmission electron

microscope (60 or 80 Kv accelerating voltage and Agfa 70 mm Rapidoline F071P

Film).

2.2.6 Selective Medium Development

Multidisk code U4 (Oxoid Ltd., Basingstoke, U.K.) was used to obtain information

on the antibiotic sensitivities of the test bacterium imd also· of a number of other· .

microorganisms. From these two sets of information, a selective medium for the

bacterium was made by including in the growth medium, an antibiotic or a

. combination of antibiotics (at the stated concentrations), that permitted growth of the

subject but decisively inhibited the growth of others tested.

2.2.7 Growth curves

Using a Pasteur pipitte, two drops (approximately 60 Ill) from the broth culture of the

test microorganism was used to inoculate 50 ml of basal medium in a 250 ml conical

flask. This was incubated in a Gallenkamp Compenstat orbital incubator, set at 37°C

and 90 r.p.m., for 18 hours. A portion of the culture was then poured into a sterile

centrifuge tube and centrifuged at 4000 r.p.m. for 15 minutes at 30°C, using a MSE

39

Mistral 6L· centrifuge. After this, the supematant was decanted and the pellet.·

resuspended in a small portion of the appropriate liquid medium. Using a sterile

pipette, some of this culture suspension was inoculated into an autoclaved 250 ml

conical flask containing 50 mls of the same liquid medium, until the required starting

optical density (D.D.) was obtained (typical D.D. = 0.06). The flask was then

incubated in the rotary incubator at the desired temperature and r.p.m. Samples of

the medium were taken out at set time intervals and the O.D. monitored on a

Unicam S.P. 500, series 2, u.v.Msible spectrophotometer, ata wavelength of 650 nm,

. using a 1 cm plastic cuvette.

2.2.8 Freeze drying

An aqueous sample in an uncapped container was slope frozen in a freezer and then.

placed inside the perspex chamber of a SB6, 3 litre condenser freeze drying unit. The

air in the chamber was evacuated until all the ice had sublimed leaving behind the

freeze-dried sample.

2.2.9 . Some comparison/acidophily tests (Strain A and Ps.cocovenenans) ..

The method described by Gwynn et al (1988) was used. . Basically, the test

microorganism was streaked out on N.A. (acidified with the addition of Hel to pH

4.0) and then incubated. Growth of strain A in standard laboratory reagent (SLR)

constituted basal medium, pH 7.2; in analar reagent (AR) constituted basal medium

from which MgQ2.6H20, had been omitted, pH 7.2 and in the former medium but this

time at pH 4.0, were also assessed. The growth assessment method was as described.

at 2.2.7. Pseudomonas cocovenans was grown in tandem for comparison.

40

2.2.10 Preliminaty detection of antifungal activity

Petri dishes were placed on a Shandon Scientific spirit-levelled, flat-top table and

molten agar (SO°C) poured into the dishes. After the agar had set, the plates were

surface dried (open and inverted) in a 37°C incubator for 20 minutes. A drop of A.

niger spore suspension was.-then put at one end of the plate. Using a sterile loop, the

test Pseudomonad was streaked onto the opposite half of the plate. A control was

made just as described above but this time no Pseudomonad was applied. After 7

days incubation at 300C, the plates were examined for invasive growth inhibition and' '

compared with the control.

2.2.11 Preliminatyextraction of water-soluble, active substances

Four agar plates were prepared: 2 of NA and 2 of PDA Using a sterile loop,

colonies of the test Pseudomonad were streaked onto 3 quadrants (3/4) of one NA

plate and then streaked onto 2 quadrants (1/2) of the other NA plate. The same was

done for the PDA plates. All the plates were then incubated at 30°C for 7 days.

After this time, a flame sterilised knife was used to carefully cut the unstreaked areas

into small cubes and these were aseptically transferred into pre-autoc1aved,

appropriately labelled universals containing 10 ml of deionised water. The universals '

were left to stand for 24 hours. Then, using a 1 in diameter membrane filtration unit,

they were filtered into sterile universals containing Whatmans discs (Whatman's Ltd.,

Maidstone, U.K.). The filtrates were then freeze-dried and redissolved in 1 ml of

deionised H20. Two seeded NA plates were made by adding one drop each of E. coli '

suspension into a universal containing molten NA (48 - S2°C). They were then gently

shaken, poured into petri dishes and allowed to set. The same procedure was

followed for ~. aureus, A. niger and C. albicans but for the latter two, molten NspA

was used instead of NA. Ethanol-flamed forceps were used to transfer the Whatman

41

discs onto the agar plates and sterile Pasteur pipettes were used to transfer samples

of the liquid into corresponding cork-bored holes (9.4 mm diameter). Finally, the

plates were incubated at 37°C for the seeded bacteria and 30°C for the seeded fungi.

They were examined after 18 hours for growth and zones of inhibition.

2.3 ANTIDACfERIAL ACTIVITY STUDIES .

2.3.1 Elicitation of antibacterial activity in liquid media

During the . search for a suitable Iiquid~edium. enabling production of the

antibacterial substance, all the media as outlined at table 2.1 were tried. ·The assay

method described below was used to test for activity.

Assay Procedure for Antibacterial Activity

i) An overnight (12 - 18 hr) culture of the organism in basal medium was grown

at 90 r.p.m., 37°C.

ii) A portion of this was then centrifuged at 4000 r.p.m. for 15 minutes at 30°C

and the pellet resuspended in some of the medium to be inoculated.

iii) The medium was inoculated (50 mls in a 250 ml conical flask) to give the

desired O.D.

iv) It was incubated in an orbital incubator at 90r.p:m., 31lC for a set time.

v) 3 ml samples were taken out at set times and theO.D. and pH measured.

42

--------------------------

vi) The samples were centrifuged at 4000 r.p.m. for 15 minutes at approximately

2°C and the supematants were membrane-filtered (1 inch diameter membrane-·

filtration unit) directly into sterile universals containing 13 mm Whatman's.

discs.

vii) Ethanol-flamed forceps were used to pick up the wet discs and these were

drained, then placed onto pre-seeded (one drop of test organism culture into

molten agar (48 - 52°C)) agar plates.

viii) A pre~incubation time of 3 hours at 4°C was allowed before incubating at 37°C

for bacteria and 30°C for fungi.

2.3.2 Pigmented inoculum eXJleriments


Colonies of strain A from a NA plate were inoculated into basal medium and

groWn overnight at 90r.p.m., 37°C to achieve pigmentation. Samples of this

was then used to inoculate the test medium so as to give the desired starting

. O.D. Procedural steps iv) to viii) of 2.3.1 were then followed.


This experiment was as described at a) but without shaking.

43

I

I

---- --- -------------------------

2.3.3 Preliminary Testing of the Active Broth

a) Heat stability

Procedural steps i) to vi) of 2.3.1 were followed and 2 samples (3 ml) from the

same flask, having detectable antibiotic activity, were taken out. One was

heated in a boiling water-bath for 30 minutes at 100°C and the other acted as

a control. Using sterile forceps, sterile Whatman's discs were placed in each·

of the universals. Procedural steps vii) to viii) of 2.3.1 were then followed.


Using a pasteur pipette, 10 drops of Ehrlichs reagent were added to 2 mls of

a membrane-filtered, biologically active filtrate in a test tube. The test tube

was shaken vigorously and then left to stand for a few minutes. A violet

colour is indicative of pyrrols.


a) Medium component single-deletion from the SLR basal medium.

This is a variation of the method used by Guirard (1970) in which instead of

amino acid single deletion, medium component single deletion (MCSD) was·

made: . one ingredient was omitted from the basal medium to constitute the .

new test medium. Procedural steps i) to viii) of 2.3.1 were than followed.

44

b) MCSD from the AR basal medium

The same procedure as at 2.3.4 a) was followed but this time chemicals of AR

grade were used instead of SLR grade.

c) 'Variation of nutrient concentration

The concentrations of the following sequence of medium components were

varied with associated screening (2.3.1) at each concentratiori and the nutrient,

concentration which gave the best antibiotic production was carried forward

along the sequence:

i) Glucose (0.0 - 1.0%)

ii) NH4Cl (0.0 - 0.3%)

d) Variation of physical parameters

, , The effects of varying the following seque~ce of parameters on antibacterial

agent production were also studied, in like manner, as at ) and here again the

optimum conditions for antibiotic production, obtained at each step along the

sequence, was carried forward to the next:

i)" pH (3.0 - 9.0)

ii) Agitation/aeration (0.0 - 180 r.p.m.)

iii) Temperature (27 - 47°C)

iv) Initial inoculum level (O.D. 650 nm; 0.0 - 0.2)

45

2.3.5 . Cultivation in a laboratoty-scale ferinentor

A 6-litre Biolafitte, type BL06/l, laboratory-scale fermentor, with temperature,

airflow, Rushton-type impellor and pH monitoring/regulating device was used (Fig.

2.1). Four litres of the appropriate medium, optimised for antibiotic production, was

poured into the fermentor and this was then autoclaved. The inoculum, a culture

already producing antibiotic in basal medium and previously grown in the orbital

incubator at 31lC, 90 r.p.m. for 18 hours, was added until an initial D.D. of = 0.06

was achieved~ The airflow rate, temperature and stirring rate were as determined by

prior experimentation. Monitoring of antibiotic production took place by collecting

samples of broth at various times and then assaying against E.coIi (agar-plate

diffusion).


Stochastic error calculations (involving coefficient of variations) for the whole

experimental procedure, starting from shake-flask cultivation to measurement of

.. zo~es of inhibition, were don~. Standard deviation error-bar calculations were· also

computed for each datum point.

2.3.7 Thin layer chromatography (TLC) plate preparation

a) Analytical TLC

A number of 5 cm x 20 cm analytical TLC plates were made with Keisegel

G60 silica of 0.25 mm thickness using a Shandon Unoplane apparatus. Prior.

to being used, the plates were put into a 110°C oven for 2 hours to activate

them. They were allowed to cool, then 'cleaned' by eluting right to the top

with methanol/chloroform (1/9) in a glass, Shandon Southern Chromatank.

46

Turn s/m i n indi cator _____

Temperatore __ i ndicator

Air fl ow ________ indi ca tor

Fig. 2.1 : A Biolaffette 6-l itre laboratory-sca le fermentor in operation.

Precautionary inlet a nd outlet air filtering devices have been fitt ed to

the fermentor and then connected in series to 2 dresder nasks .

Ai r out l et (\~l i te rubber tub ing to out side)

b) Preparative TLC

Basically the same procedure as at a) was followed but here, a single large

glass plate of 100cm x 20 cm dimension was used and the silica spreader was

set to give a thickness of 1 mm. After drying, it was put in a Shandon

activator oven, set at. 150°C for 2 hrs, to activate it. It was then 'cleaned' as

before.

2.3.8 Detection of active compound

a) Ultra-violet light observation

150 mls of methanol/chloroform (1/9) was poured into the glass Chromatank

which had placed in it, 2 (3" x 7") filter papers. After covering the tank, it was.

left for 2 hours for the solvent vapour to achieve saturation. Using a capillary

tube (601'1), an analytical TLC plate was spotted and dried (20 mm from the·

end) several times with the active broth filterate. The plate was then put into

the tank and elution allowed to commence until the solvent front had reached

a pre-determined mark (typically 15 cm from the baseline/original spot).

After this, it was taken out and dried. The position and colour of any eluted

spots visible under normal light was recorded. The position, colour and

fluorescence/colour of any spot viewed under a BTL U.V. light viewer at 253.7

nm and then at 356.0 nm were recorded.

b) Charring

The same procedure as per a) was repeated. The eluted analytical TLC plate

was then carefully sprayed in a fume cupboard, using a Universal Aerosol

spray with a 1:1 ratio of cone. H2S04 and glacial acetic acid. The plate was

48

.. finally put in a 110°C oven and left for 30 minutes to char so as to reveal spot

locations.

c) Bioautography

The method described by Aszalos & Issaq (1980) was initially tried but,

because results were inconclusive, the following procedure was used: first, the

procedure as outlined at a) was repeated. Then positions of the spots as they

. appeared at a) and b) above, on a single plate, were identified and the plate·

appropriately sectioned to include all the spots. Each section was then·

scraped into appropriatelly labelled sample tubes containing a set yolume (5

mls) of ethanol. The sample tubes were stoppered and thoroughly shaken.

The decants from these mixtures were then assayed against E.coli by using

samples from them to fill cork-bored wells (No.5) in seeded N.A. plates and

then incubating these overnight at 37°C.

2.3.9 Isolation and purification of the antibacterial principal

a) Determination of appropriate solvent systems for extraction

i) Solvent for broth extraction

An approach, based on the use of the solvent system strategy developed by

Aszalos and Issaq (1980), for identification of antitumour antibiotics (Fig. 2.2)

was followed. With this approach, however, the main object was to see which

solvent system caused best separation when the analytical TLC plates ·were

spotted with the active broth filtrate. This gave rise to solvent 1.

49

(}1 o

PrOH:3 AcOH:1

(1.)

Fig. 2.2:

MOVED IN NONE

PrOH:8 PrOH:8 H 2O:2 H2O:2

AcOH:l AcOH:2 Pyridine:l Pyrldine:l

(lb) (lc)

CHCI3

(A)

'·4 PrOH:IO H20 : 8:

AcOH:I! Pyridinru:1i

(til)'

ACTIVE BROTH FILTERATE I

EIOAc (8)

11

AcOH:l0 EIOAc:90

(3al

I MeOH:l0 (C) CHCI

3: 90

III

MOVED IN C.D

MeOH (0)

111-4

NH.t0H:5 Acetonitrile CHC13 :33

Acelone:95 (3c) Acetone:67

(3b) (3d)

tV V

MOVED IN A. B. C. D.

MOVED IN 0 MOVED IN B. C. 0 11·1 11·2 11·3 IV·1 IV·2 IV·3

Acelone AcOH:l0 NH4OH:4 MeOH:2 MeOH:4 MeOH:8 (2.)

MeOH:20 MeOH:20 Benzene:98 Benzene:96 Benzene:92

CHC1 3:70 CHC13:76 (40) (4b) (4c)

(2b) (2c)

A modified Aszalos and Issaq solvent system'flowchart (after Chan and Aszalos, 1986), originally developed for the

identification of antitumour antibiotics but used here to determine the appropriate solvent system for the active broth

filtrate.

ii) Solvent for Iiquid/liquid extraction (see b below)

Analytical TLC plates were again used in the manner described at i) to obtain

. the appropriate solvent system but this time the following solvent systems were

tried: ethylacetate, acetone/ethylacetate (1/9), hexane, acetonelhexane (1/9)

hexane/acetone/ethylacetate (9/1/9). This gave rise to solvent 2.

iii) Solvent for TLC extraction (see c below)

The same procedure as at ii) was repeated but this time using the following

solvent systems: ethylacetate, H20, methanol, methanol/chloroform (1/9).

This gave rise to solvent 3.

iv) Solvent for trituation (washing)

The same procedure as at ii) was repeated but this time using the following

. solvent systems: hexane/acetone/ethanol . (90/10/45) and (90/10/1),

hexane/ethylacetate (9/1), hexane. This gave rise to solvent 4.

b) Liquid/liquid extraction procedure

Three, 2 litres samples of solvent 1 were separately shaken with equal volumes

of the same acidified (pH 2 with 5 M HCI) broth supernatant in a 5 litre

bottle .. In each case the solventJayer was separated in a separating funnel and

reduced in volume to approximately 10 mls under vacuum, using a Rotavopor

R, type KRvr 65/45, rotary evaporator.

All 3 solvent reductates were added together then washed 5 times with 1%

NaHC03 in a 9:1 ratio (solvent:NaHC03). The NaHC03 layer was acidified

51

--- ---------------

to pH2 then extracted 3 times with solvent 1. The solvent was then washed

with 1 % NaHC03 exactly as before. After separation, it was added to the

reductates, ,completely evaporated under vacuum and the residue re-dissolved

in 50 ml of deionised water. A small amount of activated charcoal was added

and stirred before membrane filtration, using a 0.45 I'm WCN, Whatman's

cellulose acetate membrane filter. The filtrate was finally extracted 2 times

. with an equal volume of solvent 2.

Solvent 2 was discarded and the aqueous layer freeze-dried to obtain a yellow

powder.

c) A liquid/liquidfTLC extraction procedure

Two litres of solvent 1 was shaken with an equal volume of broth supernatant

in a 5 litre conical flask. Using a separating funnel, the solvent layer was

separated and reduced in volume (10 mls) under vacuum, with the rotary

evaporator. This procedure was repeated 3 times and the 3 solvent reductates

mixed. A small amount of activated charcoal was added and the mixture

stirred, then membrane filtered using a 0.45 I'm cellulose acetate filter. The

filtrate was applied (3/4" from the end) onto a Keisegel G60 preparative TLC

plate, using a System TNO Delft (Desaga Heidelberg) with a mounted, sliding,

glass, air pressure applicator. The plate was then placed in a stainless steel

preparative TLC tank containing 1.5 litres of distilled ethylacetate. After

elution, the plate was dried and first viewed under ordinary light, then U.V.

light, using a portable model UV5-11 mineral light lamp with shortwave UV

(254 nm), to identify the characteristic antibiotic band. This band was then

scraped off into a conical flask containing a magnetic stirrer. Distilled ethanol

was added and it was then stirred for 10 minutes using a Stuart Scientific

magnetic stirrer. After allowing the silica to settle, the decant was filtered

using 0.45 I'm cellulose acetate membrane filter.

52

---- _. - --------------------~-

The residual silica was resuspended in fresh ethanol and magnetic stirred. The

whole procedure was repeated once more. All 3 filtered decants were then

added together and reduced under vacuum. The concentrated solvent solution.

was allowed to slowly evaporate giving rise to crystals. Finally, the crystals .

were trituated with distilled hexane .

. 2.3.10 High pressure liquid chromatography (HPLC) of isolate

In order to check for purity, an HPLC run of the isolate was made. Initially

it was important to find out whether the unknown antibiotic would be retained

in the HPLC column thus causing column contamination problems. This was

done by spotting an analytical TLC plate with a solution (5 x 60 1'1) of the

antibiotic. The spot was then eluted with a solution A, (26 mM trifluoroacetic

acid, buffered to pH 3 with triethylamine). The same procedure was repeated

with a solution B, (49% acetilenitrile in 13. mM of tritluoroacetic acid, buffered

to pH 3 with triethylamine). Any movement of the antibiotic spots in either

of these solutions, strongly suggests that it. might safely pass through the

column.

A uv/vis scan of the purified antibiotic, between 200 to 700 nm was made, so

as to reveal a characteristic U.V. profile. The wavelength at which the most'

significant peak occurred was noted and used to set the HPLC u.v. detector.

A Gilson HPLC apparatus with 2 pumps, model 302; . dynamic mixer, model

811; manometric module, model802C; holochrome U.V. apparatus and chart

recorder, N.I., was used. The HPLC was attached to an Apple lIc personal

computer with an automatic gradient facility and programmed for a fixed

gradient between 20% solution A to 80% solution B in 20 minutes. The

column type was Altex ODS, C18 reverse phase (internal diameter, 4.6 mm., .

length, 25 cm., particle size, 5 I'm). The volume of injection of the aqueous

antibiotic solution was 100 1'1 and the flow rate was set at 1 ml/minute ..

53 I

I

.2.3.11 Dose-Response calibration cutve

A dose-response calibration cutve was made by first assaying the following antibiotic

concentrations: 1.0,0.1,0.05, 0.025, 0.0125, against E. coli, as previously (see section.

2.3.1). From this, a graph of zone of inhibition versus log of antibiotic concentration

w~s plotted and this enabled assessment of antibiotic concentration in fermentation·

broths.

2.3.12 Calculation of percentage efficiency of extraction

Using the dose-response cutve, a calculation of the percentage efficiency of the whole

extraction procedure was made.

2.3.13 Physical properties

. a) Melting point

A melting point determination of a crystal of the antibiotic was carried out using the

Reichart-]ung melting point apparatus.

b) pH/thermal stability test

The was similar to the method used by Arima et al (1965). The antibiotic crystals

were dissolved in a series of K1/Kz orthophosphate buffers which had already been

made up to the desired pHs, to give each an antibiotic concentration of 1 mg/mI. Set

volumes of these were dispersed into appropriately labelled, small sample tubes which

were then stoppered with plastic caps. Next, they were placed in a water bath at the

desired temperature for set times. Aliquots taken out after these times, were.·

individually assayed for strength of activity against E. coli (agar-plate diffusion) ..

54

c) . U.V. light stability test

Three mls of an aqueous solution, of the antibacterial substance, at a concentration

of 1 mg/ml, was put into a Unicam 10 mm U.V. silica cuvette. The cuvette was

placed in a Bassaire U.V. sterilization chamber with a TUV 15 watts U.V. fluorescent 'I light, producing a surface impact intensity of 400 lux. At set time intervals, 200 ILl

aliquots were taken out and assayed for activity against E. coli as before. A similar

'experiment using tetracycline was run in tandem for comparison.

d) Retention factor determination

Using the following solvents, the retention factors of the antibiotic, as eluted on silica

(Keiselgel 60G) were found: hexane, chloroform, pyridine, acetone, ethanol,

methanol, water, ethylacetate, methanol/chloroform (1:9).


a) Minimum inhibitory concentration (MIC) determination

i) Tube dilution method .

Two fold dilutions of the antibiotic in basal medium were used and the tubes were

incubated at 31lC for 2 - 5 days. E. coli was the test organism.

ii) Agar diffusion method

This was after the method described by Platt and Isaa~son (1973). A starting

concentration of 2 mg/ml of aqueous antibiotic solution, diluted down 2-fold to 0.0625

mg/ml, was used. The cork-bore was 9.4 mm diameter and the MIC of the

55

-------------

microorganisms tested were found by extrapolation, via a best line fit, supported by

a Pearson's correlation programme, on an ACT Sirius personal computer.

b) Assessment of microbistatic/cidal activity

E. coli was grown in basal medium to obtain an O.D. growth curve as described at

2.1.7. In the middle of log phase, a concentrated aqueous solution of the antibiotic

was added to the culture to give a final antibiotic concentration, equivalent to that of

the MIC, as determined above. Subsequent monitoring of the O.D. was continued

until no further change occurred and loopfuls taken out at this stage were streaked

out on NA plates and incubated overnight. A control growth curve was made where

no antibiotic was added ..

2.3.15 Chemical properties

Qualitative chemical analytical tests done on the antibiotic included Schiffs!Fuchsin

test for aldehydes (Nauman, et aI, 1960); ninhydrin test for amino acids (Ruhemann,

1911; Schoenberg and Moubacher, 1952); anthrone test for carbohydrates (Koehler,

1952); Erhlich test for pyrrols; Feigl test for ketones (Feigl, 1960) and

formalinJH2S04 test for alkyl or aryl halides (Berry, 1955).

2.3.16 Spectral data for antibacterial substance

a) Ultra viol~tMsible (uvMs) scan

Antibiotic crystals were dissolved in U.V. grade ethanol (96% ethanol) and

transferred into a Pye 10 mm U.V. silica cuvette. It was then scanned (200-700 nm)

using a Shimadzu UV-160 spectrophotometer.

56

~- - ------~------

b) Infra red (I.R.) scan

A KEr disc was prepared by adding antibiotic crystal to KEr granules in a 1:1000

ratio (0.1 - 0.2 mg/100 - 200 mg) and grinding with mortar and pestle under a Thorn

Reflector Display, 150 watts heat lamp~ The fine powder was then put into a die

press and compressed to a disc using a vacuum press, producing 12 -15 tons/sq in .

. The disc was scanned using a Pye Unicam SP3-100 I.R. spectrophotometer with a

pathlength of 1 cm and air as the reference.

c) Mass spectroscopy

A few crystals were used to obtain a mass spectrum, first at low resolution electron

impact, then at high resolution electron impact. The instrument used was a Kratos

MS80/05-55C spectrometer. 'I

2.3.17 X-ray ctystallography

a) Ctystal formation

Some crystals of the antibiotic were dissolved to saturation in a small volume of

various solvents, which were then left in a cupboard and the solvent allowed to

evaporate. The crystals thus formed were mounted on a microscope slide and viewed

under phase-contrast microscopy to see if twinning had occurred. (NOTE: There are

certain types of twinning which cannot be detected by phase contrast microscopy

alone and these only became apparent after the initial stages of X-ray

crystallography). To obtain the best crystals for X-ray diffraction, the following

solvents were tried: ethanol, methanol, methanol/chloroform (119), and chloroform.

57

-----------------------------------------

b) X-ray diffraction

The suitable crystal was mounted on a X-ray goniometer head. Using a series of .

oscillations, the crystal was oscillated about the crystallographic C axis and .

Weissenbe~g· photographs using CuKa radiation, enabled crystal settings, preliminary

determinations of cell dimensions and space groups to be made. The crystal was then

transferred to a 2-circle Stoc Stadi-2 Weisenberg diffractometer.The programme

used was the Shelx X-ray 72 for bonds and angles (Steward, et aI, 1972). Another

Shelx programme was used for structure solution and refinement (Sheldrick, .1976) .

. Molecular drawings were made by the ORTEP facility (Johnson, 1965).

2.4 THE EFFECT OF MAGNESIUM SALT OMISSION FROM THE

. MEDIUM RECIPE

. Though peripheral to the aims of this study, the observed ability of strain A, not only

to grow, but to grow with enhanced antibacterial substance production, in a .

chemically defined medium (basal medium) without an added Mg salt component

(Med A2), warranted further study. -

2.4.1. Cell propagation experiments

a) Strain A in Med A2 (SLR)

A 'washed' (centrifugation/decanted) culture of Strain A, previously grown in basal

rnedium, was inoculated into 50 rnls of basal medium minus the Mg salt component

(Med A2) and then grown at 371C, 90 r.p.m., to mid-log phase. A sample of this was

'washed', inoculated into a fresh batch of Med A2 and then grown as above. This;

procedure was repeated for a set number of times, constituting a set number of

propagations. Cultural characteristics were observed and noted at each propagation.

58

b) . Strain A in Med A2 (AR.)

The whole experiment as at a) was repeated but this time using analytical reagent·

basal medium and analytical reagent Med A2 ..

c) Ps. aeuroginosa in Med A2 (SLR)

The experiment at a) was repeated again but this time using Ps. aeruginosainstead

of strain A

2.4.2 Atomic absorbtion measurements of Mg2+ in Med A2

a) Calibration curve

A calibration curve was made by first diluting with deionised H20, a 1000 ppm stock

solution of Mg(N03)2.6H20 (Mg2+ source), in order to obtain a concentration series,

ranging from 0.5 ppm to 0.1 ppm, with 0.1 ppm decrements. Next, the absorbances

at these 5 concentrations were found on an atomic absorbtion spectrometer and these,

were plotted against their corresponding concentrations. The instrument used was

a Shandon Southern A3400 with air/acetylene inlets and a holocathode Mg lamp.

The wavelength chosen was 285.5 nm ..

b) Mg2+ measurements (sample)

i) The trace concentrations of Mg2+ in Med A2 of SLR grade and that ofAR.

grade were measured.

ii) The SLR medium was inoculated with cells 'washed' with SLR medium, to

give a final 0.0. (650 nm) of.= 0.065. It was then incubated for 20 minutes

59

-- ~------------------------------------------

at 3'flC, 90 r.p.m. The cells were spun down (4000 r.p.m./15 minutes) and the

supernatants assayed for Mg2+ content. The same procedure was repeated but

this time using AR medium and inoculating with cells washed with AR

medium.

2.4.3 Analysis of Mg2+ in cells of strain A grown in basal medium and in Med A2 .

A 250 ml conical flask containing 50 mls of basal medium was inoculated with

colonies of strain A from a slant. It was incubated at 37°C, 90 r.p.m., overnight. A

portion of this culture was washed 3 times with sterile saline. After resuspending in

saline, it was used to inoculate 50 mls of basal medium and 50 mls of Med A2 in

separate 250 ml conical flasks. These were then incubated as previously and after 24

hrs, taken out. The cells from the basal medium were washed 3 times with saline and

the peliet transferred to a small crucible which had previously been weighed .. The

crucible was heated until the pellet was totally charred. It was allowed to cool before

weighing again. Four mls of 5MHQ was poured into the crucible and using a glass

rod thoroughly stirred for 5 mins. The mixture was centrifuged (4000 r.p.m.; 15

minsYand the supernatantassayed for Mg2+ as previously (2.4.2).

The same procedure as above was repeated but this time using cells grown in Med

. A2.

2.4.4 Viable counts of strain A grown in basal medium and Med A2

The method described by Miles and Misra (1938) was used to monitor viable growth

. of strain A in basal medium and Med A2. A Y. strength Ringer solution was used for

the dilutions and a Finnpipette micropipette, delivering 50 1'1 quantities onto pre-dried

(20 minutes in a 37°C incubator) NA plates was also used. The plates were incubated

at 37°C for 12 hrs. Set times at which aliquots were taken out of the culture to

monitor growth were: 0,24,36,48, 72 and 84 (hrs).

60

I

2.5 ANTIFUNGAL ACTIVITY STUDIES

2.5.1 Elicitation of antifungal activity in liquid media

During the search for a suitable liquid medium capable of eliciting antifungal activity, .

all the media as outlined at Table 2.1 were tried.

. 2.5.2 Assay of antifungal activity

The method previously employed for the assay of antibacterial activity (2.3.1) was

used but here, the test microorganism of choice was C. albicans.

2.5.3 Construction of PDLM for antifungal production

a) Oxoid PDA batch No. 156 21497

3.9 gms of Oxoid PDA batch No. 156 21497 was suspended in 100 mls of deionised .

water. Using a magnetic stirrer, it was stirred for one hour, allowed to sediment then

decanted. The decant was membrane-filtered (0.45 I'm) giving rise to sterile PDLM.

The procedure as at 2.3.1 iii) to viii) was then followed.

b) Oxoid PDA Lot No. 070 40305

The procedure as at a) was repeated but this time using Oxoid PDA Lot No. 070

40305.

61

--- -- -----------------

. c) Oxoid Potato extract

0.2 gms of potato 'extract plus 1 gm of glucose were dissolved in 50 ml· of deionised

water. and this was sterilised by membrane-filtration before inoculation. The

procedure as at 2.3.1 iii) to viii) was then followed.

d) Potato liquid extract

The Commonwealth. Agricultural BureauxlIntemational Mycological Institute's

(CAB/lMI; Smith and Onions, 1983) procedure for making potato liquid extract,

including boiling of diced raw potatoes, was closely followed. Leicestershire grown

King Edwards potatoes were used. The procedure as at 2.3.1 iii) to viii) was then

followed.

2.5.4 Optimization of antifungal activity

a) Medium component single-deletion of medium q (Table 2.1).

The medium component single-detection method used at 2.3.4 a was used here.

Magnesium glycerophosphate which afforded the only buffering capacity of the ·ZIl"" original medium was eventually substituted for NaH2P04@t a concentration of 0.5 gIL.

b) Variation of nutrient concentration

Optimal concentrations for antifungal activity for the following sequence of medium

components were determined in the manner already described at 2.3.4 b): NaN03

(0.0 to 0.3%), sucrose (0.0 to 5.0%), glycerol (0.0 to 8%).

62

c) Variation of physical parameters

The optimal factors of pH, agitation/aeration and temperature were sequentially

determined in the mann~ralready described at . 2.3.4 c but in this sequence, the

. shaking speed was further increased to 210 r.p.m. and the temperature range was

22°C-42°C.

2.5.5 Cultivation in a laboratory-scale fermentor

The same fermentor used at 2.3.5 was used here. Four litres of the medium

optimised for antibiotic production was poured into the fermentor vessel and this was

autoc1aved. An optimized medium culture of strain A, previously grown in the orbital

incubator, set at 37°C, 90 r.p.m. and for 18 hours (log phase), was used to obtain an

initial OD of = 0.06. The airflow rate, temperature and stirring rate were determined

by experimentation. Monitoring of antibiotic production took place as before

(Section 2.3.5) but here, the test microorganism was C. albicans.


These were done, as previously described at 23.6 ..

2.5.7 Isolation and purification of the antifungal substances

a) Determination of appropriate solvent system f~r broth e~traction

The solvent system strategy developed by Aszalos and Issaq (1985) was initially tried

and the procedure as described ~t 2.3.9 aii) was also tried. During the latter, the

following solvents were used: dichloromethane, acetone, methanol/chloroform (1/9),

ethon.Vchloroform (1/9), ethanol/acetone (1/9),· acetone/chloroform (1/9).

63

b) Development of a solvent system(TLC strategy for extraction

Preliminary investigations to find suitable solvents for the various separation stages

were done on analytical TLC (Kieselgel P60) plates ..

After determination of the appropriate solvent system to be used, the essence of the

procedure as at 2.3.9 c) was followed for each of the following stages:

i) 1st stage - the following solvents were tried: ethanol, methanol, water.

ii) 2nd stage - the active fraction eluted at stage 1 was found to be composed of

several components, so different solvents were again tried in order to obtain

best separation. The solvents tried included, ethylacetate, acetone/chloroform

(1/9).

iii) 3rd stage -. the resultant 2 active fractions eluted at stage· 2 were also

individually found to be composed of more than one component. One fraction - . ' .

was finally separated after trying the following solvents: methanol/chloroform

(1/9) acetone/chloroform (1/9) and ethylacetate. The other one. was also

. finally separated after trying the following solvents: methanol/chloroform (1/9) .

.. acetone!chlorofonri (1/9) and ethylacetate.

2.5.8 A check for purity

a) HPLC

The same initial test procedure, involving elution of the antibiotic spot on TLC, as at

2.3.10, was used for the antifungals to see if they can be safely flushed through the

64

HPLC column. Any movement of the spots on the TLC would be indicative of safe

passage through the column ..

b) Gas Chromatography (G.C.)

0.9 ILl of the solvent in which the antibiotic was to be dissolved was injected into the

G.C. using a S.G.E. 1 ILl syringe. This resulted with a characteristic G.C. profile on .

the chart recorder and constituted the control. The same procedure was followed as

per the 'neat' solvent but this time using the solvent antibiotic solution. A single peak

. recorded on the chart is indicative of G.C. purity. The G.C. instrument was a

Pye/Unicam series 104 chromatograph-type 1 analyser with a Pye oven programmer

and an ionization amplifier. The 3 gas inlets were of air, nitrogen (carrier) and

hydrogen. The coiled column size was 1.S metres x 4 mm with a solid phase of

chromosorb W (chalk) [100 - 120 Mesh] and a liquid phase of 10% carbowax 20 M.

The recording instrument was an Omni Scribe chart recorder. The G.C. settings were

as follows: attenuator: S x 102; temperature range: SO - lS0oC; gas flow rates: air =

400 ml/min., nitrogen and hydrogen, both 40 ml/min; chart recorder speed: 2.S

----. cm/min.

2.S.9. Antimicrobial Properties

a) M.I.C. determination of an antifungal compound

Only one antifungal compound (AF1) was isolated in sufficient quantity to facilitate

wide screening. The agar diffusion method as described at 2.3.14 aii) was used. A

starting concentration of 1 mg/ml was made with a SO/SO, water/ethanol solution.

From this a series of 4, 2-fold dilutions were made using water. The MICs against

the fungi, listed at 2.1.1 b) plus E. coli,~. marcescens and~. aureus, were calculated.

65

b) Zone stability comparisons of the 3 antifungal compounds

A 13 mm Whatman disc was dipped into a 1 mg/ml solution of AFl then placed on .

a C. albicans seeded agar plate before incubating overnight at 30°C. The procedure

was repeated for the other 2 antifungal compounds. Mter overnight incubation and

measurement of zones of inhibition, the plates were further incubated for 4 days to

test for zone stability.

2.5.10 Spectral data for the antifungal substances

a) UV /vis scans

. Samples of all the antibiotics were individually dissolved in UV ethanol and each

scanned as before (2.3.16 a)).

b) I.R. scans

A drop of concentrated ethylacetate antibiotic solution was placed on one I.RNaCl

disc, under the Thorn Reflector display lamp. When the solvent had evaporated, the

above procedure was repeated 2 times. The other I.R NaCl disc was then placed

on top of the antibiotic coated face of the first disc and the 2 gently rubbed against

each other in order to obtain an even spread. They were then placed in a silica gel

dessicator and left overnight. The pair were then mounted in a cell holder and

scanned using a Pye Unicam PU 9516 I.R spectrophotometer, attached to an IBM

personal computer, with a Philips B.I.RD.S. programme, a Philips computer monitor

80 imd an Epsom FX 800 printer. All the antifungal antibiotics were scanned.

66

c) Nuclear magnetic resonance (NMR) scan

A sample of AFl was dissolved in duetriated chloroform and this was used to obtain

a proton NMR (IHNMR) spectrum. The instrument used was a 60 MHz Varian EM

360A NMR spectrometer.

67

CHAPTER 3

RESULTS

I I

I

3. RESULTS

3.1 GENERAL

3.1.1· Transmission Electron Microscopy (TEM)

An electron micrograph of an individual cell of P9897 (Strain A) is shown at Fig.

3.1. It has been negatively stained with phosphotungstic acid (PTA) and

photographed under TEM. The cell is rod shaped and slightly curved, with one

polar flagellum.

3.1.2 Selective Medium Development

.. The results of the antibiotic sensitivities of 14 microorganisms to multidisk U4 is

given at Table 3.1. From this, a selective medium for strain A was made,

comprising colistin sulphate and sulphafurazole in NA, at concentrations of 10

J.tg/ml and 500 J.tg/ml, respectively. This selective medium proved ineffective

against the 2 fungi (~. cerevisiae and £. chrysogenum )tested.

The growth of strain A on half of a NA plate and growth of Ps. cocovenenans on

the other half are shown at Fig. 3.2. The side of the plate containing colonies of

strain A appear faintly yellow in colour. This plate constituted the control. Fig.

3.3 shows some growth of strain A on the selective medium plate but no growth

of Ps. cocovenenans.

3.1.3 Biochemical/tolerance tests

Thirty one biochemical/tolerance tests, performed on both strains A and B, and

on Ps. aeruginosa, Ps. antimycetica and Ps. maltiphilia are shown at Table 3.2.

Strain A gave positive acid production results for· glucose, fructose, sucrose,

galactose and mannitol but gave negative results for lactose and maltose. Positive

results were also observed for ONPG, catalase, nitrate, urease, aesculin, lipase and

lecithinase. Of the tolerance growth tests done with strain A, only MacConkey

68

agar encouraged growth and here, it was relatively slow (36 hrs incubation at

37"C). Negative results were also observed with strain A for oxidase, methyl red,

Voges-Proskauer, indole, starch hydrolysis, deoxyribonuclease (DNase), haemolysis

and, ornithine and lysine decarboxylase activity. Of the tolerance growth tests

done, the following gave negative results: 1% TIC, 6.5% NaCI, 0.03% cetrimide,

Pseudomonas agar, 42°C and minimal medium. Strain A also gave a polymyxin

MIC of 10 ~g!mI.

Strain B gave a very similar biochemical test profile to that of strain A, the only

. exceptions being aesculin and lecithinase which were both negative, and a

polymyxin MIC of 0.1 ~g!mI.

Ps. aeruginosa differed from strain A in that it gave positive results for oxidase,

heamolysis (sheep's blood not horses'), 1 % TIC, 0.03% cetrimide, Pseudomonas

agar, fast growth in MacConkey agar (18 hrs incubation at 37°C) and 42°C; but

negative results for sucrose, ONPG, aesculin and lecithinase.

Ps. antimycetica differed from strain A in that it gave positive results for maltose,

starch hyqrolysis, 1% TIC, 0.03% cetrimide, fast growth on MacConkey agar (18

hrs incubation at 37°C) and 42°Cj but gave negative results for mannitol,

. galactose, ONPG and aesculin.

Ps. maltiphilia differed from strain A in that it gave positive results for maltose,

DNase, haemolysis (horse's blood not sheeps'), 0.03% cetrimide, fast growth on

MacConkey agar (18 hrs incubation at 37°C) and 42°Cj but gave negative results

for mannitol, sucrose. and lecithinase.

3.1.4 Some Comparison Studies between Strain A and Ps.cocovenenans

The growth profile of strain A, grown in basal liquid medium (Cruickshank's

medium) at pH 4.0 (i), at pH 7.2 (ii) and again at pH 7.2 but this time, with a

medium component single deletion of MgCI2.6H20 (A.R.) (iii) are shown at Fig.

3.4. All 3 are similar in overall shape in that they describe conventional growth .

curve profiles, however, growth in medium i was much slower and achieved a

69

lower optical density (0.0.) maximum (0.8) at 48 hrs, compared to 0.0. maxima

of 2.2 at 36 hrs for media ii and Hi. With medium ii, yellow pigmentation was

observed after 36 hrs.

. The growth profiles of Ps. cocovenenans, grown in the 3 media described above

are shown at Fig. 3.5. Medium ii gave rise to a conventional growth curve profile

but both media i and Hi exhibited long lag phases where the 0.0. of Hi rose after

24 hrs and that of i rose after 48 hrs. Ps. cocovenenans in mediumi did not attain

the growth levels achieved in the other 2 media (0.0. maxima of 1.026 for i, of

1.78 for ii and of 2.18 for Hi. In medium ii yellow pigmentation was observed after

48 hrs.

Growth of strain A on one half of a NA plate and growth of Ps. cocovenenans on

the other half, after 24 hrs incubation, at 37°C are shown at Fig. 3.6. The side of

the plate containing colonies of strain A appear faintly yellow in colour. This

constituted the control. Fig. 3.7 shows good growth of strain A on one half of a

NA plate at pH 4.0 with strong yellow pigment exudation but Ps. cocovenenans

failed to grow on the other half of the plate. The plate was incubated for 24 hrs

at 37°C.

3.1.5 Growth Curve Studies

The mean generation times (mgt) for both strain A and Band Ps. aeruginosa are

shown at table 3.3. Strain A, grown in media 1-6, gave mgts of 170, 99, 84, 134,

106, 110 (coefficient of variation, 6%) respectively and strain B, grown in media

1-6, gave mgts of 156, 97, 81, 104, 106, 161 (coefficient of variation, 5%)

respectively. . Additions of the vitamins, riboflavin and nicotinic acid at

concentrations of 0.001 % and 0.01 % respectively, made no difference to the mgts

of either strain. Ps. aeruginosa grown in media 1 and 6 gave mgts of 100 and 88

(coefficient of variation, 5%) respectively.

70

3.1.6 Invasive Growth Inhibition of Aniger by some Pseudomonas species

From table 3.4, inhibition zones of A niger on PDA by strain A, strain Band Ps.

aeruginosa were 15 mm each (coefficient of variation, 3%) but Ps. cocovenenans

produced a zone of inhibition of 7 mm (coefficient of variation, 3%). No zones

of· inhibition were seen with Ps. antimycetica and Ps. maltiphilia and infact,

invasive growth of the A niger swamped these 2 organisms. With the CzaF~k

Dox Agar (CDA) experiments, the zones of inhibition created by strain A against

A niger was 23 mm (coefficient of variation, 5%), accompanied by an intense

. yellow exudate; strain B was 18 mm (coefficient of variation, 5%) but Ps.

aeruginosa hardly grew, so results here were invalid. Ps. antimycetica and Ps.

maltiphilia showed no zones of inhibition and here again, A niger swamped the

2 pseudomonads. Figs. 3.8 - 10 shows examples of invasive growth inhibition of A

niger by strain A and Ps. cocovenenans. All the plates were incubated at 30·C

for 7 days.

3.1.7 Preliminary Extraction of Water-soluble Active Substances

After following the procedure outlined at 2.2.11, no zones of inhibition were

observed with this experiment.

3.2.0 ANTmAcrERIAL ACfIVITY STUDIES

3.2.1. Elicitation of Antibiotic Production in Liquid Media

From table 3.5, the only liquid media to give rise to zones of inhibition for

antibacterial activity were basal medium (c), strain A's medium (d), Czapek Dox

Liquid Medium (CDLM), (n) and CDLM-modified (q), all occhrring on the

secondl but not on the seventh day of incubation.

71

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3.2.2 Preliminary Testing of Antibacterial (ab) Substance/s _

a) Heat stability

After heating in a boiling water-bath for 30 mins,the activity of the broth

filtrate against E.coli, as measured by the disc diffusion assay, had dropped

by 10%.


The experiment with Erhlich's reagent on -the active broth filtrate was

negative (no colour change).

3.2.3 Pigmented Inoculum Experiment


After 24 hrs incubation at 37°C, 90 r.p.m., the basal liquid medium had

developed intense yellow pigmentation which upon assay gave a disc zone

of inhibition of 23.6 mm against E.coli. Further incubation did not increase

visual pigmentation.


Although some faint yellow pigmentation was observed, a sample of the

basal medium broth, taken out after 24 hrs incubation at 37°C, did not

register a zone of inhibition upon assay. Further incubation failed to _ .

increase visual pigmentation.

72

. 3.3.3 Optimization experiments

a) . Medium component single deletion (MCSD) [SLR]

The growth profiles (O.D.) and the antibiotic concentration profiles

(mg/~1)of strain A, at 90 r.p.m., 37°C,grown in the 'complete', MCSD

NH4CI (Med AI), MCSD-MgCI2.6H20 (Med A2), MCSD-Na2SOdMed

A3), MCSD~KH2POJK2HP04 (Med A4) and MCSD-glucose (Med AS)

media are shown at Figs. 3.11 and 3.12. The 'complete', Med A2 and Med

A3 profiles describe conventional growth curve profiles with maximum

stationary phase O.Ds. of 2.00. Med Al growth profile rises to O.D. 0.33

in 24 hrs, then levels out with increasing time and Med A4 growth profile

rises to O.D. 0.48 in 24 hrs then falls with increasing time to 0.0. 0.28 in

60 hrs. Med A5 growth profile, after inoculation, remains constant

throughout the period of incubation.

The pH profiles for all the media with the exception of Med A4, falls only

slightly with time. Med A4's pH profile falls fairly steeply to 3.5 in 36 hrs

and then levels out, reaching 3.7 in 60 hrs .

. No alltibiotic production was deteCted in Med AI, and Med A5 throughout

the period of incubation. The complete, Med A2 Med A3, all produced

detectable amounts of antibiotic during the incubation, with production

peaking at ,24 hrs but then falling away with time. Med 2 gave rise to the

greatest antibiotic concentration, 0.065 mg/ml in 24 hrs, steeply falling to

0.034 mg/ml in 48 hrs then levelling off, reaching 0.028 mg/ml in 60 hrs.

The 'complete' medium was next, with 0.03 mg/ml in 24 hrs, falling to 0.015

mg/ml in 48 hrs then continuing to 0 mg/ml in 60 hrs. Finally, Med A3 was

the smallest, with a rise to 0.018 mg/ml in 24 hrs which thereafter gradually

fell to 0 mg/ml in 48 hrs.

73

b) Medium component single deletion (AR)

Results of the MCSD (AR) experiments are shown at Figs. 3.13 and 3.14 .

. The experiment is a repeat of the MCSD (SLR) experiments but this time

using A.R. grade chemicals. All the growth profiles are similar to their

SLR counterparts except for those of Med A3 and A4. The· former rises

to about O.D. 0.3 then begins to level out, barely rising with time, and then

falls slightly after inoculation, with increasing time, to an 0.0. of 0.04 in

60 hrs.

The pH profiles were also similar to their SLR counterparts, however,

overall antibiotic production was dissimilar in that production was only

detected in the' complete' medium. Here, it was first detected after 36 hrs,

describing a peak of 0.33 mg/ml which then fell to 0.017 mg/ml in 48 hrs

before levelling off to 0.011 mg/ml in 60 hrs.

c) Glucose variation

The growth profile (O.D.; 650 nm) of strain A, grown in Med A2, at 3?-C,

90 r.p.m. and at the following percentage concentrations of glucose: 0.3,

0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 are shown at Fig. 3.15. The curves of each

concentration describe conventional growth curve profiles with O.D.

maxima of 2.0.

The continuation of the above O.D. profiles with the following percentage

concentrations of glucose: 0.3, 0.24, 0.18, 0.12, 0.06, 0.0 are shown at Fig.

3.16. The 0.3 to 0.06% curves all follow conventional growth curve profiles

but with progressive decreases in stationary phase OD's, with decreasing

glucose concentration (0.3%, O.D. 2.1; 0.2%, O.D. 1.9; 0.18%, O.D. 1.7;

0.12%, O.D. 1.1; 0.06%, O.D. 0.46). The 0.0% profile very gradually falls

away with time to O.D. 0.052 in 60 hrs.

74

I

I

The antibiotic concentration/time profiles of strain A, grown in Med A2 at

the concentrations and conditions cited at Fig. 3.15 are shown at Fig. 3.17.

All the concentrations gave rise to similar Ab concentration profiles where

production increased to a peak at 36 hrs then fell away with time. The

highest Ab concentration measured was 0.067 mg/ml and this occurred at

0.3% in 36 hrs.

The Ab concentration/time profiles of strain A, grown in Med A2 at the

concentrations and conditions cited at Fig. 3.i6 are shown at Fig. 3.18.

The 0.3% and 0.12% curves are similar in profile to those at Fig. 3.21 but

the 0.24% and 0.18% curves, though similar in profile to the rest, begin to

level at 36 hrs, reaches a peak at 48 hrs then falls away with time.

Altogether there is a progressive increase in Ab concentration with

increasing glucose percentage. Peaks occur at 36 hrs both for 0.12% (0.32

mg/ml) and for 0.3%, (0.067 mg/ml), and at 48 hrs both for 0.18% (0.04

mg/ml) and for 0.24% (0.045 mg/ml). Here again, the highest

concentration achieved occurred at 0.3% and at 36 hrs. Antibiotic activity

was not detected at 0.06% and 0.0% glucose concentration throughout the

period of incubation .

. The Ab concentration of strain A, grown in Med A2 of various percentage

glucose concentrations and measured. at set times are shown at Fig. 3.19.

The curves all indicate a rapid increase in antibiotic production with initial

increases in percentage glucose concentrations to 0.3% which then broadly

levels off thereafter. The levelling off at 24 hrs occurs at 0.035 mg/ml

antibiotic concentration and is relatively steady as the glucose

concentration increases, whereas at 36 hrs, there is a rise to 0.067 mg/ml

followed by a fall to 0.053 mg/m!. It then fluctuates between 0.047 to 0.06

mg/ml with increasing glucose concentration. At 48 and 60 hrs there are

significant drops from 0.058 mg/ml and 0.047 mg/ml respectively to 0.034

mg/ml and 0.025 mg/ml respectively at 0.5% glucose concentration. Both

profiles then level out again with increasing glucose concentration.

75

------ ------- - --- --- ---- ---.----------~-----

The growth curve (0.0. (650 nm)/time), the Ab concentration profile and

the pH profile of strain A. grown in Med A2 (0.3% glucose), at 90 r.p.m.,

37°C are shown at Fig. 3.20. The pH profile barely falls with increasing

time. From the glucose variation experiment done, this graph represents

" the optimum conditions which gave rise to the highest level of Ab

production (0.067 mg/ml) achieved so far in small (50 ml) batch cultures.

d) Ammonium chloride variation

. The growth profiles (0.0.; 650 nm) of strain A, grown in Med A2, at 90

r.p.m., 37°C and at the following NH4CI percentage concentrations: 0.3,

0.24, 0.18, 0.12, 0.06 are shown at Fig. 3.21. The curves of each

concentration are similar and describe conventional growth curve profiles

with 0.0. maxima of 2.0.

A continuation of Fig. 3.21 at the following NH4CI percentage

. concentrations: 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.0 is shown at Fig. 3.22.

All the curves follow conventional growth curve. profiles, with 0.0. maxima

of 2.0, the exception being the 0.0% curve which plateaus with an 0.0.

maximum of 0.2.

The Ab concentration profiles of strain A. grown in Med A2, at the

concentrations and conditions cited at Fig. 3.21 are shown at Fig. 3.23. All

the Ab concentration profiles show increases in Ab production with time

which rises to a peak at 36 hrs then falls away with increasing time. The

0.06% profile however, shows a tendency toward levelling before and after

the peak. The 0.06% medium which gave rise to the highest concentration

of Ab at 36 hrs (0.126 mg/ml), was closely followed by the 0.12% medium

(0.115 mg/ml) also at 36 hrs (0.115 mg/ml).

The antibiotic concentration profile of strain A. grown in Med A2, at the

concentrations and conditions cited at Fig. 3.22 are shown at Fig. 3.24.

The 0.01%,. and 0.02% profiles suggest gradual increases in antibiotic

76

production with time, to a peak of 0.03 mg/ml and 0.066 mg/ml

respectively, in 48 hrs, then a falling away with time. The 0.03% to 0.06%

curves suggest that increases in concentration coirolated with increases in

production which peaked at 0.072 mg/ml and 0.126 mg/ml, respectively, in

-36 hrs, then fell away with time. By far the greatest Ab concentration was

measured at 36 hrs from the 0.06% medium.

The Ab concentrations of strain A, grown in Med A2 of various percentage

NH4C1 concentrations and measured at set times are shown at Fig. 3.25.

From the 24 hrs to 48 hrs profiles, there appears to be a rapid increase in

. production with increasing NH4CI concentration,· all peaking at 0.06% (24

hrs, 0.115 mg/ml; 36 hrs, 0.126 mg/ml; 48 hrs, 0.112 mg/ml) and gradually

falling with increasing NH4C1 concentration. At 60 hrs, there occurs an

initial, rapid rise in production which begins to level then gradually rise to

0.06 mg/ml at 0.024% before gradually falling to 0.048 mg/ml at 0.03%.

The O.D. growth profile (O.D.; 650 nm), the Ab concentration profile and.

the pH profile of strain A, grown in Med A2 of 0.06% NH4C1 (medium 1)

at pH 7.2, 90 r.p.m., 37°C are shown at Fig. 3.26. The pH profile

produces a slight 'trough' between 24-48 hrs but barely changes throughout

the incubation period. From the NH4CI variation experiment done, this·

graph represents the optimum conditions giving rise to the highest Ab

concentration (0.126 mg/ml) achieved so far in small (50 ml) batch

cultures.

e) pH variation

The growth profiles (O.D.; 650 nm) of strain A, grown in various medial

which have been buffered to pHs 3, 4, 5, 6, 7, 8 and 9, at 90 r.p.m., 37°C

are shown at Fig. 3.27. The pH 6 and 7 profiles are similar and show

conventional growth curve profiles with O.D. maxima of 2.0. pH profiles

5 and 4 also describe conventional growth curve profiles but both occur at

lower stationary phase O.D.s. (O.D.s 0.9 and 0.53 at 36 hrs respectively).

77

After inoculation, pH profiles J and 9 fall away with time to 0.0. 0.01 and

0.005 at 60 hrs, respectively. pH profile 8, afterinoculation, does not grow

until after 36 hrs whereafter it grows rapidly, reaching a peak of 1.49 in 48

hrs, then falls away sharply with time.

The Ab concentration/time profiles produced by strain A, grown in

medium 1 at the pHs and conditions cited above are shown at Fig. 3.28.

Antibiotic activity was only detected in the pH 6 and 7 media and both

profiles are similar. The Ab concentration in both media increases with

time and peak between 36 and 48 hrs before falling away with time. Of

the two, the pH 6 medium consistently gave rise to the highest Ab .

concentration (0.178 mg/ml in 48 hrs).

The Ab concentration profiles of strain A, grown for set time periods in

various media 1,· buffered to the pHs mentioned above are shown at Fig.

3.29. All the curves have a similar profile in that after the first detection

of Ab concentration at pH 6, there is a fairly steep fall which passes

thro~gh pH 7. The highest Ab concentrations were detected in the pH 6

medium and were measured between 36 and 48 hrs (0.174 mg/ml to 0.178

mg/ml respectively).

The growth curve (0.0.; 650 nm), the Ab concentration profile and the

pH profile of strain A, grown in medium 1 at pH 6 (medium 2), at 90

r.p.m.; 37°C are shown at Fig. 3.30. The pH profile barely changes, falling·

very slightly with time. From the pH variation experiment, this graph

represents the optimum· conditions which gave· rise to·· the highest Ab

concentration (0.178 mg/ml) achieved so far in small (50 ml) batch

cultures.

78

I

I

I

I

i

I

.- --------------- ----

t) Agitation rate (RPM) variation

The 0.0. growth profiles (O.D.; 650 nm) of strain A, grown in medium 2,

at the following r.p.m.s: 40, 60, 90, 120, 150, 180, all at 37°C are shown at

Fig. 3.31. The 90, 150 and 180 r.p.m. curves are shnilar in that all describe

conventional growth curve profiles but are dissimilar in that the maximum

O.D.s achieved, progressively decrease with increasing r.p.m. The 60 and

40 r.p.m. profiles are also similar to the above but here, there is a tendency

for the stationary phase O.D. to fall slightly then begin to rise and in

addition to this, as the r.p.m. decreases, there is a drop in stationary phase

O.D. The .120 r.p.m. curve is different from all the others and describes

typical biphasic growth.

The Ab concentration/time profiles of strain A, grown in medium 2, at the

r.p.ms and conditions cited above are shown at Fig. 3.32. Ab

concentrations were detected only at 90, 120 and 150 r.p.m. At 90 r.p.m., .

the Ab concentration rises steeply to 0.174 mg/ml in 36 hrs, levels off to

0.178 mg/ml in 48 hrs before falling sharply to 0.008 mg/ml in 72 hrs. At

120 r.p.m. the Ab concentration rises to 0.072 mg/ml in 24 hrs, falls to

0.066 mg/ml in 36 hrs, barely rises to 0.063 mg/ml in 48 hrs then rises

steeply to 0.372 mg/ml in 72 hrs before falling steeply to 0.195 mg/ml in 84 .

hrs. At 150 r.p.m., the Ab concentration rises exponentially to 0.096 mg/ml

in 60 hrs, begins to rise again to 0.138 mg/ml in 72 hrs before levelling to

0.135 mg/mUn 84 hrs.

The growth curve. (O.D.; 650R .. )the Ab concentration profile and the pH

profile of strain A, grown in medium 2 at 37°C, 120 r.p.m. are shown at

Fig. 3.33. The pH profile falls gradually to 5.4 in 24 hrs, barely changing

at this level to 48 hrs before rising slightly to 5.7 in 60 hrs and maintaining

this level throughout to 84 hrs. From the r.p.m. variation experiments, this

graph represents the optimum conditions which gave rise to the highest Ab

concentrations (0.372 mg/ml) achieved so far in. small (50 ml) batch

cultures.

79


The growth profiles (O.D.; 650m¥>f strain A, grown in medium 2, at 120

r.p.m. and at the following temperatures: 27, 32,37, 42, 47°C are shown

at Fig. 3.34. The organism, grown at 42°C, describes a classic growth curve

profile, with a maximum stationary phase O.D. of 0.98 at 36 hrs, followed

by a slow decline phase. At 32°C, the profile, although fairly similar in .

shape to that at 42°C, occurs at lower stationary phase O.D.s which

exhibits fluctuations within an O.D. range of 0.328 to 0.563. At 37°C, a

biphasic growth profile is described. At 27°C, the O.D. barely rises above

that produced by the inoculum in 24 hrs but after this time, rises fairly

steeply to 0.209 in 36 hrs and levels out gradually with increasing time to

0.260 in 72 hrs. The 47°C profile fell fairly steeply to O.D. 0.023 in 24 hrs

and maintained this level throughout the incubation period. Cell

aggregation/clumping was seen, after 24 hrs, in all the flasks incubated at

27°C and 37°C.

The antibiotic concentration/time profiles of strain A, grown in medium 2,

at the temperatures and conditions cited above are shown at Fig. 3.35.

Throughout the incubation period, no significant production was obsedrved

at 47°C and 27°C. The Ab concentration profiles of the 42°C and 32°C

were similar and rose very gradually with increasing time to 0.04 mg/ml and

0.033 mg/ml in 84 hrs, respectively. At 37°C, the Ab concentration rose

to 0.072 mg/ml in 24 hrs, dipped slightly to 0.063 mg/ml in 48 hrs, rose

steeply to 0.372 mg/ml in 72 hrs then fell steeply with time to 0.195 mg/ml

in 84 hrs.

The antibiotic concentration produced by strain A in medium 2 when

grown at various temperatures and measured at set times are shown at Fig.

3.36. Ab concentrations were detected from 24 hrs to 84 hrs and only

between 32°C and 42°C. The Ab/temperature profiles at the above times

were similar in that all peaked at 37°C. The 24-48 hrs profiles we.re

similar in that they all had peak Ab concentrations of the same order ( -

0.065 mg/ml). The 60-84 hrs profiles were similar in that they all had peak

Ab concentrations of the same order (0.2 mg/ml). The 72 hrs profile had

80

by far the highest Ab concentration peak (0.372 mg/ml). The optimum

conditions for· Ab production arising from the temperature variation

experiment is essentially similar to those giving rise to the profiles at Fig.

3.33.

h) Initial 0.0. variation

The growth profiles (0.0.; 650 nm) of strain A, grown in medium 2, at 120

r.p.m., 37°C and at the following initial O.o.s: 0.200, 0.150, 0.100, 0.6,

0.060, 0.030, 0.015 are shown at Fig. 3.37. The curves of initial O.D.s 0.015

and 0.030 are similar and' follow classical growth curve profiles with

maximum stationary phase O.D.s of 1.789 and 1.992 respectively. The

decline phase O.D.s of these curves start from 48 hrs and fall to 1.029 and

1.173 in 84 hrs, respectively. The 0.203, 0.150 and 0.100 initial O.D.·curves

first rise to 0.0. 0.989, 1.447 and 1.673 in 24 hrs respectively then all fall

exponentially to a trough at 72 hrs, and thereafter begin to rise' to 0.909,

0.7 and 0.793 in 84 hrs, respectively. The 0.06 initial 0.0. curve followed

a biphasic growth profile with mid stationary phase 0.0. of 0.388 in 36 hrs

and final stationary phase 0.0. maximum of 1.34 in 72 hrs.

_. The Ab concentration/time profiles of strain A, grown in medium 2, at the

inoculum levels and conditions cited above are shown at Fig. 3.38. Only

initial 0.0. 0.06, 0.03 and 0.015 profiles gave rise to detectable Ab

concentrations. In the initial O.D. 0.015 flask, an Ab concentration of

0.096 mg/ml is first detected at 36 hrs then falls away to 0.035 mglml in 60

hrs, levelling out to 0.044 mg/ml in 84 hrs. In the initial O.D. 0.03 flask,

'. an Ab concentration of 0.012 mg/ml is first detected at 24 hrs, rises steeply

toO.091 mg/ml in 36 hrs, then gradually falls away with time to 0.065

mglml in 84 hrs. In the initial O.D. 0.06 flask, the Ab concentration rises

to 0.072 mg/ml in 24 hrs, dips slightly to 0.063 mglml in 48 hrs, rises steeply

to 0.372 mg/ml in 72 hrs before falling steeply with time to 0.195 mg/ml in

84 hrs.

81

The optimum conditions arising from the initial O.D. variation experiment

are essentially similar to those giving rise to the profiles at Fig. 3.33.

i) Summmy

A summary of the sequence of optimization experiments with

corresponding factorial increases in production, starting from the

control/complete medium is shown at Table 3.6. . An overall. factorial

increase of 9.3 was finally obtained giving rise to a yield of 0.372 mg/m!.

The greatest single increase in Ab concentration occurred during the r.p.m.

variation experiment.

j) Statistical Error Calculation

The O.D. measurements were found to have a stochastic coefficient of

variation, of 12.3%, the pH measurements, of 1.2%, the zones of inhibition

measurements, of 9.7% and the dose-response calibration curve

assessments of antibiotic concentration in broth culture, of 21.9%.

k) Scale-up fermentations

Two sequences of optimization trials for the 6-litre fermentor, culminating

in the optimum conditions of 150 tr/min, 4L/min, in 3 days and at 37°C are

shown at Table 3.7a and b. A rating system based on visual colour

(yellow) observation was used to obtain the above conditions at set times

of 72 hrs (a). These conditions' were then carried forward for another·

sequence of optimization trails, based on time (b) and here, zones of

. inhibition were used to monitor progress.

82

3.3.4 Isolation. Purification and Quantification of the Antibacterial Principal

a) Determination of appropriate solvent system for extraction /


The path taken during determination of a solvent system for broth

extraction, using the Aszalos and Issaq flowchart (Aszalos and Issaq

1980) is shown at Fig. 3.39a. This resulted in methanol/chloroform

(1/9; solvent 1).

After elution in various solvents the spots and their retention

factors (Rfs) as derived from of the above flowchart (Fig. 3.39a) are

described at Table 3.8. The vertical columns show the Rf results of

. active filtrate (AF)' spots and of fresh medium (FM) spot on

analytical G60 TLC plates, eluted with chloroform (CHCI3),

ethylacetate (EtOAc), methanol (MeOH) and methanol/chloroform

(MeOH/CHCI3; 119). Identification of any eluted spots present

were by charring at 110°C after spraying with cone. H2S04 and

glacial acetic acid (1), by visual assessment (2), and by UV light

(253.7 nm and 356 nm) (3). Two charred spots both with Rfs of 0.7

(1) wer~ found· only after methanol elution of the AF and FM

spots. A yellow active spot (2) was found for each AF spot after

elution with CHCI3, EtOAc, MeOH and MeOH/CHCI3, and these

gave rise to Rfs of 0.13, 0.01, 0.64, and 0.67, respectively. No eluted

spots were observed for the FM spots here. A fluorescent, , . .

yellow/green; non-active (by the assay method 'at section 2.3.1)

material was only observed (3) with the methanol elution of AF

spots. The FM spots acted as controls.

ii) Solvent/solvent extraction

A flowchart for the determination of solvent 2 is shown at Fig.

3.39b. The active spot from. the solvent l/broth extraction was

83

- ----------~

eluted with ethylacetate and gave a yellow spot, Rf 0.29; a <

yellow/green fluorescent spot, Rf 0.58; a dark spot, RfO.72 and··

another dark spot, Rf 0.93. It was eluted with acetone/ethylacetate

(1/9) and gave a yellow spot, Rf 0.25; a fluorescent blue spot, Rf

0.7 and a 'fluorescent yellow/green spot, Rf 0.78. It was eluted with

hexane but no movement was observed. It was eluted with

acetonelhexane (1/9) and gave one yellow/green fluorescent spot, Rf

.. 0.02. Fifnally, it was eluted with hexane/acetone/ethylacetate(9/1/9;

solvent 2) and gave a yellow spot, Rf 0.04; a fluorescent blue spot,

Rf 0.22 and a fluorescent yellow/green spot Rf, 0.46.'

All developed chromatograms were viewed under u.v. light (253.7

nm and 356 nm) and the yellow spot was the only active spot.

The constructed flowchart for the isolation and purification of the

. antibacterial substance using the solvent/solvent extraction route

. and culminating in a freeze-dried powder of the yellow antibiotic is .

shown at Fig. 3.41.

A yield of 50 mg/10L was obtained from the laboratory-scale

ferrilentor.

iii) SolventtrLC extraction

A flowchart for the. determination of solvents for TLC extraction is

shown at Fig. 3.40 .. The active spot (an analytical TLC plate)

obtained from methanol/chloroform (1/9) (solvent 1) broth

. extraction, was eluted with ,ethylacetate (solvent 2) and gave a

yellow spot, Rf 0.29; a yellow/green fluorescent spot, Rf 0.58; a

dark spot, Rf 0.72; and another dark spot, Rf 0.93. After elution

with H20, it gave a dark spot, Rf 0.23; . a yellow spot, Rf 0.56; a

fluorescent blue spot, Rf 0.62 and a fluorescent yellow/green spot, .

. Rf 0.77. Elution with methanol gave a yellow spot, Rf 0.75 and a

84

, fluorescent yellow/green spot, Rf 0.81. Finally, after elution with

methanol/chloroform- (1/9) it gave a yellow spot, Rf 0.77 and a

fluorescent yellow/green spot, RfO.85.

After prep TLC elution with ethylacetate; -a solvent for trituation

was also sought and the Rf results of' those solvents' tried were as .

follows: Hexane/acetone/ethanol (90/10/45), gave a yellow spot, Rf·

0.02; a fluorescent blue spot, Rf 0.04 and a yellow/green fluorescent

spot, Rf 0.05: hexane/acetone/ethanol (9/1/1), gave a yellow spot,

Rf 0.01; a fluorescent blue spot, Rf. 0.02 and a yellow/green

fluorescent spot, Rf 0.04: hex~ne/ethylacetate (9/1) gave a yellow

. spot, Rf 0.005; a fluorescent blue spot, Rf 0.01 and a yellow/green

spot, Rf 0.02: hexane alone gave a yellow/green fluorescent spot, Rf .

0.1 but did not move the yellowspot.

All developed chromatograms were viewed under UV light (253.7 .

nm and 356 nm). The yellow spot was the only active spot.

The constructed flowchart for the isolation and purification of the

antibacterial substance using the solvent{fLC route and culminating

in pure yellow crystals is shown at Fig. 3.42.

A diagram of the developed chromatogram of the active substance,

eluted on prep TLC and as viewed under U.V. light (254 nm) is

shown at Fig. 3.43. It also shows a Rf ofO.29±0.06 for the yellow

band, a Rf of 0.58+0.03 for the yellow/green fluorescent band, a Rf

of 0.72±0.05 for the dark band (I) and a Rf of 0.93+0.06 for the

dark band (11).

A yield of 236 mg/l0L was obtained from the lab-scale fermentor;

85

----- - ._---

b)· Calculation of percentage efficiency of extraction

The percentage efficiency of extraction from active broth to

antibiotic crystals was 62% for the solvent(TLC extraction and 13%

for the solvent/solvent extraction.

c) A check for purity

The initial experiment to find out whether the antibiotic was of a

type which was likely to be adsorbed to the silica in the HPLC

column gave a Rf of 0.64 with solution A (26 mM trifluoroacetic

acid, buffered to pH 3 with triethylamine), a Rf of 0.88 with

solution B (49% acetilenitrile in 13 mM of trifluoroacetic acid) and

a Rf of 0.83 with solution NB (50/50). The wavelength at· which

the most significant peak occurred on the uv/vis scan was 256 nm

and this was used to set the HPLC instrument. The resulting

chromatograph revealed a single peak of retention time 10.4 mins.

d) Assessment of antibiotic concentration in fermentation broth

The log-dose response calibration curve used for assessing the

amount, in mg/ml, of the antibacterial substance present in the

broth situations is shown at Fig. 3.44.

3.3.5 Physical Properties

a) Melting point

The melting point of the antibiotic was 171-173°C and melting occurred with

decomposition as indicated by a browning of the yellow crystals on the hot plate

of the Reichart-Jung melting point apparatus.

86

b) Retention factor determinations

The Retention factor (Rf) determination results are shown at Table3.9. It shows

methanol to be the best eluent with Rf of 0.67 foIlowed by pyridine with Rf of

0.63 then methanol/chloroform (1:9) with Rf of 0.6 .. Hexane, however, did not

move the antibiotic spot and chloroform barely did so (Rf, 0.07).

c) pH/thermal stability tests

. The pH/thermal stability test results are shown at Table 3.10. It shows progressive

loss in activity/stability with increasing time when subjected to concurrent increases

in temperature and pH. There is good stability at Iow pH (29% loss in· activity

after 1000 e incubation for 60 mins at pH 2) but poor stability at high pH (100%

loss activity after 60°C incubation for 60 mins at pH 9)

d) Ultraviolet light stability test

The UV light stability test results are shown at Table 3.11. There is significant

loss in activity/stability (19%) after the first day and this value remains constant

throughout the 7 days. With the tetracycline control, the loss in activity/stability

after the first day is not as pronounced (14%) as the antibacterial substance but

from then on, loss is of the same order (18%).

3.3.6 Microbiological Properties

a) Minimum inhibitOlY concentration (MIC)

The tube dilution method gave an MIC of S 0.5 /Lg/ml against E. coli in

basal medium, incubated at 37°C. The agar diffusion method gave MICs

against a range of microorganisms as shown at table 3.12. From this, C.

albicans and Cl. sporogenes were uninhibited up to 2000 /Lg/ml and the

fungi were generaIly less inhibited than the bacteria. E. coli's MIC by this

method was 1.3 /Lg/ml.

87

- -----------

b) Effect of the antibacterial substance on growth of E.coli

A graph of 0.0. against time, depicting antibiotic challenge against E.coli

growing in basal medium at 90 f.p.m., 37 Q C is shown at Fig. 3.45. The

control curve describes a conventional growth curve profile with maximum

stationary phase O.D. of 1.75. The test curve initially followed the control

curve but levelled off, achieving a mioomum stationary phase O.D. of

0.402. This occurred soon after addition of enough of the antibiotic to give

a MIC equivalent in the 50 ml culture. A1iquois taken out, at times 3, 3.5

and 4 hrs and individually streaked onto NA plates, registered growth after .

overnight incubation at 37Q C.

3.3.7 Chemical properties

All the qualitative chemical analytical tests but one, done on the antibiotic, proved

negative. The positive result obtained was for Feigl test (slight red) but negative

results were obtained for the following: SchiffsIFuschsin test (no wine purple

colouration) Formalin test, ninhydrins test (no colour change) Anthrone test (no

. colour change); Kovac's test (no colour change); Ehrlichs test (yellow colour

rather than violet).

3.3.8 Spectral data

. a) Ultra violetMsible scans

The scan of the impure (broth filtrate) and purified antibiotic are shown

at Fig. 3.46 and Fig. 3.47 respectively. At Fig. 3.46 there is a large peak

at 210 nm with absorbance 2.3, a smaller peak at 257 nm with absorbance

0.85 and a broad peak at 380 nm with absorbance 0.175. At Fig. 3.47 there

is a peak at 258 nm with absorbance 1.175 and a broad peak at 400 nm

with absorbance 0.25.

88

-------------------

b) Infra-red scan

The scan of the antibacterial substance is shown at Fig. 3.48. The main

absorption bands are at 3120, 2980, 1680, 1620, 1540, 1440, 1390, 1305,

1260, 1220; 1160, 1060, 975, 930, 880, 825, 785, 740, 725 cm· l•

c) Mass spectrometIY

The scan at Fig. 3.49 shows the first electron/mass (e/m) ratio to be.193.

Subsequente/m ratios ~f significance detected, include: 179, 165, 151, 136,

123, 109, 93, 83, 67, 56. Table 3.13 shows a list of the arithmetically

possible atomic compositions of the compound as generated by the mass

spectrometer data system.

3.3.9 X-ray cIYstallography

a) Solvent cIYstallization

Twinning was clearly evident under phase-contrast microscopy when using'

ethanol and' methanol but under this scrutiny, was not apparent with

'methanol/chloroform (1/9). Itwas'only after preliminary X-ray diffraction

that the latter was revealed to be unsuitable as twinning had occurred.

The solvent best suited for crystal formation was chloroform.

b) X-ray data

C,HP2NS' Mr= 193.1, monoclinic, PZ/C, a = 9.675 (5), b = 14.028 (5),

C = 6.873 (5), B = 116.4 (2), V = 835.24, Z = 4, Dx = 1.536 gcm,3,

(>-.(MoKa) = 0.71069 A, Jl. = 0.69 cm'l, F(OOO) = 512, T = 293K.

The positional parameters and equivalent isotropic temperature factors are

listed at Table 3.14. Bond lengths and angles are listed at Tables 3.15 and

3.16, respectively. Using the above data and an atomic numbering scheme,'

89

the molecular structure, Fig. 3.50, and the unit cell contents, Fig. 3.51, were "

draWn by the ORTEP facility of the X-ray diffractometer.

3.4.0 THE EFFECf· OF MAGNESIUM SALT OMISSION FROM THE

MEDIUM RECIPE

3.4.1 Cell Propagation Experiments

A flowchart/result, of the experiments done on strain A and Ps. aeruginosa in AR

and SLR basal medium is shown at Fig. 3.52. It showed that starting from the

'complete' (basal) medium situation, growth and an increased production of yellow .

pigment (visually assessed; + +) from strain A was perpetuated by 'washed' .

inocula transfer (initial OD -= 0.06) in a series of Med A2 (SLR) (50 ml/flask).

However, when AR grade chemicals were substituted in the same experiment, .

although pigmentation was detected (visually assessed; . +) in the 'complete'

medium, growth without pigmentation occurred in Med A2 (AR), ceasing

altogether after first generation propagation. When Ps. aeruginosa was substituted

for strain A in the SLR experimental situation, a yellow/green pigment, with no

detectable Ab or Af activity was produced after propagation from the complete

medium to Med A2 (SLR). Here, growth ceased after further propagation (first

generation) in Med A2 (SLR).

3.4.2 Atomic Absorbtion (AA) Measurements of Mg2+in Basal Media

The AA calibration curve for Mi+ (Mg(N03)2.6H20) from . which ,deflections on .

the AA instrument are read to give the concentration in ppm is shown at Fig.

3.53 .

. The Mi+ concentration· (ppm and molarity) present in Med 'A2, after the'

inoculation (OD ::: 0.06), incubation (90 r.p.m., 37°C for 20 minutes) and

membrane filteration experiment, using both SLR and AR grade chemicals are

shown at Table 3.17. SLR and AR constituted media controls were also measured

for Mg2+ content.

90

- -----------c----------c---------

All SLR constituted media appear to contain more Mg2+ than the AR constituted

media (0.095 ppm and 0.140 ppm (SLR); 0.038 ppm and 0.102 ppm (AR): 0.095

ppm and 0.038 ppm for the experiment and then 0.140 ppm and 0.102 ppm for

the controls).

3.4.3 Analysis of Mg2+ in cells of strain A grown in basal medium and in Med

A2

The results of experiments at 2.4.3 are as follows:

1) weight of charred cell pellets (basal medium) = 9.0 mg·

2) weight of charred cell pellets (Med A2) = 7.0 mg

3) [Mg2+] from pellets dissolved in 4 ml of HCL

(basal medium) = 0.7 ppm

4) [Mg2+] from pellets dissolved in 4 mls of HCL

(Med A2) ~

= 0.2 ppm

. Mg2+ in cells of strain A basal medium = 0.7 x 1 = 0.31ppm/mg of cells 9.0 1

Mg2+ in cells of strain A basal medium = 0.2 x 1 = 0.11ppm/mg of cells 7.0 1

3.4.4 Viable Growth Curves

The viable growth curve profiles (log viable cell count/time) of the complete

medium and of Med A2, plotted on the same axis is shown at Fig. 3.54. They are

generally similar but the MedA2 profile appears to have grown to a log viable

cell count (L VCC) of 11.8 in 36, hrs immediately followed by a steep fall, whereas

the fall from a LVCC of 11.1 in 36 hrs, in the complete medium profile, is gradual

at first, reaching a LVCC of 10.8 in 48 hrs, finally followed bya steep fall.

91

3.5.0 ANTIFUNGAL ACfIVITY STUDIES

3.5.1 Elicitation of Antibiotic Production in Liquid Media·

From Table 3.5, the only liquid media which gave rise to detectable (disc diffusion

. assay) antifungal activity against C. albicans were Potato Dextrose Liquid Medium·

-1 (PDLM-1!medium v) CzapG!.k Dox Liquid Medium - modified (medium q).

Activity 'was detected for all, after day 7 but with medium q activity was also

detected after day 2.

3.5.2 Preliminaty Testing of Aritifungal (AD Substance/s

Heat stability

After heating in a boiling water-bath for 20 mins, the activity of the broth

filtrate against C.albicans, as measured by the disc diffusion assay, had

dropped by 5%.

3.5.3 Pigmented Inoculum Emeriment

a) Orbital Incubation

After 2 days incubation at 90 r.p.m., 37°C, the medium q had developed

an intense yellow pigmentation with antifungal (Af) disc zone of inhibition.

of 24.2 mm against C. albicans and antibacterial' (Ab) disc zone of

inhibition of 25.0 mm against E. coli. Further incubation failed to increase

pigmentation.

The O.D. growth profile, the zone of inhibition profile and the pH profile

of strain A, grown in PDLM batch number (No.) 15621497 (PDLM-1), at

90 r.p.m., 37°C are shown at Fig. 3.55. The O.D. describes a conventional

growth curve profile with good growth (O.D. 2.0 in 24 hrs, maintaining this

level to 288 hrs). The zone of inhibition profile rose steeply from about.

92

130 hrs to 36.1 mm in 144 hrs, began to level, reaching a peak of 41.6 mm

in 264 hrs before dropping to 35 mm in 288 hrs. The pH profile started

. at 5.6, dropped steeply in 24 hrs, remained at this level to 144 hrs then

gradually rose again, eventually reaching 7.7 in 288 hrs.


After a 10 day ambient (20°C) incubation, the medium q culture had

developed yellow pigmentation, giving Af disc zone of inhibition of 20.2

mm and Ab disc zone of inhibition of 23.4 mm. Further incubation

increased pigmentation.

The O.D. growth profile, the zone of inhibition profile and the pH profile

of strain A, grown in PDLM batch No. 15621497, at static condition and at 37°C,

against time are shown at Fig. 3.56. The O.D. rose to about 0.7 in 24 hrs,

plateaued before gradually rising to 1.7 in 288 hrs. The only zone of inhibition

detected was at 288 hrs and was only of 14.5 mm diameter. The pH profile rose

slightly before dropping to 3.8 in 48 hrs then maintained this level throughout the

period of incubation. A similar experiment with PDLM of the same batch No.

gave no activity with Ps. aeruginosa.

3.5.4 Optimization Eweriments

a)' Medium component single detection (MCSD) OF medium q

Results of the MCSD experiment are as shown 'at Fig. 3.57-59 inclusive.

They show the O.D. growth profiles, the antibacterial (Ab) and Af zone of

inhibition profiles of strain A, grown in complete, MCSD-NaN03 (Med

B1), MCSD-Mg glyP (Med B2), MCSD-FeS04 (Med B3), MCSD-KCl,

(Med B4), MCSD-sucrose (Med B5), MCSD-glycerol (Med B6) and

MCSD-carbon source (Med B7) media.' The complete and Med B4 growth

profiles are very similar and describe conventional growth curve profiles

with stationary phases which after 12 hrs at about O.D. 0.6 increased

93

exponentially to about 1.2 in 96 hrs. Med B3's CUIVe is also similar but

occurs with lower stationary phase O.D., from 0.33 in 12 hrs to 0.839 in 96

hrs, as is also Med B7's CUIVe which occurs with even lower stationary

phase O.D., from 0.227 in 12 hrs to 0.29 in 96 hrs. Med B2's CUIVe is fairly

similar to the Med B3's CUIVe except that it rises gradually to 0.648 in 36

hrs then rises exponentially to 0.960 in 96 hrs. Med B6's growth CUIVe

initially rises steeply to 0.838 in 12 hrs but thereafter falls away

exponentially. Med B5's profile appears to have a biphasic character with

a mid-stationary phase occurring between 40 to 60 hrs at O.D.s 0.218 to

0.228. Finally, Med B1's CUIVe initially shows a very small rise in O.D. to

0.140 which plateaus and then very gradually falls away with time to 0.106

in 96 hrs.

The pH profiles of Med B4 and Med B7 showed a similar trend, rising

gradually to peak at about 7.6 in 36 hrs and barely maintaining this level

in 96 hrs. Med B5's profile also followed this general trend but here,

.described a more even keel, whereas Med B6's pH profile which also

showed this general trend, was more accentuated. The complete medium

pH profile was also similar but displayed a steep initial rise to 7.5 which

levelled out after 12 hrs and had no marked dip at 60 hrs. The pH profile

of Med B3 dipped initially to 5.8 but after 12 hrs rose back to the original

level (6.8), maintaining this throughout the incubation period. Med Bl's

profile also had an initial drop but was much more steep, falling to 3.9

then levelling· out after 12 hrs and maintaining this throughout the

incubation period. Med B2's pH profile was more erratic. Initially it fell

to 4.5 in 12 hrs, rose gradually back again to 7.6 in 48 hrs then fell to 6.5

in 60 hrs, levelling out with time.

No zones of inhibition were detected in Med B1, Med B3, Med B5 and

Med B7. Of all the antibiotic producing media here which were able to

trigger production of both Ab (E. coli) and Af (C,. albicans) substances,

only Med B2, initiated the production of the antibacterial substance first

(with the exception of Med B6) whereas it was the other way round for the

94

rest. Med B6 appeared to have initiated the production of both antibiotics

about the same time (12 hrs). From Fig. 3.58 the Ab zone of inhibition

profile, produced by the complete medium, describes a semi-circle between

12-72 hrs with an apex of 17.3 mm in 36 hrs. Med B4's Ab profile rises

from 24 hrs to a peak of 19 mm at 36 hrs then falls fairly steeply to 14 mm

in 48 hrs. Med B2's Ab profil~ rises from 24 hrs to 17.6 mm, levels out at

72 hrs then rises again to 22.3 mm in 84 hrs before falling to 17.3 mm in

96 hrs. Med B2's peak zone of inhibition was by far the largest Ab zone

of inhibition and occurred at 84 hrs.

During the MCSD experiments, Af zones of inhibition were detected in the

complete medium, Med B4 and Med B6 at 12 hrs only. Of these, Med B4

gave the largest zone of inhibition, (19 mm) whilst the other 2 zones were

of the same order (.=.17 mm) Fig. 3.59 Med B2 Af zone of inhibition

profile however, was first detected at 84 hrs and continued to rise from

17.5 mm to 18.5 mm in 96 hrs. The last detected zone size here was

comparable to that of Med B4.

b) Sodium nitrate variation

The growth profiles (0.0.; 650 nm) of strain A, grown in Med B2 plus

NaH2P04 (0.5 gm/L; medium 3) at 37°C, 90 r.p.m. and at the NaN03

percentage concentrations of 0.00, 0.05, 0.10, 0.15, 0.20, 0.25 and 0.30 are

shown at Fig. 3.60. As the concentration of NaN03 increases there seems

to be a tendency towards the formation of biphasic growth curves between

0.0 to 0.15% and between 0.25 to 0.3%. This biphasic tendency is best

expressed at the lower concentrations. The best overall growth occurred

at 0.2%. However, although best initial growth occurred in the 0.3%

medium, achieving an 0.0. of 1.15 in 24 hrs, this fell away to 0.0. 0.618

in 96 hrs and only later rose again to 0.0. 1.125 in 192 hrs. It should be

noted that the 0.0% curve rose very gradually to 0.0. 0.147 in 96 hrs, but

fell away with time to 0.095 in 192 hrs.

95

The Ab (E. coli) zone of inhibition/time profiles of strain A, grown in

medium 3 at the NaN03 concentrations and at ~he conditions cited above

are shown at Fig. 3.61. No Ab zones of inhibition were detected in the 0.2

and 0.0% media throughout the incubation period. The 0.3% profile first·

appears with a zone of 16.6 mm at 96 hrs, rises fairly steeply to 19.1 mm

in 120 hrs then maintains this level throughout to 192 hrs. This same trend

is followed by the 0.25 to 0.1% profiles. However, it is to be noted that

the 0.1 % zone of inhibition profile begins to rise after 168 hrs to 20.3 mm

in 192 hrs, that the 0.15% zone of inhibition profile first appears at 72 hrs.

and that the 0.25% profile falls away after 168 hrs to 16.1 mm in 192 hrs.

The 0.05% profile appears quite different from the rest in that it first

appears with a zone of 15.7 mm at 120 hrs and continues rising steeply to

24.1 mm in 192 hrs. The last detected Ab zone size here, was the largest

so far produced by any medium in the present series.

The Af zone of inhibition/time profiles of strain A, grown in medium 3, at

the concentrations and conditions cited above are shown at Fig. 3.62. No

Af zones of inhibition were detected in the 0.25, 0.2, 0.15 and 0.0% media

throughout the incubation period. The 0.05 % profile first appears with a

zone of 19.4 mm at 120 hrs, rises to a peak of 23.3 mm at 144 hrs then

falls away steeply to 16.6 mm in 192 hrs. A similar though lower profile

level is seen with the 0.1 % curve but here, it starts with a zone of 18.7 mm .

at 96 hrs and rises steeply only after 120 hrs, to 21.5 mm in 144 hrs before

falling steeply to 14 mm in 192 hrs. The 0.3% profile also appears similar

but occurs at a lower profile level than that of 0.1 %. Here, it starts with

it zone of 14.6 mm at 72 hrs and again only rises steeply after 120 hrs to

19.1 mm in 144 hrs, falls steeply to 15 mm in 168 hrs before plunging to

zero hi 192 hrs.

A graph of Ab zone of inhibition profiles for strain A, measured at 0 hr,

24 hrs, 96 hrs, 120 hrs, 144 hrs, 168 hrs, 192 hrs, against NaN03 percentage.

concentrations is shown at Fig. 3.63. No zones of inhibition were detected

in the 0.2% medium and, also, at all NaN03 concentrations up to 24 hrs

96

incubation. The largest zone, 24.1 mm, was detected at the lowest

concentration, 0.05%, only after the longest time, 192 hrs. It should also

be noted that apa.rt from the. 72 hrs profile, and at times beyond this,

increasing the concentration beyond 0.2% gave rise to production of the

antibacterial substance/s which increased with increasing concentration.

The zone of inhibition profiles of 96 hrs and 120 hrs are similar, though

the former occurs at lower levels. The profiles of 144 hrs, 168 hrs and 192

hrs are also similar to the above two, but register zones of inhibition

occurring at 0.05%, which all appear to increase with time from 15.7 mm

at 120 hrs, to 24.1 mm at 192 hrs.

The graph of Af zone of inhibition profiles, measured at the same times

and conditions cited above is shown at Fig. 3.64. No zones of inhibition

were detected from the media containing 0.15, 0.2 and, 0.25%

. concentration of NaN03 and, also, at all NaN03 concentrations up to 24

hrs. As with the Ab profiles, the largest Af zone, 23.3 mm, was detected

at the lowest concentration,0.05%, falling as the NaN03 concentration is

increased. However, unlike the Ab profile, the largest Af zone occurred

at 144 hrs. Here, increasing the NaN03 concentration beyond 0.25%

resulted in detectable production of the antifungal substance/s and it is to

be -noted that before 72 hrs,. ill concentrations lower than 0.3%, no

significant production was detected. Production was also only detected at

96 hrs in the 0.1% and the 0.3% media.

The growth profile (0.0.; 650 nm), the Af and Ab zone of inhibition

profiles imd the pH profile of strain. A, grown in medium 3 of 0.05%

NaN03 (medium 4), at pH 6.8, 37°C and 90 r.p.m are shown at Fig. 3.65.

The pH profile initially falls to 3.9 in 24 hrs; plateaus and begins to rise

again to 6.2 in 96 hrs, levelling out to 6.4 in 192 hrs. The biphasic

character of the growth curve is clearly evident. It should be noted that

production of both the Af and Ab substances appear to begin during the

second log phase of the growth curve and concurrent with the increases in

pH from 4 in 72 hrs to 6.4 in 120 hrs. From· the NaN03 variation

97

experiment, this represents the optimum conditions for the largest zones

of inhibition achieved so far: Ai, 23.3 mm; Ab, 24.1 mm.

c) Sucrose variation

The growth profile (O.D.; 650 nm) of strain A, grown in medium 4, at

. 37°C, 90 r.p.m. and at the sucrose percentage concentrations of 0, 0.5, 1,

2, 3, 4 and 5 are shown at Fig. 3.66. The media containing 0.5 and 1 per

cent sucrose gave conventional growth .curves with the 0.5% curve

occurring at lower stationary phase O.D.s. The 0.0% curve which has only

glycerol as a carbohydrate source, rises steeply to O.D. 0.60 in 48 hrs then

falls to O.D. 0.402 in 72 hrs before gradually rising with increasing time to

.O.D. 0.75 in 192 hrs. From 2.0 to 3.0%, there is a tendency towards

biphasic growth which has its best expression at 3.0%. The 4 and 5%

media gave rise to similar growth profiles: there is an initial O.D. rise to

about 0.116 in 24 hrs, followed by a further, gradual rise with increasing

time to about O,D. 0.350 in 192 hrs. These last two media gave by far the

poorest overall growth.

The Ab zone of inhibition/time profiles of strain A, grown in medium 4, at

the sucrose concentrations and conditions cited above are shown at Fig.

3.67. No zones of inhibition were detected at the 0, 0.5 and 5.0%

concentrations throughout the incubation period. The zone of inhibition

profiles of the 2 and 3% media are similar in that production seems to

increase with time but are different in that the first detected zone of

inhibition for the 2.0% medium occcurred at 120 hrs, and that of the 3.0%

medium occurred after 144 hrs. The 4.0% and 1.0% profiles appear at the

same time (192 hrs) but much later than the 2 and 3% media .. Of the

above four profiles, the 4% zone of inhibition curve is comparatively

greater and rises to 21.5 mm in 192 hrs, followed by the 3% and 2% which

rises from 15.7 mm in 144 hrs to 19.7 mm in 192 hrs and from 16 mm in

120 hrs to 19.1 mm in 192 hrs, respectively (of the same order). Last of

all comes the 1%, which rises to 14.4 mm in 192 hrs.

98


the sucrose concentrations and conditions cited above are shown at Fig ..

3.68. No zones of inhibition were detected at 5.0% concentration

throughout the incubation period and at 4.0%, evidence of antifungal

production was only detected after 192 hrs. Zones of botli the 0% and

0.5% sucrose media, which were of the. same order (15 mm), were first

detected at 168 hrs. Production in the latter appeared to increase with

time whereas the former's zones of inhibition remained at the same level

throughout. The 1 % and 2% concentrations, with similar profiles, also

gave zones of inhibition of the same order. (17.5 mm) which when they

once appeared, stayed level with increasing time. However, though similar,

the 1% zone of inhibition appeared much earlier, at 48 hrs and the 2%

. first appeared at 96 hrs. The initial zones of the 1% medium were also

noticably larger: 15.8 mm, compared to 14.3 mm. In contrast to the

general trend, the 3% profile starts with a zone of inhibition of 17.2 mm

at 120 hrs, rises with increasing time to a peak at 19.3 mm in 144 hrs then

gradually falls to 17.7 mm in 192 hrs. As can be seen, zones here (3%),

are much larger than the zones of all the other profiles.

The growth profile (O.D.; 650 nm), the Ab and Af zone of inhibition

profiles and the pH profiles of strain A, grown in medium 4, at 90 r.p.m.,

37°C are shown at Fig. 3.69. The graph is similar to that of Fig. 3.

(medium 3), the only significant difference being the Af zone ·of inhibition

profile which appears 24 hrs later ..

d) Glycerol variation

The growth curve profiles (O.D.; 650 nm) of strain A, grown in medium

4, at 37°C, 90 r.p.m. and at the glycerol percentage concentrations of 0, 1,

2, 3, 4, 5, 6, 7 and 8 are shown at Fig. 3.70. The 0 and 5% profiles are

similar and describe biphasic growth profiles where the final stationary

phase falls away with time. However, final stationary phase O.D. of the

0% curve occurs at a lower level (O.D. 0.401 as opposed to O.D. 0.608 for

99

5%, all at 144 hrs). Profiles 1 to 4% also exhibit some biphasic growth

. where the initial log phase is followed by a fall in O.D. to a 'trough',

between 120 hrs - 144 hrs, after which time they rise again with increasing

time .. After an initial log phase, rising to O.D. 0.56 in 48 hrs, the. 6% .

profile attempts a stationary phase but this begins to fall from 72 hrs, to

0.0. 0.295 in 120 hrs. This fall is followed by a second short log phase,

rising to O.D. 0.416 in 144 hrs which then falls to O.D. 0.233 in 192 hrs.

The 7% profile is not unlike the 6% but manages a longer stationary

phase, reaching O.D. 0.56 in 96 hrs, after an initial log phase rise to O.D.

0.416 in 144 hrs. Here, it also fails to achieve any form of secondary log

phase but falls fairly steeply to O.D. 0.208 in 192 hrs. As can be seen, this

profile occurs at lower O.D.s than the 6% profile.


the glycerol concentration and conditions cited above are shown at Fig.

3.71. Throughout the incubation period, no zones of inhibition were

detected for the 0, 1, 2, 3% concentrations and at 4%, evidence of Ab

production was detected only after 192 hrs. With the remaining . ,

concentrations, there appears a general increase in Ab production with

increasing time, though the 8% profile levels out to 19.7 mm in 192 hrs .

. Other differences lies in the first detection of zones which often occurred

at different times: the 7 and 8% profiles first appear with zones of the

same order (=. 16 mm) at 72 hrs; the 6% profile, at 120 hrs (15.8 mm) and

the 5%, at 144 hrs (14.9 mm). The 8% medium gave rise to the largest

zone of inhibition (20.2 mm) at 168 hrs.


the concentrations and conditions cited above are shown at Fig. 3.72. No

zones of inhibition were detected for the 0 and 1% concentrations

throughout the incubation period and at 2% concentration, evidence of Af

production was detected only after 192 hrs. The 3% concentration profile

first appeared with a zone of 19.6 mm at 168 hrs and rises with increasing

time to 23.6 mm in 192 hrs. The appearance of the 4 to 7% profiles do

100

not genera\Iy continue to rise with increasing time but rather tends to

stabilize between a 17-20 mm zone of inhibition band and here, the initial

rise of the 7% profile above 20 mm, is the exception. First detection ofAf

activity for each of the 4-7% media occurred at different times: the 7 and

8% profiles, first occurs at 48 hrs, the 6% profile at 96 hrs, the 5% profile

at 120 hrs and the 4% profile at 144 hrs. The 2% and 3% mediadearly

produced the largest zones of inhibition after the longest recorded time

(24.4 mm and 23.6 mm respectively after 192 hrs).

A graph of Ab zone of inhibition profiles measured at 0 hr, 4~ hrs, 72 hrs,

96 hrs, 120 hrs, 168 hrs, 192 hrs, against percentage glycerol concentration

is shown at Fig. 3.73. No zones of inhibition were detected up to 48 hrs

at aU concentrations. There is a general trend of a gradual increase in

production with increasing percentage concentration, at a\I times, after 72

hrs. The 192 hrs profile however, shows increases in zone of inhibition

with increasing concentration to 6% (19.3 mm) then levels out, reaching

19.7 mm at 8%. The largest zones of inhibition were exhibited at 8% after

144 hrs to 192 hrs. Up to 144 hrs, there is a progressive increase in zone

sizes with increasing incubation time, mainly from 72 hrs to 120 hrs.

A graph of Af zone of inhibition profiles measured at the same times,

glycerol concentrations (medium 4) and conditions cited above is shown at

Fig. 3.74. Zone size measurements between 2 to 8% concentration, and

after 72 to 168 hrs, tend to lie between a 16-20 mm zone of inhibition

band. The zones of inhibition, measured in ascending glycerol percentage

concentration media, at 48 hrs, were first detected at 7% and thereafter,

zone size feU with increasing glycerol concentration. The zone of inhibition

profiles at 192 hrs, first appears with a zone of 24.4 mm at 2%

concentration, faUs graduUy with increasing concentration to 19.5 mm at

4% then levels out within the zone of inhibition band described above as

the glycerol concentration is increased further. The 7 and 8% profile first

appears at 48 hrs, the 6% at 96 hrs, the 5% at 120 hrs, the 4% at 144 hrs,

the 3% at 168 hrs and the 2% at 192 hrs. No zones of inhibition were

101

detected in the 0 and 1% media throughout the incubation period. The

largest zone of inhibition, 24.4 mm, was detected in the 2% medium at 192

hrs, closely followed by that detected in the 3% medium (23.6 mm) also at

192 hrs.

The O.D. growth profile, the Ab and Af zones of inhibition profiles and

the pH profile of strain A, grown in medium 4 of 2% glycerol (medium 5),

at 90 r.p.m., 37°C are shown at Fig. 3.75. The pH profile falls very

gradually from 6.8 to 5.5 at 96 hrs and then very gradually rises to 6.6 in

192 hrs. The biphasic character of the O.D. profile is clearly evident and

here again, the time of detectable production of the antifungal substance/s

coincide with the rise of the second log phase. No antibacterial activity

was detected throughout the incubation period. From the percentage

glycerol variation experiment, this represents optimum conditions for the

largest Af zone of inhibition, 24.4 mm, achieved so far.

e) pH variation

The growth profiles (O.D.; 650 nm) of strain A, grown in medium 5, at

37°C and at initial pHs of 3,4, 5, 6, 7, 8 imd 9 are shown at Fig. 3.76" At

. pH 3 there was hardly any change in the initial O.D. throughout the

incubation period. The pH 4, 5, 6 and 7 profiles all exhibit some biphasic

character. The pH 4 and 5 profiles have a relatively short mid-stationary

phase (24 to 96 hrs) followed by a gradual rise in O.D. but the pH 6 and·

7 profiles, after the initial log phase; exhibit a longer mid-stationary phase

. (24 to 144 hrs). Growth in media of pH 8 and 9, follow a conventional

growth curve profile with a hint of biphasic character in early stationary

phase (72 hrs) for 9 and mid-stationary phase (120 hrs) for 8.


the pHs and conditions cited above are shown at Fig. 3.77. Only the pH

3 media registered zones and this started with 18.6 mm at 48 hrs and

gradually rose to 29.1 mm in 192 hrs.

102


the pHs and conditions cited above are shown at Fig. 3.78. No zones of

inhibItion were detected for pHs 3 to 5 throughout the incubation period.

The remaining pHs alI gave rise to zones of inhibition but only at 192 hrs .

. By far the largest zone of inhibition, 25.4 mm,occcurred with the pH 8

medium, folIowed by pH 7, 20.5 mm; pH 9, 17 mm and pH 6, 15 mm.

The growth profile (0.0.; 650 nm), the Ab and Af zone of inhibition

profiles and the pH profile of strain A, grown in medium 5, at pH 3, 90

r.p.m., 37°C are shown at Fig. 3.79. The pH profile remains steady, barely

faIling with time. No antifungal zones of inhibition were detected

throughout the period of incubation. Peak Ab zone of inhibition (29.1

mm) occured at 192 hrs.

The 0.0. growth profile, the Ab and Af zone of inhibition profiles and the

pH profile of strain A, grown in medium 5 of pH 8 (medium 6), at 90

r.p.m., 37°C are shown at Fig. 3.80. The pH profile initially falIs to 4.4 in

48 hrs then levels out to 72 hrs, climbs to about 5.8 and maintains this level

throughout. A statisticaIly significant though small biphasic character is

evident as shown by a rise in 0.0. from 144 hrs which then levels off

reaching at 192 hrs. Antifungal activity was only detected at 192 hrs and

Ab activity was detected throughout the incubation period. From the pH

variation experiments, this represents the optimum conditions for the

largest Af zone of inhibition (25.4 mm) achieved so far in smaU 50 ml

batch cultures.

f) Agitation rate (RPM) variation

The growth profiles (0.0.; 650 nm) of strain A, grown in medium 6, at

37°C and at the r.p.ms of 0, 40, 60, 90, 120, 150, 180 and 210 are shown

at Fig. 3.81. The 180 and 150 r.p.m. curves describe conventional growth

curve profiles and although the 210 r.p.m. curve is similar to the above

two, it differs in that there is a 24 hrs lag phase. The 120 r.p.m. is also

similar to the above but dips to 0.0. 1.507 at 168 hrs before gradually

103

rising. to O.D. 1.549 in 192 hrs. The 90 r.p.m. is also similar but exhibits

, a statistically significant, though small biphasic tendency, with mid-

. stationary phase around 120 hrs. The 60, 40 and 0 r.p.m. curves, though

initially following a conventional growth curve profile, begin to gradually .

fall away after 72 hrs, and here, stationary/decline phase O.D. increased

with increasing r.p.m.


the r.p.ms and conditions cited above are shown at Fig. 3.82. No zones of

inhibition were detected at 210 r.p.m. throughout the period of incubation.

The 0 r.p.m. (static incubation) profile was first detected with a zone of 16

mm at 24 hrs. It then remainsvirtually constant until after 144 hrs where

it begins to rise, peaking at 17.8 mm after 168 hrs, then falls to 16.1 mm

at 192 hrs. The 40 r.p.m. profile is similar but after a zone of 15.7 mm at

24 hrs, it rises to the higher level of 19.5 mm in 72 hrs before levelling out

and following a similar profile to that of 0 r.p.m .. The 60 r.p.m. profile is

similar to the 40 r.p.m's, up to 17.8 mm in 144 hrs where it dips slightly to

17 mm in 168 hrs before rising to 20.9 mm in 192 hrs. Zones of inhibition

were at first not detected during the period of incubation at 90 r.p.m. until

at 192 hrs where a zone of 25.4 mm was detected. The profiles of 120

r.p.m., 150 r.p.m. and 180 r.p.m. are all similar to each other in that, with

the exception of 180 r.p.m. which starts late at 72 hrs, they were all first

detected after 24 hrs and generally rose with increasing time. By far the

largest zone of inhibition (29.8 mm) was detected at 120 r.p.m. (192 hrs)

followed by 25.9 mm at 150 r.p.m.; 25.4 mm at 90 r.p.m. and 24.9 mm at

180 r.p.m., all at 192 hrs.

A graph of Af zone of inhibition profiles of strain A, measured at 0 hrs, 24

hrs, 72 hrs, 120 hrs, 144 hrs, 168 hrs, 192 hrs, against the shake rate

(r.p.m.) is shown at Fig. 3.83. At 24 hrs, a zone of inhibition of 16 mm was

registered at 0 r.p.m. and this remained constant with increasing r.p.m. to

60 r.p.m. It then dropped to zero at 90 r.p.m., rising again to 16 mm at

120 r.p.m., falling to 14.5 mm at 150 r.p.m. before finally falling to zero at

104

---- - ------------------

180 r.p.m. This general profile pattern is repeated after 24 hrs to 168 hrs

with small differences, that is to say, from 0 to 40 r.p.m., there occurs an

increase in antifungal production with increasing r.p.m., followed by a fall

at 60 r.p.m. It is to be noted here that the final fall in zone of inhibition,

occurs at 210 r.p.m. (not 180 r.p.m. as per the 24 hrs profile) and, from

120 r.p.m. to 180 r.p.m., zone of inhibition profiles progressively increase

in level with increasing time. The zone of inhibition profile measured at

192 hrs, starts to increase with increasing r.p.m. to a peak of 29.8 mm at

120 r.p.m., begins to fall to 25.9 mm at 150 r.p.m., then falls to zero at 210

r.p.m. It is to be noted that unlike the rest, the zone of inhibition profile

here, does not fall at 90 r.p.m.

The growth profile (O.D.; 650 nm), the Af zone of inhibition profile and

the pH profile of strain A, grown in medium 6 at 120 r.p.m., 37°C are

shown at Fig. 3.84. The pH profile begins at 8 and gradully falls to 5.7 at

144 hrs, levels out thereafter to 168 hrs before gradually rising to 6.1 in 192 .

hrs. From the r.p.m. variation experiment, this represents the optimum

conditions for the largest Af zone of inhibition (29.8 mm) achieved so far

in small 50 ml batch cultures.

g) Temperature variation··

. The growth profiles (0.0.; 650 nm) of strain A, grown in medium 6, at 120

r.p.m. and at 22°C, 27"C, 32°C, 37°C and 42°C are shown at Fig. 3.85.

The 22°C profile rises gradually to O.D. 0.571 in 216 hrs. The 32°C and

27°C curves describe conventional growth curve profiles with the latter occurring at a lower stationary phase 0.0. (2.1 and 1.55 in 216 hrs,

respectively). The 37°C profile is similar to that of 32°C but dips to 0.0.

1.507 in 168 hrs and gradually rises to O.D. 1.737 in 216 hrs. Of the two,

the 32°C profile has overall the best growth. The 42°C growth profile

initially follows the 32°C profile, up to 24 hrs, falls steeply to O.D. 0.281 .

in 72 hrs then levels out to O.D. 0.280 in 216 hrs. Cell

aggregation/clumping was seen, after 72 hrs, in all the flasks incubated at

105

I

22°C and 27°C. A malodour was emitted, after 96 hrs, from flasks

incubated at 42°C.


the temperatures and conditions cited above are shown at Fig. 3.86: No

zones of inhibition, were detected at 22°C and 47°C throughout the

incubation period. At 37"C the production generally increases with

'increasing time. At 27°C the profile initially increases to 22.4 mm ill 72

hrs, but levels out to 120 hrs, rises to 24.6 mm in 144 hrs, then after 168

hrs, begins to fall away to 20.2 mm in 216 hrs. At 32°C, there is rapid production at the early stages of the incubation period with a rise to 25.8

mm in 24 hrs which levels out and then begin to rise again to 30.6 mm in

192 hrs, levelling to 31.4 mm in 216 hrs. The largest zone of inhibition,

35.5 mm, was detected at 37°C and at 216 hrs. It should also be noted

that at 192 hrs, both the 32°C and 37°C situations gave rise to comparable

zone sizes.

The growth profile (O.D.; 650 nm/time), the Af zone of inhibition profile

and the pH profile of strain A, grown in medium 6, at 120 r.p.m., 32°C are

shown at Fig. 3.87. The pH profile falls steeply within 24 hrs to 5.9 and , ' ,

levels out, maintaining this value throughout the remaining period of

'incubation. The Af zone of inhibition profile indiCates high initial levels of

production.

The growth profile (O.D.; 650 nm/time) of the Af zone of inhibition profile

and the pH profile of strain A, grown in medium 6, at 120 r.p.m., 37°C are " shown at Fig. 3.88. It is similar to Fig. 3.84 but shows further increases in

, antifungal production with increasing time (35.5 mm at 216 hrs).

h) SummaI)'

A summary of the optimization experiments with corresponding percentage

increases in zone of inhibitions sizes, starting from the complete medium

is shown at Table 3.18. An increase of 109% was finally achieved and the

106

. greatest single increase in zone size occurred during the r.p.m. variation

. experiment.

i) Statistical error calculations·

The 0.0. measurements were found to have a stochastic coefficient of

variation of 33%, the pH measurements of 7.3%, the Ab zones of .

inhibition measurements of 11.2% and the Af zone of inhibition

measurements of 12.4%. The stochasic error of variation experiment,

repeated for the final medium and conditions, gave rise to the.foIIowing

variations: OD, 6.9%; pH, 2.8% and Af zone of inhibition, 4%, done at

pH 8 and 120 r.p.m.

j) Scale-up fermentations

Two sequences of a simple scale-up optimization trial (variation of

agitation turns/min (tr/min) and aeration, Jitres/min (Umin)) in the 6-litre

laboratory scale fermentor are shown at Table 3.19(a) and (b). Optimum

conditions o~ 100 tr/min, 4 Umin, at 37°C in 6 days, were eventuaIIy obtained, using visual colour assessments, based on a rating system of +;

++; and ++:+- (where the intensity of yeIIow colour corresponds with

activity of the broth filtrate).

3.5.5 Isolation and Purification of the Antifungal Principals

. a) Determination of appropriate solvent systems


Results of the first tier of the Aszalos and Issaq solvent system strategy

(Aszalos and Issaq, 1980) were as at Table 3.20 a) and b) Fig. 3.89. It

shows at the first tier, some 'rocket' movement of the antibiotic spot on

analytical TLC plate for the MeOH/CHCI3 (l/9) elution and subsequent

107

sparse movements at the second tier. Of the solvents tried at Fig. 3.90,

only. AcOH/CHCI3 (1/9) gave best separation and subsequently, best

immiscibility for the aqueous broth.

ii) Solvent for TLC extraction

A stage by stage development of solvents for the TLC extraction of the

antibiotics is shown at Fig. 3.91. This in essence constitutes the skeleton

of the 'isolation of antifungal antibiotic flowchart' and may therefore be

read in conjunction with Fig. 3.92.

A flowchart for the isolation and purification of the antifungal and

antibacterial substances using a liquidffLC route is shown' at Fig. 3.92.

Three, light yellow, amorphous, antifungal substances AF1, AF2 and AF3

were extracted and a yellow, crystalline, antibacterial substance, AB1, was

also extracted. Yields of 50 mg/lOL, 19 mg/lOL and 19 mg/lOL were

obtained for AF1, AF2 and AF3, respectively, from the lab-scale

fermentor.

b) A check for purity

i) HPLC

The initial test on analytical TLC plate (3.3.4; C), to ascertain whether the

antibiotic substances were likely to pass safely through the HPLC column

showed some 'rocket' movement of the antibiotic spots in solution B but

no movement in solution A.

ii) Gas chcromatography

The G.C. antibiotic/ethanol solution scan, substracted from the control

ethanol scan, indicated a single peak of retention time 12.8 mins for AF1.

108

- .- -----c--:----

Single peaks were also obtained in the same way for AF2, retention time,

16.3 mins and for AF3, retention time 21.8 mins.

3.5.6 Microbiological Properties

The minimum inhibitory concentrations (MICs)of AF1 against 9 microorganisms

are shown at Table 3.21. The antibiotic generally shows strong antifungal activity

but is also. potent against ~. aureus. It appears most potent against T.

mentagrophytes.

Four sets of inhibition results left over from the agar diffusion MIC experiments,

featuririg T. mentagrophytes; A. niger, f. chasogenum and ~. aureus, are shown

at Figs. 3.93, 3.94, 3.95, 3.96, respectively; After daily observation to monitor

stable zone size, the photographs were taken after 5 days incubation at 30°C for

fungi and at 37°C for bacteria. T. mentagrophytes gave the largest stable zones

per concentration (mg/ml) followed by~. chasogenum than A. niger.

Thirteen millimetre Whatman's disc zones of inhibition produced by the 3

antifungal antibiotics, against seeded C. albicans which had been left incubating

at 30°C for 4 days are shown at Fig. 3.97. After 24 hrs, the zones of inhibition of

AF1, AF2 and AF3 were 21.3 mm, 24.6 mm, 23.3 mm respectively. After"the 4

days, AF3's zone had been completely overcome, AFl's zones nearly overcome

but AF2's zone remained intact.

3.5.7 Spectral Data

a) uvMs spectra

The uvMs scan of AF1 is shown at Fig. 3.98. There are two large peaks

at 218.5 nm and 243 nm with absorbances of 1.88 and 1.86 respectively,

and a smaller peak at 287 nm with an absorbance cif 0.934.

The uvMs scan of AF2 is shown at Fig. 3.99. The peaks which occur one

after the other at 216.5 nm, 244 nm,· 269 nm, 290 nm, 328 nm, 348.9 nm

109

have corresponding absorbances of 0.981, 0.650, 0.437, 0.248, 0.165 and

0.115.

The uv/vis scan of AF3 is shown at Fig.3.100. There is one peak at 209 nm

with an absorbance of 0.783.

The uv/vis scan of ABl was identical to that at Fig. 3.42 ..

b) I.R. Spectra

The IR spectra of AFl is shown at Fig. 3.101. Salient peaks include those

at 3540, 3428, 3060, 3024, 2924, 1710, 1582, 1478, 1460, 1434, 1272, 1180,

1154, 754, 700.

The IR scan of AF2 is shown at Fig. 3.103. Salient peaks include thoseat

3364, 2920, 2852, 1732, 1508, 1454, 1376, 1362, 1066, 672.

The IR scan of AF3 is shown at Fig. 3.104. Salient peaks include those at

3300, 2956, 2920, 2852, 2332, 1732, 1566, 1520, 1462, 1420, 1308, 1260,

1152, 1120, 854, 796.

The IR scan of ABl was identical to that at Fig. 3.55.

c) NMR Spectra

The NMR scan of AFl is shown at Fig. 3.102. Salient peaks are to be

found at 7.4 a (d), 7.2 a (d), 3.8-4.3 (m) 1.9 a (d), 1.25 a (m) 0.75 a (d).

d) Mass spectrometIY

A low resolution mass spectrometry scan of ABl was similar to that at Fig.

3.49.

110

Table 3.1: Zone of inhibition results (mm) of multi disk code U4, tested against strain A (P9897), strain B (P9898) and. a range of microorganisms.. These. results were used in the development of selective media. The antibiotics, with their corresponding concentrations,' are as listed. Bacteria were incubated on NA at 37°C and fungi on NspA at 30°C.

Table 3.1: SELECJ1VE MEDIUM DEVELOPMENT

MULTIDISK CODE U4

.

ORGANISM/AN P 9897 P 9898 Ps.aeruginosa· Ps.antim~cetica Ps.malti~hilia . Ps.cocovenenans E.coli TIBIOTIC

TE 17 mm 21 mm Omm Omm Omm 24 mm 23 mm CN 21 mm 23 mm 33 mm 25 mm 31 mm 12 mm 29 mm CR Omm Omm Omm Omm Omm Omm 23 mm CT Omm Omm 15 mm 17 mm 19 mm Omm

'.' 15 mm

SF Omm Omm Omm Omm 13 mm 28 mm 10 mm AMP Omm Omm Omm Omm Omm Omm 27 mm CAR 19 mm 27 mm 15 mm Omm 15 mm Omm >40 mm SXT Omm 13 mm Omm Omm Omm 26 mm >40 mm

ORGANISM/ Sal.abon~ S.aureus S.e~idermis B.cereus S.marcescens S.cerevisiae P.chmogenum ANTIBIOTIC .

.

TE 29 mm >40 mm >40 mm 35 mm 23 mm Omm 'Omm

CN 13 mm >40 mm 25 mm 16 mm 18 mm Omm Omm CR 16 mm >40 mm >40 mm 21 mm 13 mm Omm Omm CT 11 mm 15 mm 28 mm ·Omm 14 mm Omm Omm SF ,21 mm 19mm· 31 mm 13 mm 35 mm Omm Omm . AMP 33 mm >40 mm >40 mm 23 mm 20 mm . Omm Omm CAR

-33 mm >40 mm >40·mm· 23 mm >40 mm Omm Omm .

SXT 18 mm >40 mm 33 mm Omm 37 mm Omm , Omm

KEY: Abbreviation Antibiotic Concentration

TE Tetracycline 50,,8 CN Gentamicin 10,,8 CR Cet!'alOridine 25 ,,8 CT Co ' tin sulphate 10 ,,8 SF Sulphafurazole 500,,8 AMP Amgicillin 25,,8 CAR Car enicillin 100,,8 SXT Co-Trimoxawle . 25 ,,8

ORGANISM P 9897 P 9898 P,aeruginosa P.antimvcetica P.maltiEhitia TEST

OXlDIFERM (OiF)

~ ~ ~ r--:----: ~ GLUCOSE

FRUCTOSE I~+ ~+ ~ + F~ + F~ + SUCROSE I~+ ~+ ~ - F'-Z. + F~ + GALACTOSE I~+ .~+ ~- + I~ - I~ + LAcroSE ~ - ~ -~ r _ - ~ - ~ -MALTOSE ~- I~ - ~ - ~ + ~ + MANNITOL N + I~ + ~ + ~ 1". _. - N - :

OXIDASE + · ·

ONPG + + · · + .

CATALASE + + + + +

MR · ·

VI> · · · · INDOLE · · · · ·

NITRATE + + + + +

STARCH HYDRO' · · · + ·

UREASE + + + + +

AESCULIN + · · · +

LIPASE . + + + + +

LECITIIlNASE + · · + ·

~ BOXYLASE

ORNmtINE · · · · · LYSh'ffi · · · DNase · · . · +

HEAMOLYSIS · · + · +

TOLERANCE TTCl% · · + + · NaC16.5% · · · · 4Z'C · · + + +

Polymy.aa MIC ID"' 10'" 10-' 10-' ID"' [mymI]

· · + + + Cetrimide 0.03%

· · + · · Pseudomon8ll agar

+ + + + + MacConkg: agar

+ + + + + Minimal Med

Table 3.2: Biochemical/tolerance test results of strain A and B with 3 pseudomonads as controls (Cowan & Steel, 1974; MacFaddin, 1983; Palleroni, 1984).

112

Fig. 3.1: An electron-micrograph (T.E.M.) of a negative stained individual cell of P9897 (strain A; magnification, X 30K).

Fig. 3.2: Control for the selective medium experiment showing growth of strain A (top half) and growth of Ps. cocovenenans (bottom half), on NA, after 24 hrs., at 37°C.

Fig. 3.3: A test of a selective medium showing growth of strain A (top half) but no growth of Ps. cocovenenans (bottom half), on NA impregnated with sulphafurazole (500 /Lg/ml). .

Fig. 3. 1

Fig. 3.2

Fig. 3.3

Fig. 3.4: Optical density (650 nm) growth profiles of strain A, obtained from the comparison studies done, showing growth in basal medium (SLR) at pH 4.0 _-_ (i); growth in basal medium (SLR) at pH 7.2 v-v (ii); and growth in Med A2 (AR) at pH 7.2 0-0; (iii). Alf were grown at 31oC, 90 r.p.m. . .

Fig.3.5: Optical density (650nm) growth profiles ofPs.cocovenenans, obtained from the comparison studies done, showing growth in basal medium (SLR) at pH 4.0 0_' (i); ~owth in basal medium (SLR) at pH 7.2 b.-b. (ii); and growth in Med A2 (AR) at pH 7.2 0-0 (iii). All were grown at 31o C, 90 r.p.m.

~ ~

§ ~

GJ • I

f ~

r rJJ? • n I err ~. ~".~,- .. .- .... ::--.... ~ " ......... :::::::::::-,.. ::,.....

'. '" ..... """, """,

S! d·

q (lUUOSS) Aj!SUaa IO~!!dO • M .

Cl .~

IJ..

114

/ I

;; 0

" S!

" ..

" ..

" ..

~

"

" S!

" ..

" ..

'? '-' ., E' !=

:5 ., ,,~ ..

" ...

" . -" o.

Fig . 3.6

Fig. 3.6: A control for the comparison studies (acidophyly) done, showing growth of strain A (top half) and growth of Ps.cocovenenans (bottom half), on NA at pH 7.2, after 24 hrs. incubation at 37°C.

Fig. 3.7

Fig. 3.7: Test for the comparison studies (acidophyly) done, showing profuse growth of strain A (top half) and no growth of Ps.cocovenenans (bottom half), on

A at pH 4.0, after 24 hrs. incubation at 37°C.

Table 3.3: Mean generation times (mgt) (min) of strain A (P9897) and B (P9898) and Ps.aeruginosa (control), grown in various constructed media to obtain their respective chemically defined medium (c.d.m.) ..

Table 3.4: Invasive growth inhibition of A.niger by strain A, B and other pseudomonads, grown on potato dextrose agar (PDA) and on Czapek dox agar, for 7 days, at 3D·C.

Table 3.3

GROwm CURVE STUDIES

MEDIA OGRANISM MEAN GENERATION TIME [MINS)

1 [A) Cruickshank's P 9897 170 + 6

basal P 9898 150 + 5

medium f··aeruginosa .. 100 + 5

2 [B) A + His. P 9897 99 + 6

Phenyl P 9898 104 + 5

3 [e) A + Arg. Orn P 9897 134±6

Lys Mdk. P 9898 97. + 5

4 P 9897 110±6 B+C P 9898 101 + 5 ..

5 A + Yeast P 9897 84±6 Extract P 9898 81 + 5

6 A + Ala, leu P 9897 106 + 6

.

Lys, His. P 9898 106 + 5

Orn ~.aeruginosa 83 + 5

Table 3.4

INVASIVE GROwm INHIBITION OF A. NIGER

ORGANISM . ZONE OF INHIBITION [MM)

POTATO DEXTROSE CZAPEK DOX AGAR AGAR

P 9897 15 ±..3 23 + 5

P 9898 15 ±3 18 + 5

U .. aeruginosa 15 .

Ps. antim~cetica 0 0

Ps. inaltiphilia >

0 O·

Ps. cocovenanans

Ps. cocovenanans 7+3 . .

116

Fig. 3.8:

Fig. 3.9:

Control for the invasive growth inhibition experiment, showing invasive growth of Aniger from the apex of the top half of the PDA plate, after 7 days incubation at 30·C.

A test experiment for invasive growth inhibition, showing inhibition of Aniger invasive growth, by strain A which had been streaked onto the bottom half of the PDA plate and incubated for 7 days at 30·C.

Fig. 3.10: A test experiment for invasive growth inhibition, showing inhibition of Anigerinvasive growth by Ps.cocovenenans which had been streaked onto the bottom half of the PDA plate and incubated for 7 days at 30·C.

Fig. 3.8

Fig. 3.9

Fig. 3.10

.

ANTIBIOTIC ACTIVITY/INCUBATION TIME [DAYS] ·

TEST BACTERIA FUNGI MEDIUM·

DAY 2 DAY? DAY 2 DAY 7

a · · · · ..

b · · · · c + · · · d + · · · e · · · · f · · · · g · · · · h · · · · i · · · · j · · · · k · · · · I · · · ·

·m · · · · n + · · · 0 · · · · p · + · + q + · + + r · · · · s · · · · t · · · · u · · · · v · · · + w · · · · x · · · · y · · · +

.

KEY: + ACTIVITY • NOACTMTY

Table 3.5: Results of the various media assayed for antibiotic activity: . c = basal medium; d = constructed c.d.m. for strain A; n = Czapek Dox liquid medium (CDLM); q ':" CDLM • modified and v = PDLM·1. Test· microorganisms used for the assay include E. coli (antibacterial) and C. albicans (antifungal).

118

Fig. 3.11:

Fig. 3.12:

A summation of optical density,(650 nm) growth profiles of strain A, grown in the basal medium (SLR) MCSD experiment: basal/complete medium, _-_; Med A1, 0-0; Med A2, 0_0; Med A3, 0-0; Med A4, .6.-.6.; .. Med AS, v-v; all at 37°C, 90 r.p.m., with an initial OD of.= 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

A summation of antibacterial (E.coli) antibiotic concentration (mg/ml x 1O"2)/time (h ) profiles, produced by strain A in the basal medium (SLR) MCSD experiment: basal/complete medium, ---; Med A1, 0-0; Med A2, 0_0; Med A3, 0-0; Med A4, .6.".6.; Med AS; v-v; all grown at 37°C, 90 r.p.m., with an initial OD. of .= 0.06. Standard deviation computed for each datum point are shown, where applicable, as error bars.

. M . "" .-'.l.

N

• M . Cl .-u..

5!

I

" I ..

•

...

~ . ..,..

~\

I

'"

-o-<l

\ / -o:r

~ ~

/\ <l-o-'

.,;

(WUOS9) ~!SuaQ IOO!ldO

I ..

119

I ...

'i>-

I 'i>-

I N

;; .,;

o

., ..

., .,

., ~

z '-'

., CD

... E ;::

., "

5!

.,

z '-'

., CD

... E ;::

Fig. 3.13:

Fig. 3.14:

A summation of optical density (650 nm) growth profiles of strain A, grown in the basal medium (AR) MCSO experiment: basal/complete medium, _-_; Med A1, 0-0; Med A2, e_e; Med A3, 0-0; Med A4, ~_~; Med AS, .... v;all at 37°C, 90 r.p.m., with an initial 00 of.=:. 0.06. Standard deviations computed for each datum point are shown, where . applicble, as error bars .

. A summation of antibacterial (E.coli) antibiotic concentration (mg/mI x 10 -z)/time (h) profiles, produced by strain A, grown in the basal medium· (AR) MCSO experiment: basal/complete medium, ---; Med A1, 0-0;

Med A2, e_e; Med A3, 0-0; Med A4, ~-~; Med AS, v-v; all grown at ... 37°C, 90 r.p.m., with an initial 0.0. of = 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

• M

· til ,~

LA ...

· en ,~

"-

•• ITT I • o O<i

~ \'1/ - o=t:r=-

\.~

>-!>-<

/ -!>o

I >-!>o

I -i>'

!jl.

0 .,

0 ...

'? ...... o CD ... E

1=

0

'"

!l

[:

~ 6:

o CD ... E

1=

r'----~'----~,----_., ----_., -----_~,-----.,------.o n ~ ~ N n_, ., 0 ~ N 0

120

Fig. 3.15: A summation of optical density (650 nm) growth profiles of strain A, grown in Med A2 of various percentage glucose concentrations: 0.3%, ___ ; 0.4, 0-0; 0.5%, ._.; 0.6%,0-0; 0.7%, t.-t.; 0.8%, .,.-.,.; 0.9%, v-v; 1.0%, +-+; all at 37°C, 90 r.p.m., with initial O.D. of = 0.06. Standad deviations computed for each datum point are shown, where applicable, as error bars.

Fig,3.16: A summation of optical density (650 nm) growth profiles of strain A, grown in Med A2 of various percentage glucose concentrations: 0.3%, ___ ; 0.24%, 0-0; 0.18%, ._.; 0.12%,0-0; 0.06%, t.-t.; 0.0%, v-v; all at 37°C, 90 r.p.m., with initial O.D. of =. 0.06. Standard deviations,

. computed for each datum point are shown, where applicable, as error bars.

.

.' .

• 0 .. 0

• '"

\ 0 ~ ..

. \.~ ~

o ., .., E ;:::

0 .. g

g 1.0 . (V) . Cl .-LL..

lIJe 0 <J --1>--. 0 OD

1/ I I / 0 "' . .:le 0 <J ~r:-

\/ I I I 0 ..

-~ <J 0;>-0

/ -:? ~

o ., .., E

;:: O~<J

0 .. 0 ~

<0

• '" . Cl g .~

LL..

~~~~~--~~~~~~--~~~~~----+o o ci

121

Fig. 3.17: A summation of antibacterial (E.coli) antibiotic concentration (mg/ml x lO-~/time (h) profiles, produced by strain A in Med A2 of various percentage glucose concentration: 0.3%, _-_; 0.4%, 0-0; 0.5%, 0_0; 0.6%, 0-0; 0.7%, .10.-.10.; 0.8%, ,,-,,; 0.9%, v-v; 1.0%, +-+; an grown at 37°C, 90 r.p.m.,· with initial 0.0. of =- 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

_ Fig. 3.18: A summation of antibacterial (E.coli) antibiotic concentration (mg/ml x lO-Z)/time (h) profiles, produced by strain A in Med A2 of various percentage glucose concentrations: 0.3%, ---; .0.24%, 0-0;0.18%, 0_0; 0.12%,0-0; 0.06%, .10.-.10.; 0.0%, v-v; an groWn at 37°C, 90 r.p.m. with initial. 0.0. of =- 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

• M

• Cl'> .-

• M

· Cl'> .-LL

o .. o .,

? ~

o CD ., E >=

.------r------.------r------.------r------.-----~O .. ., ... ., N·

122

o

o .. o .,

o ...

? ~

o CD ., E >=

Fig. 3.19: A summation of antibacterial (E.coli) antibiotic concentration (mg/ml x 10-2)1 glucose concentration profiles, produced by strain A in Med Al, at various times (h): 0 h, _-_; 24 h, 0-0; 36 h, 0_0; 48 h, 0-0; 60 h, 1>-1>;

all wown at 31oC, 90 r.p.m., with initial O.D of =- 0.06. Standard deVIations computed for each datum point are shown, where applicable, as error bars.

en . M . Cl .-Lo..,

---~..L

en o

co o

'" Cl) .m o 0

(.J ::l (3

Ill. Cl) • Cl

o C ..... C Cl)

e 'I; Cl) 00..

-o

r-----_r------,-----~r_----_r------,_------r_----_IJ0

~-OLX) (lw/6w) 'ouo:) ogO!QUu'V

123

- o

'E' c o t.()

e >--'r;;; c Q)

c 13 o

!;:C-

·0

10

•

0.1

16

.---...... ---

/ .r '0 .-c. 0

-E. 0)

g, •

0 C 0

U 0

:;:: 0 .-

..0 :;:: c

" .-.-- <C

a

---"""?"'-~-.+---+---+ -'----

O.Olr---r-----'".----.... ---,,----.----I-o 6 o 10· ~. ,30 ~ 50 60

Fig. 3.20:

Time Ch)

Optical density (650 nm) growth profile, - --; antibacterial (E. coli) antibiotic concentration (mglml x 1O-~ profile, A-A and pH profile, +-+ of strain A, grown in Med A2 (0.3% glucose concentration), at 37°C, 90 r.p.m., with an initial O.D. of =- 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

124

Fig. 3.21: A summation of optical density (650nm) growth profiles of strain A,. grown in Med A2 of various percentage NH4CI concentrations: 0.3%,-_; 0.24%, 0-0; 0.18%, e_e; 0.12%, 0-0; 0.06%, A-A; all at 37°C, 90 r.p.m., with initial 0.0. of.=:. 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

Fig. 3.22: A summation of optical density (650 nm) growth 'profiles of strain A, grown in Med A2 at various percentage NH4Cl concentrations: 0.06%, _-_; 0.05%, 0.0; 0.04%, e_e; 0.03%,0-0; 0.02%, A-A; 0.-01%, y-y; 0.0%, v-v; all at 37°C, 90 r.p.m. with initial 0.0. of.=:. 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

• 0

'"

l 0 ., • ~ 0 .. • ~

Z '-'

o CD ..... E i= ......

0 .. S!

N · •

Ol .-u..

•

• ~ • \\

N N

• '" • S! Ol .-u..

125

Fig .. 3.23: A summation of antibacterial (E.coli) antibiotic concentration (mg/ml x 1O"2)/time (h) profiles, produced by strain A in Med A2 of various percentage NH40 concentrations: 0.3%, _-_; 0.24%, 0-0; 0.18%, 0_0; 0.12%, 0-0; 0.06%, 4-4; all grown at 37·C, 90 r.p.m., with initial O.D. of =. 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

Fig. 3.24: A summation of antibacterial (E.coli) antibiotic concentration (mg/ml x 1O-2)/time (h) profiles, produced by strain A in Med A2 of various percentage NH4Cl concentrations: 0.06%, ---; 0.05%, 0-0; 0.04%, 0_0; 0.03%,0-0; 0.20%,4-4; 0.01%, T-T; 0.0%, v-v; all grown at 37°C, 90 r.p.m., with initial O.D. of· =. 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

i

M N . .

Cl .~

w..

"

od' N

• M

• Cl .~

w..

o '"

o

'"

~ o CD ., E

i=

r------r------r-----,------.------,-----~r_--~~O .. .. .. o

( OLX) (lwj6w) ·~UO:> ~!I0!q!fUV %-. .

0

'"

0

'"

0 .. ~

0 " .., ~

0 .. S

r_----,-----~r_----,------.-------r------r_--~ .. O o

126

Fig. 3.25: A summation of antibacterial (E. coli) antibiotic concentration (mglml x _ 1O-2)/percentage NH4CI concentration profiles produced by strain A in Med A2, at various times (h); 0 h, _._; 24 h, 0 0 0; 36 h, 0.0; 48 h, 000; 60 h, ,\,.,\,; all grown at 37°C, 90 r.p.m., with initial O.D. of.=:. 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars. .

~-------.---

""' N o

M

o .., o

(])

0-0 N°.... o 0

..c ()

E :::J 01: o

~ E o E

« (]) 0') c

"E g e ocf

III o o

o en ' r-----~----_r----_r----_.------r_----~~~~g

00 .-

N - o to -(r_mx) (lw/Bw) °Ouo::> O!tO!q!WV

127

'N

10 32

.---------------------.r-10

1'. ..... 0

c o LO e >... -'in c Cl)

Cl

"6 o ~ o

-::::-

~ Cl

.§. • 0

C 0

U 0 :;:: 0

0.1 :0 :;:: c «

8

----+----+------+- -

O.Ol~---r__--_r"---r_--__r---...,_--_l_O 6 o 10 20 30 . 40 SO 60

Time (h)

Fig. 3.26: Optical density (650 nm) growth profile, ---; antibacterial (E.coli) antibiotic concentration (mg/ml x 1O-~ profile, A-A and pH profile, +-+ of strain A, grown in Med A2 of 0.06% NH.CI concentration (medium 1), at 37°C, 90 r.p.m., with an initial OD of -=. 0.06. Standard deviations . computed for each datum point are shown, where applicable, as error bars.

128

Fig. 3.27:

Fig. 3.28:

A summation of optical density (650 nm) growth profiles of strain A. grown in medium 1, buffered at various pHs: 3, ---; 4, 0-0; 5, 0_0; 6, 0-0; 7, A-A; 8, '1'-'1'; 9, v-v; all at 37°C, 90 r.p.m., with initial O.D. -=- 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.·

A summation of antibacterial (E. coli) antibiotic concentration (mg/ml x . 1O-2)/time (h) profiles, produced by strainAin medium 1, buffered at . various pHs: 3, _-_; ·4, 0"0; 5, 0_0; 6,0-0; 7, A-A; 8, '1'-'1'; 9, v-v; all grown at 37°C, 90 r.p.m., with initial OD. -=- 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars. .

-------- --

..... N · '" · Ol .-u...

co N

• CV)

• Ol .-u...

kf 1\ <>~. \\ \ \ o ~ .. -0-. ~

129

• t> 0 .. /1 0

on • t>

I 0 .. • t>

11 '? ~

o ID .., E ;:::

0 «

g

Fig. 3.29: A summation of antibacterial (E. coli) antIbiotic concentration (mg/ml x. 1O·2)/pH, produced by strain A in Medium 1, at various times (h): 0 h, ___ ; 24 h, 0-0; 36 h, ._.; 48 h, 0-0; 60 h, A-A; all grown at 3JOC, 90 r.p.m., with initial C.D. of =. 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars. .

'" N •

'" . Cl ,~

u..'

'"

<o:c a.

r-----------.-----------~----------_r----------~~ 10 ~

o ~

10 o

130

--------------------~------~---- -- ----

'E' c: o Lt)

e >-

== CIl c: Q)

Cl

"5 0 :;: c.. 0

10 32

11 11 11

......... 1

0 ...... 0 c-

'E "'" Ol E

16 '= • 0 c: 0 u 0

1 :;: 0 .-

0.1 ..c 1 :;:

c: <

----'~------+------~~----+.------~

0.01l¥----,---.,----,----r----,----l- 0 o 10 20 30 40 50 60

Time (h)

7

::t CL

5

Fig. 3.30: Optical density (650 nm) growth profile, ---; antibacterial (E. coli) . antibiotic concentration (mg/ml x 10.2) profile, ~-~ and pH profile, +-+ of strain A, grown in medium 1 at pH 6.0 (inedium 2), at 3rC, 90 r.p.m., with an initial O.D. of =- 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

131

Fig. 3.31: A sUIIlIl}ation of optical density (650 nm) growth profiles of strain A,. grown in medium 2, at various r.p.m.: 40 r.p.m., ---; 60 r.p.m., 0-0;

90 r.p.m., 0_0; 120 r.p.m., 0-0; 150 r.p.m., A-A; 180 r.p.m., x-x; all at 37°C, with initial O.D. of =- 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

Fig. 3.32: A summation of antibacterial (E.coli) antibiotic concentration (mg/ml x 1O·2)/time (h) profiles, produced by strain A in medium 2, at various r.p.m.: 40 r.p.m., _-_; 60 r.p.m., 0-0; 90 r.p.m., 0_0; 120 r.p.m., 0-0;

150 r.p.m., A-A; 180 r.p.m., x-x; all grown at 37°C, with initial O.D. of =- 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

..-

I

'" • '" • '" .~

u.

N

'" · '" • '" .~

u.

o 5l

o ..

o .. z '-"

'" • E ~~ ;::

I r'

r---~----~--~----~----~--~----~--~O o .,. on

'" o

'" on N

132

o N

o

l' c

0 LO ~ >-....

'00 C III

Cl

""6 ,g .... a.. 0

10

0.1

, T T,/'"'-. • 1 '/1

• T ~

~.--T

64

,....., ",.

'0 ..--.0 c--€.. Cl

E 32~

.0 c o u o . B

=0 :0 :;: c <

_____ J . . L V+-t-+. i-+'-T/ .. ~---l---~ . .

0,"/ , o 100

4 o

Fig; 3.33:

20 40 60 BO TIme Ch)

Optical density (650 run) growth profile, .-.; antibacterial (E.coIi) antibiotic concentration (mg/ml x 1O.~ profile, 4-4 and pH profile +-+ of strain Po. grown in medium 2 at 120 r.p.m., 37°C, with an initial O.D. of =- 0.06. Standard deviations, computed for each datum point are shown, where applicable, as error bars.

133

Fig. 3.34:

. Fig. 3.35:

A summation of optical density (650 nm) growth profiles of strain Po. grown in medium 2 at various temperatures: 27°C, ---; 32°C, 0-0; 37°C, ._.; 42°C, 0-0; 47°C, A-A; all at 120 r.p.m., with initial 0.0. of =- 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.. . .

A summation of antibacterial (E.coli) antibiotic concentration (mg/ml x lO-~/time (h) profiles,produced by strain A in medium 2 at various temperatures: 27°C, _-_; 32°C, 0-0; 37°C, ._.; 42°C, 0-0; 47°C, A-A; . all grown at 120 r.p.m., with initial 0.0. of =- 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars. -

... ~ OD <l

I /\ I .... 0 CJ • <l

\ / . / \ I ~CJ\r'

<l

I o ___ []I <l

I \/ \ I 000 • • ......:J-

\// ~. 1 ~

~/ g .; ...

'" (WUOS9) AI!SUaO ICO!ldO · C',

· 01 .~

L'-

· 01 ,~

u..

134

, -0 .;

0 g

0 ..

0 .. 0 ..

0

'"

0

o '"

'? ....-CD

.5 .....

Fig. 3.36: A summation of antibacterial (E.coli) antibiotic concentration (mg/ml x lO'2)/temperature (0C), produced by strain A in medium 2 at various times: 0 h, _-_; 24 h, 0-0; 36 h, e_e; 48 h, 0-0; 60 h, .1.-.1.; 72 h, T-T; 84 h, v-v; all grown at 120 r.p.m., with initial 0.0. of =- 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

ID CV) , CV)

, 01 ..... u..

o '<t

o t<)

, , r------.-------r,------"-------r,------"-------,r------.,-------rl ~ 0 III '<t t<)

o ~ 0 III Q III 0 tor) ~.. N ,.... ~

(t_OLX) (lLUjBw) 'ouoJ 0aO!qaU'v' 135

Fig. 3.37:

Fig. 3.38:

A summation of optical density (650 nm) growth profiles of strain A, grown in medium 2 at various initial O.Ds.: 0.200, ---; 0.150, 0-0;

0.100, e_e; 0.060, 0-0; 0.030, A-A; 0.015, v-v; all at 37°C, 120 r.p.m. Standard deviations compued for each datum point are shown, where applicable, as error bars. .

A summation of antibacterial (B.coli) antibiotic concentration (mg/ml x 1O-2)/time (h) profiles, produced by strain A in medium 2 at various initiaIO.Ds.: 0.200, _-_; 0.150, 0-0; 0.100, e_e; 0.060,0-0; 0.030, A

A; 0.015, v-v; all grown at 37°C, 120 r.p.m. Standard deviations . computed for each datum point are shown, where applicable, as error bars.

· 01 .-u..

00 CV')

• CV')

• 01 .-""

~--~----~~~-----r----~,----~--~----~o o .. '" ., o

" !!! o

136

Table 3.6: Sequence of optimization experiments with corresponding factoral increases in production, starting from the controVcomplete medium. MCSD stands for medium component single deletion of the complete medium.

Table 3.7(a): Summary of a simple scale-up optimization trial (variation of agitation . (tr./min) and aeration (Umin)) in the laboratory-scale fermentor.

Corresponding visual colour assessments where increase in intensity relates to increase in antibiotic activity are based on a rating system of + 1 to + 6 which was used to monitor sequential progress, each at 72 hrs.

Table 3.7(b): Summary of a further optimization trial (variation of time (days)) on optimal conditions achieved at table 3.7(a). An antibiotic assay against E. coli (zone of inhibition (mm)) was used to monitor progress.

Table 3.6

SEQUENCE OF OPTIMIZATION FACTORlALINCREASE EXPERIMENTS EXPERIMENTS IN PRODUCTION

1 Medium A 1 ..

(Control)

2 MCSD 1.8 Experiment (Medium B)

3 Variation of glucose • (Medium B)

4 Variation of 2.8 NH,C1 (Medium C)

5 Variation of pH 4.3 (Medium E)

6 Variation of r.p.m. 9.3

7 Variation of temperature •

8 Variation of initial O.D. " .

Table 3.7

a) b)

OPTIMIZATION IN THE LAB-SCALE 150 tr./min. 4l)min FERMENTOR

AGITATION AERATION VISUAL TIME ZONE OF COLOUR I INHIBITION OBSERVATION

(TURNS/MIN) (LITRES/MIN) (RATING) (DAYS) (MM)

100 1 + 1 0.0

200 1 ++ 2 18.5

150 1 +++ 3 21.5

150 2 ++++ 4 19.0

150 3 +++++

150 4 ++++++

150 5 +++

137

Fig. 3.39(a): Determination of an appropriate solvent system for broth extraction, showing pathway taken when using the Aszalos and Issaq solvent system strategy (Aszalos and Issaq (1980), resulting in MeOH/CHCI3 (1/9) (Solvent 1) [See table 3.8) .

. Fig. 3.39(b): Determination of an appropriate solvent system for the Iiquid/liquid extraction route resulting in HexlAcOH/EtOAc (9/1/9).

Fig. 3.40: Determination of appropriate solvent systems for the IiquidffLC· extraction, resulting in:

(i) EtOAc (Solvent 3)

(ii) Hex. (Solvent 4).

Fig. 3.39 a: !JE!W1INATI(}I Cf .APPROPRIATE SQVENT SYSTEMS

LlOJID EXTRACTION

.

EtOJIc

Broth SI rematent

'---- ---

(~lvent 1)

rw-vCl-tl (1/9) 3

, j

1 I

(A)

Fig. 3.39b: .--____ ....-____ -+=1-:::....;;,-...:-...:-;,.;-:....::.-.:..-.:,-.:..-...:-;,.;-...:-...:-;,.;-;,.;-;,.;-;,.;-:..;;;- I , I. I

E

Fig. 3.40: LIQJID/TLC EXrnJ!CTI(}I

rw-vcrcLJIiltibiotiC concentrate .

(~l~~ 1) . : I I , I 1-- ----.

.

EtoJlc

kO-I (1/9)

, (Solvent 3)

I

fe</ EtoJlc (9/1/9) Solvent 2

(B)

.. - - - - - - - - - - - - - - - - - -I

fe</ kO-!/Eta-I fe</ kO-!/Eta-I (90/10/45) (9/1/9)

138

I I

I I :

fe</EtOAc (9/1)

!-'ex (~lvent 4)

Fig. 3.41: Constructed flow chart for the isolation and purification of the antibacterial substance using a liquid/liquid extraction route.

'1

Fig. 3.4~1 :==~I~SOLA;;;J,;;:I(J.I~JIN)=PIJ~R;,IF~ICA=J=I(J.I=OF=IWT=I=BJlC=TER~I=AL=SUBST==:::fJf[=E ===

(X 3)

RESIIl..V\I.. BrolH

SUPffi'JATENT OF JlCTIVE BROlH

r PCIDIFY TO p-i 2 with 5 rtCl

1 EXTRPCT WIlH SOLVENT A

SOLVENT A

! EXTRPCJ WIlH 1% NaJ-ro3

(X 5)

( i) SOLVENT A EVAPORATE (if) DISSOLVE IN d. H_O ( ii i) CJ-IIl.RCQI\L' WASH" <!

PCIDIFY TO i1i2

, (X 3)

EXTRPCJ WIlH SOLVENT B __ -----......

139

JlQJECXJS lJ\YER

J EVAPORATE ,

(freeze dry) __ -I"'~CRYSJALS

Fig. 3.42: Constructed flow chart for the isolation and purification of the antibacterial substance, using a IiquidffLC extraction route.

~~-- ~-----

Fig. 3.42: ISOlATIOO I'm PURIFICATI(JIJ OF IV'ITIBJlCTERIAL SUBSTMtE

JlCTIVE B~lH

CEi;"fRIFUffiTION [6000 r.p.m. 15 min. 1°C]

1 ~ EXlRPCT SUPERNATENr WIlH s:x.VENT 1

X5

s:x.v LAYER PQJEaJS LAYER

WASI-i WIlH D10LCXJRIZING OWIDIL fID M:MlRJINE-FIL TER

l PREP TLC ELUTED WIlH s:x. VENT 3

~ SCRJIPE OFF JlCTIVE LAYER·

fID ELUTE WIlH s:x. VENT 3

1 EVJIJlORATE TO DRYNESS

fID REDISSOLVE IN. CJ-lOROFm1

J SLOtI EVJIJlORATIOO

TRIllVITE TO Fm1 CRYSTJIJ..S ~ WIlH 5n.VENT 4 I> PURE CRYSTJIJ..S

140

Table 3.8: Retention factors of active broth filtrates (AF) and fresh, uninoculated medium/control (FM), obtained from analytical TLC elutions, viewed under UV light (253.7 - 356 nm) and charred after spraying with conc. H2SOJglacial acetic acid (see Fig. 3.39 (A)).

Fig. 3.43:· Developed pr~p TLC of an a~tive concentrate cif solvent A, eluted with· solvent 2 imd as viewed under UV light (254 nm).

Table 3.8

SOLVENT/ ELUTED CHCl3

SUBSTANCES

AF FM AF

Charred spot 0.00 . 0.00 0.00

(Active) 0.13 0.00 0.09 Yellow pigment

(yellow/green) 0.00 0.00 0.00 Fluorescent material

.

Fig. 3.43

EtOAc

FM AF

0.00 0.70 .

0.00 0.64

0.00 0.22

SOLVENT FRONT

. PIGMENT 5 dark PIGMENT 4 dark PIGMENT 3 yellow/green

(fluorescent) ANTIBIOTIC 2 yellow PIGMENT 1 dark

[start]

MeOH MeOH/CHI3 (1/9)

FM AF FM

0.70 0.00 0.00

0.00 .. 0.67 0.00

0.00 0.00 0.00

Table 3.9: Retention factor de terminations of the antibacterial substance, using.a selection of solvents which cover a cross-section of solvent, polarities.

Fig. 3.44: Antibiotic concentration/zone of inhibition log-dose response calibration curve, used for assessing the amount (mg/ml) of the antibacterial substance present in active broth filtrates. Standard deviations computed for each datum point are shown, where applicable, as error bars.

Table 3.9

SOLVENT RETENTION FAcrOR (Rf)

Hexane 0.00

Chloroform 0.07

Pyridine 0.63

Acetone 0.53

Ethanol 0.46

Methanol . 0.67

Water 0.54 '

Ethylacetate 0.15

MeOH/CHCI3 0.60 (1/9)

Fig. 3.44

40

J5

'E' g JO c:

.2 :3 :;: S

(' -0 .. 25 c:

:: '). ~

20

15-1----.,----.-----~---__, o 0.5 1 1.5 2

Log Cone (mg/ml) (Xl0 )

142

Table 3.10: The effect of pH/temperature (QC) on the activity/stability of the . antibacterial substance with increasing time (mins). The microorganism

used for the antibiotic assay was E.coli.

Table 3.11: The effect of UV light (sterilization chamber with TUV 15 watts U.V. fluorescent light) on the activity/stability of the antibacterial substance with increasing time (days). The microorganism used for the antibiotic assay was E.coli. Tetracycline was used for comparison.

Table 3.12: Minimum inhibitory concentration (MIC) determinations of the antibacterial substance, against a range of microorganisms, grown on NA at 37°C (bacteria) and on NspA at 30°C (fungi). Assessment was by the agar diffusion assay ..

Table 3.10

1CMPERATURE TIME (MINS) PERCENTAGE LOSS IN ACTIVITI ("Cl

pH:z.O pH 7.0 pH 9.0

10 0 I 33

60 30 0 4 SS

60 0 16 lOO

10 12 33 lOO

80 30 26 S2 lOO

60 32 S9 . lOO

10 21 39 lOO

lOO 30 28 S6 lOO

60 29 60 . lOO

Table 3.11

TIME (DAYS) . . . PERCENTAGE LOSS IN ACI1VITY

ANTIBACTERIAL SUBSTANCE 1CTRACYCLINE

0 0 0

I 19 14

3 19 18

5 19 18

7 19 18

Table 3.12

MICROORGANISM MIC ("GIML)

Escherichia coli , 1.3

Salmonella abony 1.0

Bacillus cereus ,26.6

Clostridium sporogenes '. >2000

Staphyloccus auenu 11.9

Staphyloccus epidermis 10.8 .

Saccharomyces cerevisiae 621.3

Serratia marscescens 540.6

Candida albicans >2000

Penicil/ium cluysogenum 238.7

143

'E' c o 1[)

e >-.-'iii c Q)

Cl

"0 o :a. o

10

A

I / JZi'

/rzf /

/

,.)a-El J2i"

0.1 "I'----..... -----,--'----;..------r------, o 1 2 3 4 5

TIme (h)

Fig. 3.45: The effect of mid-log phase, antibacterial antibiotic, MIC challenge against E.coli, grown in basal medium, at 37°C, 90 r.p.m.: control optical density

. (650 nm) growth profile, 0-0; test optical density (650 nm) growth profile, .-•. 'A' denotes point at which the antibiotic was added.

144

Fig. 3.46

+2.~Ar-------------------------------~

0.500 (NDIV)

O.OOA ~------;'M'7~mmIT======-mI 200 ~~

Fig. 3.46: A uv/vis spectrum of the antibacterial, basal medium broth filtrate, after 36 hrs incubation at 37"C, 90 r.p.m.

Fig. 3.47

+ 2.5OA r-----------------~.,

0.500 (NDIV)

I

.

. i'J '------- ~ O.OOA 2/;vOO;-----------:-:100t:".-=0-;(7':"f'.f-V:-:::D~IV",.)--'--........ ----7...100 I'M

Fig. 3.47: A uv/vis spectrum of the purified antibacterial substance, dissolved in UV ethanol. .

145

Fig. 3.48

I I

--r·····

%0 '~--j'-+--i.c.,+:"+~+·"':'-:~··l~

I I

0 ..... .1 . :4(1O()cJ!' .. ~_ ... ___ 3500

,.

L

, ·~·i· .. - ~ .. -. I·: ::" . \ : .• , .

.. ' ."

.;;: I .• !

':"1' .: ..... :." .!"

.. , .

. L. . , .!:' .. ::."

.. ".'--" .1.:-_ .

i: ".-.

1 :;;····:,!l··· i'

Fig. 3.48: A KBr. disc infra-red scan of the antibacterial substance.

,;

•• ! •

· Fig. 3.49: A high resolution mass spectrum of the antibacterial substance.

Table 3.13: Probable elemental ratios (emperical formular) of the antibacterial substance, as generated by the mass spectrometer data system.

Fig. 3.49·

'" ~ Z < 0 I J ~ (

'tl > ,: j "

" S

:DB ~

.. J S.

?a

••

5.

••

3.

2.

,.

Table 3.13

C 3a

12 11 11 14 13 13 7

17 6 9 9 e

113 4 4 3

.,

CI3 H N I 5

El 7 3 I 6 3 a 5 4 a 9 El 1 S 0

.0 7 I I 8 4 El 7 5 I 6 5 1 la I a 9 :1 1 :3 2 El 9 £)

1 113 ~ ~

El 9 4 1 B 4

IO.

i 03 I

I

:I. 1"

'" 11

I ..:r

... 11 11''''1'

'00 ,., "0 , ..

0 DE'! ~ns I'."lSS <)PTS ~IHT

5

a -4.1 193.6599 71 laa.ea a 9.3 El 8.4 I -5.5 I -1.0 I 7.1 2 -8.2 2 -6. ! 1 2 4.3" 3 -9.5 3 -1.5 :; 3.8 "'- 9.:3 5 -5.5 5 2.G 5 7.£1

147

Table 3.l4: Positional parameters and equivalent isotropic temperature factors arising from the x-ray diffraction of the antibacterial crystal. N, C, 0 represents nitrogen, carbon and oxygen, respectively and the numbers in parenthesis represents the atomic numbering used for the construction of the molecular structure. [See Fig. 3.49].

Table 3.15: Bond lengths (A) arising from the x-ray diffraction of the antibacterial crystal. N, C, 0 represents nitrogen, carbon and oxygen, respectively and the numbers in parenthesis represents the atomic numbering used. for the construction of the molecular structure. [See Fig. 3.49].

Table 3.16: Bond angles (0) arising from the x-ray diffusion of the antibacterial crystal. N, C, 0 represents nitrogen, carbon and oxygen, respectively and the numbers in parenthesis represents the atomic numbering used for the construction of the molecular structure. [See Fig. 3.49].

Table 3.14

• UEQ = '. ',ATOM X' Y Z UEQ

N (l) .312 .( 3) 57'36 ('31 7312 ( 5) 51 ( 4l CC" 1645 ( 4) 5961 .(':3)' 7166 (·'.n 53 ( 5) N(2) 1592 ( 3) 6392. ( .3) 5285 ( 6)' 52 ( 4) C (2) 259 ( 51 6657 <..:n 3517,(.71 53 , 5) C (31 -1177 .( .4) 6430 .(. 31 3721 .{. 6)· 48 ( 5~ C(4) ·991 ( 4) 5993 ( 31 5692 .( 6). 46 ( 5): K(3) -a.340 ( 3) 5847 .( 3). 5836· ( 6)· 54 ( 4) K (4) .3735 ( 4) 6037. ~;.31 4209 (. 6) 67 ( 5) C(5) -3760 (·5) 6395 (' 4) 2460 ( 8) 69 ( 6) jH51 -2512 ..L 41 6637.·'(' .3) 2122 ·C· 6) 61 ( 51 0(' , 2909.( 3) 518' ( 3) 8645 <. 5) 75 ( 5) C (6) 3084 ( .. n 666.3, (.6) 5343 (14) 79. ( '9) .0 (2) 258· ( 4) 7022 ( 2)· 19<7 ( .5). 69 ( S) C(7)' -2.306 ( 7)' 5469 , 6) 7848 ( 9). 75 ( 8)

Table 3.15

CC, ) NC·1) '.'374 ( 5) C(4) COl 1.423· ( 6). C (4) - N<·' ) '~308 C 5) NCSl - C(3) 1.302 ( 5) H(2)' C(., l ,_407 .. ( 5) NC.3) - C(4) ",368 ·C 5·) OC'1) - ( (Il 1.2'9·( ft.) N(4) . N(3) "'·34' C " C (2)' N(2) 1.372 , 5) ccn - N(3) '';'467 ( 6) C<6) N<2) '.476 (. 6) CCS) - NC4l 1.2S3.C 6l C (3) C(2) '.491·.C 6) NC5l - C (S) 1.370 C 6) o (2) C(2) '.207 C S)

Table 3.16

. C (4). -' N (1) - C·(ll 117.0 C 4) HCS) -. C(3)' -C·(2) ".9'.-2 ( . .4) N(2) - CC,) ~ NC·ll 120.9 ( 3) N(51 . C(3) - C(4) '23.8 .( 4) OC') - .. C·('.l· .,. H (·1) 121~2. , 4) C(3) - ~'4) - N·C 1) 126.5 ( 3) . O(1) - C (,) - N(2) 11-7.9 ( 4) N(.n - CI4) · NC') ~ 1-9.,1 ( 4) C(2) - N(2) .,. CCll 124.4 (. 3) N(3) - C(4) - C(3) 11·4 .. 4 ( 3) C (6)· _. N(2) .,. C(1) '16.8 ( 4l N(4) - N(3) - C (4)' 123.·2 ( 4). C(6) - H(2) .,.' C (2) 118.4 C 5). C(7) - N(3)· • C (4) 120.0 ( 4) C (3) .,., C c;n ". N·(2) 114.0 C 4) c(n - NO) - N(4) ~16.8 C 4) 0(2) - C (2) ::- N(2) 122.6 C .4) C(S) - NI4) · N(3) .116.06 ,'(.4) 0(2) - C (2) .,.' C (3). '23.4 ( 4i N(S) - C(5) - N(4) . ,26.·9_(,4). C(4) - C(3) - C(2) 110.9 .( 3) CC';) - N CS) - C(3) " 4.8 C 4)

148

c

01

Fig. 3.50: Molecular structure of the antibacterial crystal, as generated by the x-ray diffractometer, from bond lengths and angle, and from an· atomic numbering scheme.

Fig. 3.51: Unit cell contents of the antibacterial substance, as generated by the x-ray diffractometer.

150

Fig. 3.52: . A) The effect of cell propagation on growth of strain A. from basal medium, along:

i) a series of Med A2 (SLR).

ii) a series of Med A2 (AR).

B) The effect of cell propagation on growth of Ps.aeruginosa, from basal medium, along a series of Med A2 (SLR)

Strain A and Ps.aeruginosa were grown at 37°C, 90 r.p.m., with initial OD levels of =- 0.06. Static, overnight incubation to obtain colonies on NA was at 37°C. The times (h), inbetween the 'blocks', represents duration of incubation before. propagation. A visual colour assessment of. the pigments produced were obtained using a rating system of + 1 to + 2.

... <.11 -

Fig, 3.52

(A)

(B)

GROWI'll P 9897 COLONIES ON N.A.

GROWI'll Ps.aerugiDosa COLONIES ON N.A.

GROWI'll BASAL MED

ill!!.... (SLR)

l!.!!!!..

YEllOW PIGMENTATION [+ I)

GROWI'll BASAL MED

. (SLR) NO PIGMENTATION

I)

11)

.l!!!!..

GROWTH BASAL MED. (SLR) YELLOW PIGMENTATION [+ I)

GROWTH BASAL MEDIUM (AR) YEUOW PIGMENTATION [+ I)

GROWTH BASAL MED (SLR) NO PIGMENTATION

GROWI'll MEDAl

.!!l!!! (SLR) l!1!!! YELLOW PIGMENTATION [+ 2)

ISh.. (AR) ~ NO PIGMENTATION

GROWI'll MEDAl

.l!!!!.. (SLR) ~ YEllOW,GREEN PIGMENTATION

.

~~)Al .

YEllOW PIGMENTATION [+ 2)

NO GROWI'll MEDAl (AR)

NOGROWTII MEDAl (SLR)

!il!!

Fig. 3.53: Atomic absorbtion calibration curve for Mg2+: Absorbance (285.5 nm); Mg2+ concentration (p.p.m.). Standard deviations computed for each datum point are shown, where applicable, as error bars.

Table 3.17: Atomic absorbtion measurements of Mi+ concentration in

a) SLR and AR basal media

b) SLR and AR basal media, previously inoculated with strain A (0.0. (650 nm) 0.06), incubated for 20 min at 37°C, 90 r.p.m., then membrane filtered. .

. Fig. 3.53

Table 3.17.

? c: c: "

C.5

L'; Co."; co ~ o

" c: X ;: O.~ o ~

<1

0.:

• . 0.1

"".C'~--'---~r-__ ~ ____ .-__ ~ ", . .1',.- i. i 0.0 0.1 0.2. C.3 0.4 0.5

!.Ig'. C~nc. (ppm)

ATOMIC ABSORBTIONMEASUREMENTS

BASAL .. MAGNESIUM ION MEDIUM CONCENTRATION

. PARTS PER MILLION MOLARITY

SLR 0.095 3.9 X 10-6 a) .

AR 0.038 1.6 X 10-6

SLR FILTRATE 0.140 5.8 X 10-6

b) AR FILTRATE 0.102 4.2 X 10-6 .

.

152

I

I

I

I

13

.11

~ ..... C :J 0 ()

(j). ()

Q)

::c Cl '> Cl o·

..:.i 9 , \ \ \ \ \

1 7~----------------~----------------------~ o 20 40 60 80 100

Time (h)

Fig. 3.54: Viability of strain A, grown in basal medium,"'" and in Med A2, •.• ; all at 37°C, 90 r.p.m. Standard deviations computed for each datum point are shown, where applicable,· as error bars. .

1.53

Fig. 3.55: Optical density (650 nm) growth profile, ---; antifungal CC.albicans) zone of inhibition (mm) profile, A-A and pH profile, +-+ of strain A, grown in PDLM-1, at 37°C; 90 r.p.m., with an initial O.D. of = 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars.

Fig. 3.56: Optical density (650 nm) growth profile, ---; antifungal (C.albicans) zone of inhibition (mm) profile, A-A and pH profile, +-+ of strain A, grown in PDLM-1 at 37"C, 0 r.p.m. (static) with an initial O.D. of.::::. 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars.

lid .. '" , , ,

(ww) uomQ!4UI }O auoz .. ~

~ ~ 00 ID •

0 • /<i' +: ., I I' • --<1-'-0 +'

I \ \ 0

'" '"

• <:i '+

:\ 0 0

'" '? ~

o Cl) • <I ' + ~ E , ;::

0 5!

• -El + " '" I 1 , .- /+

0

5! cl 0 cl

Lt") (WUOS9) -4!SUaa ID~!ldO

Lt")

• er>

· 'Cl .~

......

HJ .. '" , , ,

(ww) UO!~!Q!4UI }O auoz .. J;i ;; ", 0 0

ID

0

• <30' + .,

\ \. ! • 4 +

\ I \ 0

'" '" • .<j +

\ 0 0

'" ~

o Cl) • ~ + ~ E ;::

0 5!

ID , 0 Lt") • f /+ '" · \ er> .- <I + • I \ 10 Cl -.~ ,. , , .!.' I • 1

, ,!." ,. , <;I I, ...... S! ;; cl

cl

154

/

'E' c o lC)

e ~ 'in c Q)

Cl

"8 o =s.. o

10

. 0.01+-------.--------.-------.-------.-------, o 20 40 60 SO 100

Time Ch)

Fig. 3.57: A summation of optical density (6 50 nm) growth profiles of strain A, grown in the Med q (Med B 1) MCSD experiment: complete medium ___ ; Med B1, 0-0; Med B2, e_e; Med B3, 0-0; Med B4, A-A; Med B5, Med B6, T-T; Med B7, +-+; all at 37°C, 90 r.p.m., with initial O.D. = 0.09. Standard deviations computed for each datum point are shown, where applicable, as error bars.

155

Fig. 3.58: A summation of antibacterial (E.coli) zone of inhibition (11lm)/time (h) . profiles, produced by strain A in the Med q (Med B1) MCSD experiment:

complete medium _-_; Med B1, 0-0; Med B2, 0_0; Med B3, 0-0; Med B4, to·to; Med B5, x-x; Med B6, y-y; Med B7, +-+; all at 37<C, 90. r.p.m., with initial 0.0. =. 0.09. The antibiotic disc used was 13 mm in. diameter. Standard deviations computed for each datum point are shown, . where applicable, as error bars.

Fig. 3.59: A summation of antibacterial (C.albicans) zone of inhibition (mm )/time (h) . profiles, produced by strain A in the Med q (Med B1) MCSD experiment: . complete medium _-_; Med B1, 0-0; Med B2, 0_0; Med B3, 0-0; Med

B4, to-to; Med B5, x-x; Med B6, y-y; Med B7, +-+; all at 37°C, 90 r.p.m., with initial 0.0. =. 0.09. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars. . .. ... ..

[NB. Symbols for Med B5, Med B6 and Med B7, all lie superimposed upon each other, along the 13 mm time axis],

co ll') .

• en .~

u..

-.- t-'

'" ll') . .

en

(ww) UO!I1Q!4UI 10 8UOZ

,0\ T

'·'~i • I • I • I • I ••

·~I ~i

156

..

• N 0

0: S!

o S!

o ..

" N

>---'en c: <I>

Cl

'0 ()

![ o

\ 10

O.Ol+-------.------r------.-----. o 50 100

Time (h) 150 200

Fig. 3.60: A summation of optical density (650 nm) growth profiles of strain A, grown in Med B8 (medium 3) of various NaN03 percentage concentrations: 0.0%, ---; 0.05%,0-0; 0.1%, 0_.; 0.15% 0-0; 0.2%, A-A; 0.25%, v-v; 0.3%, y-y; all grown at 37°C, 90 r.p.m., with initial O.D. of = 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars ..

157

Fig. 3.61: A summation of antibacterial (E.coli) zone of inhibition/time (h) profiles, produced by strain A in medium 3 of various NaN03 percentage·· concentrations: 0.0%, _-_; 0.05%, 0-0; 0.1%, 0_0; 0.15%,0-0; 0.2%, v-v; 0.25%, A-A; 0.3%, ,.-,.; all grown at 37°C, 90 r.p.m., with initial O.D. = 0.06. The antibiotic disc used was 13 mm in diameter. Standard . deviations computed for each datum point are shown, where applicable, as error bars.

Fig. 3.62: A summation of antibacterial (C.albicans) zone of inhibition/time (h) profiles, produced by strain A in medium 3 of various NaN03 percentage concentrations: 0.0%, _-_; 0.05%,0-0; 0.1%, 0_0; 0.15%,0-0; 0.2%, v-v; 0.25%, A-A; 0.3%, ,.-,.; all grown at 37°C, 90 r.p.m., with initial O.D. = 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars.

. " Cl .~

u..

N <.D . CV') . Cl .~

u.. '" N

, o N

(ww) UO!!!q!4ul la suoz

on

(ww) UO!I!Q!4UllO suoz

158

----------~~-----------------.--

o o N

z ~

o Cl)

S! E 'i=

o "

.0 o N

z ~

o Cl)

'.9 E

o

o on

i=

Fig. 3.63: A summation of antibacterial (E. coli) zone of inhibition (mm)/percentage NaND3 concentration profiles, produced by strain A in medium 3, at various times (h): 0 h, ---; 24 h, 0-0; 72 h, ._.; 96 h, 0-0; 120 h, ~-~; 144 h, x-x; 168 h, v-v; 192 h, +-+; all grown at 37°C, 90 r.p.m., with initial D.D. = 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars.

Fig. 3.64: A summation ofantifungal (C.albicans) zone of inhibition (mm)/percentage NaND3 concentration profiles, produced by strain A in medium 3, at various times (h): 0 h, ---; 24 h, 0-0; 72 h,·_·; 96 h, o~o; 120 h, ~-~; 144 h, x-x; 168 h, v-v; 192 h, +-+; all grown at 37°C, 90 r.p.m., with initial O.D. = 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars.

25

20

'E' -5. ,5 c o

:B ., ~ -o ,0

CD

~

5

Fig. 3.63

o'4---------.r~------~--------~·~-------.~------~--------~·,·~ 0.00 0.05 0.'0 0.15. . 0.20 0.25 0.30

25

20

'E' -5. c ~ .2

Percentage Sodium nitrate

Fig. ·3.64

:is ·,'.::.------1(------ ~! -==::::..]11----181----. .--

~ -0 CD

10 c ~

5

. o. 0 0.05 0.10 0.15 0.20 0.2S 0.30

Percentage Sodium nitrate

159

'? c

0 L() lO '-"

>---Vi ; c <I)

Cl

"0 (J .--a.. 0

10 64

.--.~ .

·~r 'l-3

'? • g J

C 0

• =+= /~ :c

:c .£

• • --0 CD

!,/Y:::~<" c 0

:I. • N

. 5rA \1 9

~-1!l.v.. -- -- -- ---------- '3

.~ ;j--+-+--+ I

+/ 5= C-

0.011-1-:-: --'-----r--.:..-----,-----__ :------+ 0 o . 50 100 150 200

Time Ch)

Fig. 3.65: Optical density (650 nm) growth profile, ---; antibacterial (E.coli) zone of inhibition (mm) profile, .to-.to; antifungal (C.albicans) zone of inhibition (mm) profile, v-v and pH profile, +-+ of strain A, grown in medium 3 of 0.05% NaN03 concentration (medium 4), at 37°C, 90 r.p.m., with an initial 0.0. of =. 0.06. The antibiotic disc used was 13 mm diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars.

160

O.ot+-----r------r------.------, o 50 100

Time Ch) 150 200

Fig. 3.66: A summation of optical density (650 nm) growth profiles of strain A, grown in medium 4 of various sucrose percentage concentrations: 0.0%, ---; 0.5%, 0-0; 1%, e_e; 2%,0-0; 3%, A-A; 4%, v-v; 5%, T-T; all at 37°C, 90 r.p.m., with initial OD of =. 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

[NB. All variants of medium 4 in this series contain glycerol].

161

Fig. 3.67: A summation of antibacterial (E. coli) zone of inhibition (mm)/time (h) profiles, produced by strain A in medium 4 of various sucrose percentage concentrations: 0.0%, ---; 0.5%, 0-0; 1%, ._.; 2% 0-0;' 3%, A-A; . 4%, v-v; 5%, T-T; all ~own at 37°C, 90 r.p.m., with initial O.D. of =: 0.06. The antibiotic dISC used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, . as error bars.

Fig. 3.68: A summation of antifungal (C. albicans) zone of inhibition (mm)/time (h) .. profiles, produced by strain A in medium 4 of various sucrose percentage

concentrations: 0.0%, _-_; 0.5%, 0-0; 1%, ._.; 2% 0-0; 3%,.11.-.11.; 4%, v-v; 5%, T-T; all ~rown at 37°C, 90 r.p.m., with initial O.D. of =: 0.06. The antibiotic dIsc used was 13 mm in diameter. Standard· deviations computed for each datum point are shown, where applicable, as error bars. .

· Ol .~.

lL.

co <.0 .

• "M

· . Ol .~

lL.

~.o_ .• It

~I "="' • . ~ I

~., . \ ""I -0,-- •.

"~I i • I • I •

o .~

o ...

'" '"

I r-------r-------r-_.----r-------r-------to

o '"

162

z '-'

0 .. . S! E

o

'"

;:

10

• • . .......--.......... . . "" ./ ..•

1

£ Cl)

s::: Q)

Cl

64

"3 'E' E

'-'" s:::

·0 :;:: :c

, :2 .E -/---------

8 •

:a I o <D s:::

o 0.1. .

~~~~~ ./ ~~ Ji----':;.;'l ·Zf-· 7l-]K-~---:"--------

~ +-+-+-+ t . ~ . /-+-+. . .

. +

;I.,

13

0.0~ .•. --------~.11----------r---------.-----------r0 o 50 100 150 2.00

Time Ch)

~

. 9

1 .

Fig. 3.69: Optical density (650 nm) growth profile, ---; antibacterial (E.coli) zone of inhibition (mm) profile, A-A; antifungal (C.albicans) zone of inhibition (mm) profile, and pH profile, +-+ of strain A, grown in medium 4 at 37°C, 90 r.p.m.,with initial O.D. of = 0.06 The antibiotic disc used was 13 mm diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars. ..

163

'E' t:

0 L()

.s. >" --'(ij t: Q)

a "'6 .2 -0. 0

0.1

I

11

0.01-1---.:....---.-----.------.--------, o 50 100

Time Ch) 150 200

Fig. 3.70: A summatIOn of optical density (650 nm) growth profiles of strain A, grown in medium 4 of various glycerol percentage concentrations: 0%, ---, 1%, 0-0; 2%,0_0; 3%,0-0; 4%, A-A; 5%, x-x; 6%, v-v; 7%, +-+; 8%, 0 - 0; all at 3rC, 90 r.p.m., with initial 0.0. = 0.06. Standard deviations computed for each datum, point are shown, where applicable, as error bars.

[NB. AIl variants of medium 4 in this series contain sucrose].

164

Fig. 3.71: A summation of antibacterial (E.coli) zone of. inhibition (mm)/time (h) profiles, produced by strain A in medium 4 of various glycerol percentage concentrations: 0%, ••• , 1%, 0.0; 2%, ••• ; 3%,0-0; 4%, A·A; 5%, x.x; 6%, 'l"V; 7%, +.+; 8%, 0-0; all at 37°C, 90 r.p.m., with initial 0.0. = 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars.

Fig. 3.72: A summation of antifungal (C.albicans) zone of inhibition (mm)/time (h) profiles, produced by strain A in medium 4 of various glycerol percentage concentrations: 0%, ••• , 1%, 0.0; 2%, ••• ; 3%, 0-0; 4%, A·A; 5%, x.x; 6%,'I"V; 7%, +.+; 8%, 0-0; all at 37°C, 90 r.p.m., with initial· 0.0. = 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars.

-...... · '" · Cl .~

u.. .

N ......

• '" • Cl .~

u..

~\ ~",r i+~~ i <>'\ i\ ~"" i

-<>-\-\~~i -<><+ a ,L .. I

• •

.. (ww) uomQl4uI )0 suoz

. S!

(ww) UOl!!Ql4ul )0 auoz

165

.. ..

'"

0 0 N

0 l2

:b o ., SJi

0 ..,

l-

? c-o " S E

;=

'" '"

Fig. 3.73: A summation of antibacterial (E. coli) zone of inhibition (mm)/percentage glycerol concentration profiles, produced by strain A in medium 4, at various times (h): 0 h, ---; 48 h, 0-0; 72 h, e_e; 96 h, 0-0; 120 h, A-A; 144 h, x-x; H;8 h, v-v; 192 h, +-+; all grown at 37°C, 90 r.p.m., with . initial O.D. = 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars.

Fig. 3.74: A summation of antifungal (C.· albicans) zone of inhibition (mm)/percentage glycerol concentration profiles, produced by strain A in medium 4, at various times (h): 0 h, ---; 48h, 0-0; 72 h, e_e; 96 h, 0-0;

120 h, A-A; 144 h, x-x; 168 h, v-v; 192 h, +-+; all grown at 37°C, 90 r.p.m., with initial O.D. = 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars ..

0~~2 -----=:::~-3 . . .'

Percentage CIYCero~ . S 7 8

Fig. - 7 ~. 4

.' 20 , ~ <

'E' E . ""15 .§

':a ", .,...-~ . '0 10

5

o : o •

1 2 i . P • • ercentag8 Cl 5 ycerol

7 a

166

E' .1: 0 L() <.0 '-' >--'Cij I: Q)

Cl

"'6 ,g -a. 0

64

0.1

:i!t:'--_. -]K--!\ll ~-jK--~-lll!: b- 13

----+ . ---+---+--+---+---+---+

0,01+-----:-. -----,-------,-----!-o 50 100 150 200

Time Ch)

9

5:1: 0..

1 .

Fig. 3.75: Optical density (650 nm) growth profile, ---; antibacterial (E.coli) zone of inhibition (mm) profile, .6.-.6.; antifungal (C. albicans) zone of inhibition (mm) profile, v-v; and pH profile, +-+ of strain A, grown in medium 4 of 2% glycerol concentration (medium 5), at 37°C, 90 r.p.m., with initial 0.0. of.= 0.06. The antibiotic disc used was 13 mm diameter. Standard

. deviations computed for each datum point are shown, where applicable, as error bars.

167

'E' s:: o L() to

. '-'

>---'(ij s:: Q) 0·

"0 .~ --a. o

10

0.011-1-------r------,--------,---_--, o 50 100 150 200

. TIme Ch)

Fig. 3.76: A summation of optical density (650 nm)growth profiles of strain A, grown in medium 5, at various pHs: 3, .~.; 4, Cl-Cl; 5, ._.; 6% 0-0; 7, A-A;

8, x-x; 9, v-v; all at 37°C, 90 r.p.m., with initial 0.0. of =. 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

168

I

I

Fig. 3.77: A summation of antibacterial (E.coli) zone of inhibition (mm)/time (h) profiles, produced by strain A in medium 5, at various pHs: 3, ---; 4 0-0; 5, e_e; 6,0-0; 7, A-A; 8, x-x; 9, v-v; all grown at 37°C, 90 r.p.m., with initial O.D. = 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where

. applicable, as error bars.

[NB. Symbols for pHs 4, 5, 6, 7 and 8, all lie superimposed upon each other along the 13 mm time axis].

Fig. 3.78: A summation of antifungal (C. albicans) zone of inhibition (mm)/time (h) profiles, produced by strain A in medium 5, at various pHs: 3, ---; 4 0-0; 5, e_e; 6, 0-0;. 7, A-A; 8, x-x; 9, v-v; all grown at 37°C, 90 r.p.m.,

with initial O.D. = 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars.

,..., ,..., •

M

· Cl

co ,..., •

M

• Cl .~

u...

- .... ~-

.~ \ ---"'---\

• I • I • I • I .. \ ••

\ I

• I • .. . ~ . I

o o ..

o .!l .

z ....., o CD $! E

;::

o VI

r-----~------r_----_r~._--~----~------TO o ... (ww) UO!l!QJ4UI 10 auoz

~~ I • I • I

I!I

I • I 1:1 ,

•

VI o

o o ....

o .\Q

z ....., g CD

.- E i=

. I r-----~~~-----o~-----.. _~~-_~--~-----.. ~·------o+o

:s: N N ~

(ww) u0!l!Q!4UI }O .auoz

169

'E c o LO ~ >

;!:: en C ID

Cl

"'8 o ~ o

0.1 64

.~."'.---.---.---.---. / . "" • • •

'1-3

-+--+-+1---+-+---+-+-+

0.0';------" -----..-----...,....,r------+O o 50 100 150 200

Time (h)

'E -.S ·C

o :;:: :c :I:: ..5 ...... o

5

3== Q.

1

Fig. 3.79: Optical density (650 nm) growth profile, - --; antibacterial (E. coli) zone of inlubition profile, b.-b. and pH profile +-+ of strain A, grown in medium 5, at pH 3, 37°C, 90 r.p.m., with mitial 0.0. of = 0.06. The antibiotic disc used was 13 mm diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars:

170

'E' c o

_ U1

e >--'Vi c Q)

Cl

"6 t) .--_ a. o

10

. 0.1

• .J

T T .,.._JI -----. '/' .. ,/J f l--f __ :l.· • • 1 . -. 1

64

'+3

21

O.OH-----.,------;------;--"--__ +O o 50 100

Time Ch) 150 200

'E' g c o

:;:: :c :c £ -' o III C

~

9

5 :z: Q,.

Fig. 3.80: Optical density (650 nm) growth profile, _._; antifungal (C.albicans) zone of inhibition profile, v-v and pH profile of strain A,. grown in medium 5. at pH 8 (medium 6), 37°C, 90 r.p.m., with initial O.D. of = 0.06. The antibiotic disc used was 13 mm diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars.

171

I I

Fig. 3.81: A summation of optical density (650 nm) growth profiles of strain A, grown in medium 6, at various r.p.ms.: 0 r.p.m., ---; 40 r.p.m., 0-0; 60 . r.p.m., ._.; 90 r.p.m., 0-0; 120 r.p.m., A-A; 150 r.p.m., T-T; .180 r.p.m., v-v; 210 r.p.m., +-+, all grown at 37°C, with initial O.D. of = 0.06. Standard deviations computed for each datum point are shown, where applicable, as error bars.

Fig. 3.82: A summation of antifungal (C.albicans) zone of inhibition (mm)/time (h) profiles, produced by strain A in medium 6, at various r.p.Ins.: 0 r.p.m., -_; 40 r.p.m., 0-0; 60 r.p.m., ._.; 90 r.p.m., 0-0; 120 r.p.m., A-A; 150 r.p.m., T-T; 180 r.p.m., v-v; 210 r.p.m., +-+, all grown at 37°C, with initial O.D. of = 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where· applicable, as error bars.

~

CO •

M

· 0'> ,~

w...

N CO · M

· 0'> ,~

w... o ..,

~ 'OJI-.

I \\\\ 1/ \ t> -lE! 0 «J-tI'"

L[\LLJ.\\ ~ A \ I

WT7~ ~

172

o

? ......-o CD 2 E

;:

o on

? ~

o CD 2 E

i=

o on

Fig. 3.83: A summation of antifungal (C. albicans) zone of inhibition (mm)/r.p.m. profiles, produced by strain A in medium 6, at various times (h).: 0 h, ___ ; 24 h, 0-0; 72 h, 0_0; 120 h, 0-0; 144 h, 1.-.1.; 168 h, x-x; 192 h,

. v-v; all grown at 37°C, with initial O.D. of = 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars.

M CO

° M

° Cl .~

U.

! • •

• Ii

0

'" '" 0 0

'" 0 al -0 CD -o (]) -t_ - ::l

c °E

0 ... . !::! (]) Co Cl!

o c 0.2 --::l

"0 > o (])

ale::

o CD

o -t

o N

r-------.--------r~~~~~~----r_------_r------~o 0 t<)

o N

(LULU) uOjUqlyul JO suoz

173

o

l' s:: o L()

. !.O '-" >-'in s:: Cl)

Cl

"6 o ![ o

10 64

____ B-IiI--Ii-. /a . •

0.1

10

13

-+---~---+-------+--......,+1--+----:+

2 0.01+-----r------r------r-----t-0 o 50 lOO 150 , 200

TIme Ch)

Fig. 3.84: Optical density (650 nm) growth profile, _._; antifungal (C.albicans) zone of inhibition profile, ".,,; and pH profile, +.+ of strain A, grown in medium 6, at 120 r.p.m., 37°C, with initial O.D. of.= 0.06. The antibiotic disc used was 13 mm diameter. Standard deviations computed for each datum point are, shown, where applicable, as error bars.

174

Fig. 3.85: A summation of optical density (650 nm) growth profiles of strain A, grown in medium 6, at various temperatures (0C): 22°C, .-.; 27°C, a-a; 32°C, '-'; 37°C, 0-0; 42°C, 1.-1.; all shaken at 120 r.p.m., with initial O.D. of = 0.06. Standard deviations computed for each datum point are shown, where. applicable, as error bars.

Fig. 3.86: A summation of antifungal (C. albicans) zone of inhibition (mm)/time (h) profiles, produced by strain A in medium 6, at various temperatures (0C): 22°C, .-.; 27°C, a-o; 32°C, ._.; 37°C,0-0; 42°C, A-A; all grown at 120 r.p.m., with initial O.D. of = 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars.

0 on N

• '@-. ..... <l

\ \\ \ I 0 0

• "1Ill'""' -.- <l N

\ \I \ 1 • m- -.- <l \ / \ . \1 0

on .0-0-- '1- - ...,. \ 1 1 6

CD .OV oS

\ \\ \ 0 ....

5!

·V<l ~\ . 0 on

--~~ 0

5! 0 0 U'> .,; 00 (WUOS9) ,(!lSUaO IC~l!dO .

(V') . 01 .-

l.L.

~----~

0 on N

..

~ .Q. • / 0

0 -.0:- .0 • ·N

.~/ ' .

.. CJIJ:: I-

'" 1, , 0 ,

..-m- • on -...,. \'\ 6

• CD

T\~ .S

0 ....

5!

---- -<>-~: 0 on

'" -.~ co . cV') , , , , , 0 . ' ,

" 0 on 0 '" 0 !!l ~ 5! '" 0> .. ., ., N N

. -L.I.. (ww) uOll!qlyul to auoz

175

'E s::

0 10 to '-"

>--'[i; s:: Q)

Cl

0 .2 -c.. 0

10 64

11 ____ -11-11- 11 - 11

1 / 43

•

~

~

A-~ V l

l-~/ 1

0.1 ~,

!\..-.-.. -~- - -- -- -- --- - ---- -- ----- ---- - - -- -- - -_._. ~ + +--+--+--+--+

13

0.01+----.------,----r----..----+0 o 50 100 150 200 250

Time Ch)

'E' g,

s:: 0

:;:: :c :.2 .£ .... 0 Q) s:: 0

N

9

5 :::: 0.

1

Fig. 3.87: Optical density (650 nm) growth profile, ••• ; antifungal (C.albicans) zone of inhibition (mm) profile, 10.-10. and pH profile, +-+ of strain A, grown in medium 6, at 32°C, 120 r.p.m., with initIal O.D. of:= 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars.

176

'E' c

.0 I.!")

~.

>. 0+-

'Uj C Q)

Cl

'0 .2 0+-

CO

10 64

43

'E'

• g, c 0

:;:: :c :c ..5 '>-0 CD C 0

0.1 21 N

9

-- '1='-------------------------------------- 13 + , "1' __ I 1"':'-+-+

0.011~-------_r------~------_.------_.~------rO 1 50 100 150 200 250

TIME(h)

Fig. 3.88: Optical density (650 run) growth profile, --_; antifungal (C.albicans) zone of inhibition (mm) profile, 10.-10. and pH profile, +-+ of strain A, grown in medium 6, at 37°C, 120 r.p.m., with initial O.D. of = 0.06. The antibiotic disc used was 13 mm in diameter. Standard deviations computed for each datum point are shown, where applicable, as error bars.

177

Table 3.18: Sequence of optimization experiments with corresponding percentage increases in antifungal (C. albicans) zone of inhibition, starting from the control!complete medium/med q (Czapak dox liquid medium modified). MCSD stands for medium component single deletion of the complete

. medium.

Table 3.19(a) Summary of simple scale-up optimization trials (variation of . agitation (tr./min) and aeration (Umin)) in the laboratory-scale

Table 3.19(b)

. fermentor. Corresponding visual colour assessments where increase in intensity relates to increase in antibiotic activity are based on a rating system of +1 to +3, was used to monitor.· sequential progress, each at 72 h.

Summary of further optimization trials (variation of time (days)) on optimal conditions achieved at table 3.19( a). An antibiotic assay using (C. albicans) (zone of inhibition (mm)) was used to monitor P!"ogress.

Table "3.18

. .

SEQUENCE OF OPTIMIZATION PERCENTAGE EXPERIMENTS EXPERIMENTS INCREASE IN

ANTIFUNGAL ZONE OF . INHIBmON

1 MediumB1 0 .. (Control)

2 MCSD Experiment 9 (Medium 3)

.. 3 Variation of NaNO, 37

(Medium 4)

4 Variation of sucrose 37 (Medium 4)

5 Variation of glycerol 44 (Medium 5)

6 Variation of pH 49 (Medium 6)

7 Variation of r.p.m. 109

8 . Variation of temperature 109

Table 3.19

1 ' .. (a) (b)

OPTIMIZATION IN THE LAB-SCALE FERMENTOR (100 tr/min) (4Umiri)

AGITATIO AERATION VISUAL TIME ZONE OF N (LITRES/MI COLOUR (DAYS) INHIBmO (TURNS/MI N) . ASSESSME I N) NT

N (MM)

(RATING)

150 4 ++ 2 0.0

200 4 + 4 . 15.0

100 4 +++ 6 20.5

50 4 ++ 8 15.6

100 3 ++

100 5 ++

178

(a)

RETENTION FACTOR RESULTS (First tier)

SOLVENT/ CHCl3 EtOAc MeOH :1 MeOH OBSERVATION CHCl3 :9

VISUAL 0.00 0.00 0.00 0.00

ULTRA- 0.00 0.00 0.00 Yellow VIOLET fluorescent

.. 'rocket'

CHARRING 0.0 0.0 0.0 Faint 'rocket'

(b)

RETENTiON FACTOR RESULTS (Second tier)

SOLVENT/ 1-11 11-2 11-3 OBSERVATION

VISUAL 0.00 0.00 0.00

ULTRA-VIOLET Small yellow Slight elution Small yellow 'rocket' 'rocket'

CHARRING 0.03 0.08 Slight elution

Table-3.20: Retention factor results for the active broth, showing progress through a) first tier, b) second tier of the Aszalos and Issaq solvent system strategy (Aszalos & Issaq, 1980), done on analytical TLC plates, viewed under U.V.light (253.7 - 356 nm) and cBarred after spraying with conc. H~OJglacial acetic acid (see Fig. 3.89).

179

Fig. 3.89 (A):

Fig. 3.90: (B)

Determination of appropriate solvent system for broth extraction, showing pathway taken when using the Aszalos and Issaq solvent system strategy (Aszalos & Issaq 1980) .

Determination of an appropriate solvent system for broth extraction,using an array of solvent systems of high polarity and resulting in AcOHlCHCl3 (1/9) (solvent A).

Fig. 3.89:

Fig. 3.90

DElER<1INATIOO OF JlPPRa'RIATE SOLVENT SYSTEMS

SOLl(ENT /BROTH EXTRAC!IOO

Broth su lematent

EtQlI(:

A~ : 10 Mal-! : 20 OCl3

11-1 11-2

Broth Supematent

(A)

Mal-I/OCl (First tier solvent) (1/9) 3

4(}l : 4 (Secald tier solvents)

Mal-! : 20 OCl3: 76

11-3

(B)

. r-----.------.--~·~-~--~-~-~--=--~--~-~-=--T-~-~-=--~-=--~--i , ,

lOl"ClTeth E r : 10 E

OCl3 : 90 /J(;(}l

180

:10

:90

/J(;

I I , , ,: 10

C}[lo:9O ~

(Solvent A)

Fig. 3.91: Flow chart arising from determination of appropriate solvent systems for the liquidfTLC extraction. 'x' denotes second 'dip' or 2-dimensional TLC separation. [See Fig. 3.92].

Fig. 3.91: - DETm-1INATI(XII OF APPR(PRIA1E gJLVENT SYSTEMS ------~~~~~~~~~~~-------

EtOA.c

: 1 fWi ._..,---~_ QCl

3 : 9 _.

,

SOLVENT/TLC EX1RACTI(XII

Solvent A Antibiotic concentrate

Etlli (Solyent B)

f20

ka1/QCI3 [1/9] . (Solvent A)

Acai : 1 Et ~

(Solvent A)

X

Meai : 1 _ Etlli QCl3 : 9

(Solvent C)

M:<H : 1 AdH : 1 CI{l3 : 9 _ . _ QCl3 : 9

(Solvent A)

Etlli ~'eOi : 1 EtJ.!\c QCl • 9

3 (Solvent C)

181

. !

EtOA

Fig. 3.92: Constructed flow chart for the isolation of the 3 antifungal antibiotics AF1, AF2 and AF3, using a liquid{fLC extraction route. AF = antifungal activity; AB = antibacterial activity and x = second 'dip' TLC separation denotes separation of active layer on fresh (except for 'x') prep TLC plate, using the preceding solvent. (See Fig. 3.91).

co N

3JLVENT A BROlll

Fig. 3.92:

3JLVENT B

,------, ,

• ~ ... - - - - .-

JlJl lXM'O I (IM>URE)

FLoo-JART FOR THE lSOlJ\TICl'l IF JlNTlFUNGIIL JlNTIBIOTICS

.- --. ~. -- . r-----: F •

E ~VENT A.

D 1- •• _- ••

C

r - - - - . B • •

I

3JLVENT A X A

L.. __ I .. - . - .

-E1 AF1

~ ~

r·~---- -, , X -.. I

gJLVENT C Y

:,Z -- ~ ' .. L _______

~ ('")

-------, AFL Y3 :

AF2 Y2 --_+ __ ~ Y1 I

--- ... ---t

------------------------------~--------~--. .-

ANTIFUNGAL ANTIBIOTIC 1

MICROORGANI§M MIC ~g!ml

E.coli 207

S.aureus 110

S.marsescens 276

S.cerevisia 119

C.albicalls 107

A.lliger 72

S.schenckii 156

P.chrysogenum 83

T.mentagrophytes 12

Table 3.21: Minimum inhibitory concentration determinations of the antifungal antibiotic AFl, against a range of microorganisms, grown on NA, at 37°C for bacteria, and on NspA,at 30°C for fungi. Assessment was by the agar diffusion assay.

183

Fig. 3.93: Stable zones of inhibition, created by antifungal antibiotic AF1, against . T.mentagrophytes, incubated in NspA for 5 days fit 30·C. The . concentration of antibiotic solutions placed into the cork-bore plugs (9.4

mm diameter) were as follows: 1 = 1 mg/ml; 2 = 0.5 mg/ml; 3 = 0.25 mg/ml; 4 = 0.125 mg/ml; 5 = 0.0626 mg/ml; 6 = 0.0 mg/ml (control).

Fig. 3.94: Stable zones of inhibition, created by antifungal antibiotic AF1, against Aniger, incubated in NspA for 5 days at 30·C.. The concentration of antibiotic solutions placed into the cork-bore plugs (9.4 mm diameter) were as follows: 1 = 1 mg/ml; 2 = 0.5 mg/ml; 3 = 0.25 mg/ml; 4 = 0.125 mg/ml; 5 = 0.0626 mg/ml; 6 = 0.0 mg/ml (control).

Fig. 3.95: Stable zones of inhibition, created by antifungal antibiotic AF1, against P.ch!),sogenum, incubated in NspA for 5 days at 30·C. The concentration of antibiotic solutions placed into the cork-bore plugs (9.4 mm diameter) were as follows: 1 = 1 mg/ml; 2 = 0.5 mg/ml; 3 = 0.25 mg/ml; 4 = 0.125 mg/ml; 5 = 0.0626 mg/ml; 6 = 0.0 mg/ml (control).

Fig . 3 .

Fi g . 3. 94

Fi g. 3. 95

164

Fig. 3.96 Stable zones of inhibition, created by antifungal antibiotic AFl, against S.aureus, incubated in NA for 5 days at 37°C. The concentration of antibiotic solutions placed into the cork-bore plugs (9.4 mm diameter) were as follows: 1 = 1 mg/ml; 2 = 0.5 mg/ml; 3 = 0.25 mg/ml; 4 = 0.125 mg/ml; 5 = 0.0626 mg/ml; 6 = 0.0 mg/ml (control).

Fig. 3.97: Stable disc zones of inhibition, created by antifungal antibiotic AF2, against C.albicans, incubated in NspA for 5 days at 30°C. Antibiotic discs (13 mm diameter), dipped in AFl and AF3, initially gave zones after 24 h but have now, after 5 days incubation, virtually no zones.

Fi g . 3.

Fi g . 3. 97

16 5

Fig. 3.98: A uv/vis spectrum of the antifungal antibiotic, AF1, dissolved in U.V. ethanol.

Fig. 3.99: A uv/vis spectrum of the antifungal antibiotic, AF2, dissolved in U.V. ethanol.·

Fig. 3.100: A uv/vis spectrum of the antifungal antibiotic, AF3, dissolved in U.V. ethanol.

Fig. 3.98 -' __ ~ _____________ --, -I< 2.50 Ar

0.5000 (NDIV)

+O.OOA,~~_~====~~~~ __________ ~ 200.0 100.0 (tWDIV) 700 I'M

Fig. 3.99 +2.50A~------__ ~ ______ -.

0.500 -(NDIV)

-+ 0.00 A.I-----'---...::::..::::......---:--'----~--""""'ift700 I'M 200.0 100.0 (tWDIV)

Fig. 3.100 + 2.00 A-"------------------,

0.500 (NDIV)

+ 0.00 AL---...::::=----:--:--'-----'----'rl-I/lu

I'M 200.0 100.0 (tWDIV)

186

90.0

70.0

Fig. 3.101: An infra-red spectrum of antifungal antibiotic, AFl.

Fig. 3.102: A 60MH. INMR spectrum of antifungal antibiotic, AF1, dissolved in CDCI3•

co co

Fig. 3.102

20ppm

10ppm

5ppm

2ppm

1ppm

O.5ppm

I .....

c: w I-w 2 0 c: I-U w a. Vl

c: 2 2 N :r: 2 0 c;:>

C <!l r·' , 2 L1.!

63, '/

xr

40,0

. Fig. 3.103: An infra-red spectrum of antifungal antibiotic, AF2.

40.3

xT

25.e

Fig. 3.104: An infra-red spectrum of antifungal antibiotic AF3.

CHAPTER 4

DISCUSSION/CONCLUSION

4.0 DISCUSSION/CONCLUSION

4.1 The microorganism

Two strains, A and B, of a Pseudomonas spp. (NCIB 9897 and 9898 respectively),

_ both of which are small, rod shaped, Gram negative bacteria with 1-2 polar

fiagelli, have recently been- proffered by Attafuah and Bradbury (1989) as

members of a new species of Pseudomonas.

The classification of novel microorganisms has always proved contentious in the

field of taxonomy and in the case of the Pseudomonas, a significant point of issue

has been its close resemblance to the genus Xanthamonas (MacFaddin J. F., , 1983). However, morphological (optical/electron miscroscopy),

biochemical/tolerance and antibiotic producing properties (antibacterial and

antifungal) of the organism, as revealed by the present study, supports the

contention that - it is a new species of Pseudomonas, to be known as Ps.

antimicrobica (Attafuah and Bradbury 1989; U.S.B. 1990). Important

characteristics which tend to distinguish it from Xanthamonas spp. include:

positive nitrate to nitrite; simple minimal growth requirements; inability to

hydrolyse starch, and the production of diffusible (extracelluar) yellow pigments.

BiochemicaLcharacteristics_ of Xanthamonas spp. are. summarised. in Bergey's_

Manual, (Bradbury, 1984).

The only deviation in biochemical test results of the present study from the

_ published data (Attafuah _ and Bradbury, 1989), is found in the

-oxidative/fermentative test for sucrose which was positive for the former test. This

is supported by the fact that the organism grows fairly well in Czapek Dox

Medium which has sucrose as the sole carbon source (Tables 3.2 and 3.4).

Hitherto unpublished biochemical/tolerance tests include positive results for

ONPG, catalase, urease, MacConkey agar, minimal medium, growth at pH 4

(NA), and negative results for MR/VP, ornithine and lysine decarboxylase, DNase,

haemolysis (sheep's and horse's blood), TIC (1%), cetrimide (0.03%), and

Pseudomonas agar. Furthermore, the two strains may also be distinguished by a

positive aesculin test for strain A but negative for B; a positive lecithinase test for

191

strain A but negative for B; a polymyxin MIC of 10-2 mg/ml for strain A but an

MIC of 10-4 mg/ml for B; no growth in medium (NA) containing co-trimoxazole

(25 /Lg) for strain A but growth in the same medium for B (Table 3.1).

Pseudomonas aeruginosa (tYPe species), Ps. antimycetica (antifungal production)

and Ps. tnaltiphilia (yellow pigment producer) were also tested at the same time

and acted as controls. •

Some of the test results bear a resemblance to those of Ps. cocovenenans which '

has recently been described by Gwynn et al (1988) as a source of antibiotics and

there was therefore a particular need to confirm separate species status for strain

A Subsequent comparison tests revealed a number of differences. These included

. an inability of Ps. cocovenenans to grow in a constructed selective medium for

strain A. containing sulphafurazole (500 /Lg/ml in N.A; Fig. 3.3). Here, this may

suggest that strain A may be impermeable to the drug or that it lacks (or has an

ability to bypass) a biosynthetic pathway for folic acid, and, like lactobacillus, may

be able to obtain its requirements exogenously (Woods, 1962; Wolf and

Hotchkiss, 1963; Hitchings and Burchall, 1965; Edwards, 1980). Very slow

. growth of Ps. cocovenenans in Med A2 at pH7.2 (24 hr lag phase) and at pH 4.0

(48 hr lag phase) was observed whereas strain A gave excellent (pH 7.2) and fairly

good (pH 4.0) growth (Figs. 3.4 and 3.5). Very good growth of strain A (with

strong yellow pigment exudation) on NA at pH 4.0, after 24 hrs, was observed, .- - . --- -, -~- --- --,- -~ , ,--

compared with no growth of Ps. cocovenenans, after the same time (Fig. 3.7).

Growth however, does occur for the latter but only after 48 hrs, as verified by

Gwynn et al (1988). Other differences include growth of Ps. cocovenenans in

medium containing carbenicillin (100 /Lg) but strain A did not grow (Table 3.1);

positive sucrose oxidative/fermentative test for strain A but negative for Ps.

cocovenenans; growth at 42°C was negative for strain A but positive for Ps.

cocovenenans; nitrate to nitrite was positive for strain A, but negative for Ps.

cocovenenans. (Gwynn et aI, 1988; Table 3.2).

From all the above, it seems that these two organisms are indeed different.

The comparison tests done from the present study Fig. 3.7 has also revealed that

Ps .. antimicrobica NCIB 9897 should now be included in a select group of

Pseudomonas, all of which share an ability to grow at pH 4.0. These include

192

Ps. cepacia NCIB 9085, Ps. acidophila ATCC 31363, Ps. mesoacidophila ATCC

31433 Ps. cocovenenans NCIB 9450 and a strain of the latter, designated 326-328 .

(Kintaka et aI, 1981a; Kintaka et aI, 1981b ; Imada et aI, 1982; Gwynn et aI,

1988).

Conditions for optimal growth in chemically defined media (CDM; different

amino acid content for each strain) indicate the organisms to be non-fastidious,

though growth of strain A was stimulated by phenylalanine and histidine and

. similarly for strain B, methionine, ornithine, arginine and lysine encouraged growth

(Table 3.3). With the exception of methionine and phenylalanine, the group of

amino acids chosen for this study, came from Ps. aeruginosa's amino acids

. requirements as defined by Guirard (1974). They were chosen mainly because Ps.

antimicrobica, was once misclassified as a local strain of Ps. aeruginosa (Y.

Tanada, quoted from Attafuah, 1965). Methionine, however, was chosen because

it is a key amino acid involved in many of the biological methylation processes

(Lehninger, 1980). Phenylalanine was chosen because, apart from the fact that

its metabolism is important in Pseudomonas classification (MacFaddin, 1983), it

ultimately gives rise to fumarate or acetyl Co A which enters the tricarboxylic acid

cycle,leading to increasing ATP production for metabolism (Lehninger 1980).

Non~fastidiousness was demonstrated by an inability to alter growth rates with the

vitamins, riboflavin and- nicotinic acid (constituents_ of yeast extract) and this

finding is supported by the observations of Attafuah and Bradbury (1989).

Compared to Ps. aeruginosa, both strains A and B are slow growers in the basal

medium used, with a mean generation time (mgt) of 170+6 mins for strain A, a

mgt of 150+5 mins for strain B and a mgt of 100+5 mins for Ps. aeruginosa.

(Table 3.3). The construction of their specific respective chemically defined

medium (CDM) however, culminated in a mgt reduction of 77+6 mins for strain

Aand a reduction of 53+5 mins for strain B. This compares very favourably with

a mgt reduction of 12 +Smins for Ps. aeruginosa, under the same conditions and

with its own established amino acid requirements (Guirard, 1974; Palleroni,

1984).

193

A preliminary experiment to confIrm inherent antimicrobial activity of both strains

of the microorganism was performed against A. niger. Pseudomonas aeruginosa,

Ps. antimycetica and Ps. maltiphilia were also run in tandem, acting as controls

and for comparison (Table 3.4). Pseudomonas aeruginosa and Ps. antimycetica

were chosen because both had previously been reported as having antifungal

activity (Waksman 1945; Thaysen and Thaysen, 1955). The fact that Ps.

antimycetica did not inhibit fungal growth on Potato Dextrose Agar (PDA) here,

may have been due to a lack of essential trace elements/precursors in the medium,

necessary for the production of its antifungal principal. This observation

emphasizes the need for good medium design in antibiotic production.

The intense yellow pigmentation produced by strain A on Czapek Dox Agar

. (CDA), was accompanied by an inhibition of A. niger which was greater than that

achieved on PDA (Table 3.4). This may suggest that with CDA, a cheinically

defIned medium specifIcally developed for fungi (Smith, and Onions, 1983), the

bacterium was under particular stress due to a scarcity of essential nutrients and

or a h9stile environment (Frobisher, 1959; Holliman, 1961). A similar test with

the comparison organism, Ps. aeruginosa, could not be pursued successfully

because this organism is not able to metabolise sucrose which is the only carbon·

source of CDA (MacFaddin, 1983). This fact also reinforces the present study's

positive· results obtained for the oxidative/fermentative test for sucrose on Ps ....

antimicrobica, as opposed to a negative result obtained by Attafuah & Bradbury

(1989). It is to be noted that under the same test conditions, Ps. aeruginosa gave,

as expected, a negative oxidative/fermentative test for sucrose (Table 3.1).

4.2 Antibacterial activitv studies

The quest for a medium able to elicit consistent antibacterial activity was mostly

influenced by the work of Hellinger (1951) and by Holliman (1961) where a

considerable number of parameters, both medium components/concentration and

environmental, were sequentially altered, in an attempt to elicit production of

secondary metabolites in liquid media. This quest resulted in a basal medium

(Cruickshanks medium; Cruickshank, 1970) which was preferred to Czapek Dox

Liquid Medium (CDLM) mainly because it was simpler and gave a longer lasting

194

production/detection of antibacterial substance at 90 r.p.m., 37°C. (Fig. 3.17 and

Fig. 3.74).

The preliminary heat stability experiment which still showed activity after 20 mins

at 100°C, (Section 2.3.3a) was done in order to establish that the active substance

was probably not an enzyme, a bacteriocin, a toxin or a similar proteinaceous

substance. The test for pyrrols on the active broth which was negative, was done

in order. to establish whether a similarity existed between the antibacterial

substance and two other, well known, pyrrol containing Pseudomonas antibiotics,

. namely pyrrolnitrin and pyolu.teorin (Takeda, 1958; Arima et ai, 1964). The

results of the above two experiments served to establish at this early stage, a

likelihood that the active principal may indeed be of some interest and therefore

worthy of pursuit.

Cultures initiated with a "pigmented inoculum" (Section 2.3.2) showed greater

antibiotic production after 24 hrs than the normal "washed inoculum" situation,

both grown at 90 r.p.m., 37°C. Wrede and Strack (1924) and Swan et ai, (1957)

have reported that the Pseudomonas pigment, pyocyanine, which can also be

reduced to leucopyocyanine, can in this latter form, readily absorb oxygen. It is

possible that oxygen uptake may have occurred during rotary incubation

(aeration/agitation) and upon metabolising the pigment, oxygen was given up for

cell use. Alternatively, or in addition, it is known that many of these pigments are

toxic and can act as bypasses of the cytochrome system resulting in toxic end-e

products of oxygen metabolism (principally peroxides). Consequently, certain cells

which are catalase abundant, protect themselves by splitting H20 2, thus liberating

O2 (Latuassan and Ber. ands, 1961) and this is likely to have a net effect of

increasing the availability of O2, Since many workers agree that good/adequate

aeration is essential for enhanced pigment production (Elema and Sanders, 1931;

Hellinger, 1951; Holliman 1961; Mudgett, 1980), this may explain why antibiotic

production was significantly greater in the "pigmented inoculum" situation than in

the "washed inoculum" situation.

In contrast to the 'shaken' situation described above, the static "pigmented

inoculum" experiment showed no detectable activity and again this suggests that

195

good/adequate aeration is essential for the development of the antibiotic in this

medium.

As an aid to understanding the biochemistry/genetics of the regulatory mechanism

of antibiotic production and of growth during the optimization study, a probable

schematic diagram is proposed. (Fig. 4.1). It has been constructed in light of all

the antibiotic studies undertaken and from current knowledge of the

biochemistry/genetics of antibiotic regulation (Bushell, 1989; Hunter and

Baumberg, 1989). It takes into cognizance Atkinson's energy charge (E.C.)

formulation (Atkinson et aI, 1969) and is predominantly based upon NADH as the

intracellular effector (Behal, et aI, 1969).

Allthe antibacterial optimization studies were initiated with 'washed' inocula, to

give an initial optical density (O.D.) of = 0.06. This was done because trace

metals, unused carbon and· nitrogen sources remaining in the former growth

medium, though diluted after inocular transfer, may still have interacted

metabolically through enzyme repression, induction, inhibition or activation with

substances in the new medium. Together with this metabolic interaction, toxic

end-product transfer and high cell packed volumes may also have inconsistently

affected antibiotic production. (Brown and Zainudeen, 1978; Young, 1979) .

. During optimization, a medium component single deletion procedure was initially_ .

adopted and this was followed by sequential variation of the concentrations of

individual medium constituents, essential for antibiotic production and growth. It

was found that omission of Mg2+ (MgQz.6HzO) from the standard laboratory

reagent (SLR) grade medium recipe, leaving only trace amounts (3.9 x 10-6M),

significantly increased production (Fig. 3.12). This is surprising because

magnesium is termed a macronutrient, essential for maintaining the integrity of

the ribosomes, for membrane stability and also for the function of many enzymes

(Strange and Hunter, 1967; Mandelstam and McQuillen, 1973). In view of this,

one would not have expected much growth let alone antibiotic production. It is

possible here, that strain A may have already evolved an uptake mechanism with

high affinity for Mg2+ and, an affinity for substitute divalent cations, all present as

trace metals in the medium (see appendix. Though these trace element values are

specifically for AR grade chemicals, one would expect higher values for the SLR

196

situation). Examples of similar uptake mechanisms have been reviewed by

Tempest and Neijssel (1978). A similar experiment, performed as above, using

purer analar reagent (AR) grade chemicals in the preparation of the medium,

stopped antibiotic production though similar growth ensued (Fig. 3.13 - Fig. 3.14).

All this strongly ,suggests some involvement of essential trace elements in the

antibiotic production and this finding is supported by other work done by Foster

et al (1943) on penicillin, Chesters and Rolinson (1951) on streptomycin; Katz

et al (1958) on actinomycin, Kurachi (1958) on pyocyanine andWeinberg and

Tonnis (1966) on bacitracin production.

From Figs. 3.15 - 3.18 it can be seen that the minimum glucose concentration for

achieving maximum growth and antibiotic production was 0.3% and this coincides

with the set value obtained for. optimum growth of bacteria during the

construction of this defined medium. (Cruickshank, 1970). Altogether, this

suggests a direct relationship between growth and antibiotic production and

between glucose concentration and antibiotic production (Fig. 3.19), below a

threshold value of 0.3% .. From this it can be argued that in this fermentation, the

antibiotic concentration may directly relate to the number of actively dividing cells:

a feature often reported by other workers in the field and is known as "growth

associated mode antibiotic production". This production is nearly always 'low

level'/incidental as opposed to produc~ion in the i~iophase which i~ comparatively

. 'high level' (Shehata, et aI, 1971; Drew and Demain, 1977; Aharonowitz and

Demain,1978). Increasing the glucose concentration furthe~ (above 0.3%), from

flask to flask, resulted in a depression in both growth and antibiotic production

and this may have been due to substrate and end-product inhibition for the

former and glucose catabolite repression for the latter (Gallo and Katz, 1972; Hu

et aI, 1984; Revilla et aI, 1984; Lebrihi et aI, 1988; Bushell, 1989).

Unlike glucose variation, no such simple relationship exists with nitrogen (NH4Cl)

variation; that is to say, between growth/concentration of nutrient and antibiotic

production (Fig. 3.21-3.25). Growth here, apparently continued noiTnally with

decreasing NH.Cl concentration, right down to 0.01% where no antibiotic

production was detected. However, peak antibiotic production was detected at

0.06%. From all this, it appears that as the NH4Cl concentration was decreased

197

from flask to flask, strain A might have, while under stress, favoured the glutamine

synthetase~glutamate aminotransferase (GS-GOGAT) enzyme system over that of

the glutamate dehydrogenase (GOH) enzyme system, resulting in increasing

production of the antibiotic (Aharonwitz, 1979). Such a property in the wild,

would give strain A a distinct survival advantage '" .

Variation of the initial pH of the medium showed that in general, as the pH was

i;, creased from flask to flask from a point (7.0), there was a fall in both antibiotiC

production and growth (Figs. 3.27 and 3.29). Furthermore, decreasing the pH

from the usual medium pH of 7.2 (normally associated with good growth of most

bacteria; Miller and Churchill, 1986; van Demark and Batzing, 1987), to pH 6.0,

a significant increase in antibacterial production occurred (Fig. 3.29). It is possible

that decreasing the pH may have imposed stress upon the organism, due to just

enough undissociated weak acids entering the cell to cause a stimulation in

general metabolism and thereby enhancing antibiotic production. (van Demark

and Batzing, 1987). In the wild, this property would be of advantage especially

in a crowded environment where acidic metabolic end-products and contents of

cell lysis such as gluconic, citric and anthranilic acids may abound (Takeda, 1959;

Martin and Demain, 1980) .

.. Increasing or decreasing the pH to excess adversely affected both growth and

antibiotic production and this may have been due to the formation of excess

undissocciated weak acids and bases, both of which are known to readily enter

cells, disrupting the metabolism and physiology (van Oemark and Batzing 1987).

Variation of agitation/aeration of different cultures, incubated at different r.p.ms.,

showed that as the r.p.m. was increased from 90, there was in general, a fall in

both growth and antibiotic production (Figs. 3.31 and 3.32). However, at 120

r.p.m., a tremendous increase in antibiotic production occurred after 60 hrs

incubation (Fig. 3.32). The growth profile for cultures incubated here, was

surprisingly biphasic (Fig. 3.33), since such profiles are normally associated with

2 carbon source media (Monad, 1942; Stainer et aI, 1984; Ooull and Vining,

1988).

198

Cultures incubated at 120 r.p.m. would have received an increase in oxygenation,

(relative to the 90 r.p.m. situation) which may have increased cellular

concentrations of NAD+, leading to feedback inhibition of the electron transfer

chain (ETC), via an allosteric enzyme (Fig. 4.1). Eventually, an increase in

celluhir concentration of cAMP, coupled with a decrease in ATP concentration

(low E.C.) would have resulted. Up to this stage, exponential growth (steady

increase in cell mass) would have been directly proportional to "Iow level"

antibiotic production on account of an already triggered antibiotic biosynthesis at

start of primary log phase (such low level/incidental antibiotic production has been

observed by Hormouchi and Beppu, .1987, and Martin and Demain, 1980).

However, at this stage, when the growth cycle had entered mid-stationary phase,

an antibiotic activator protein may have. combined with the cAMP, forming a

. complex. This complex would then have attached itself to a promotor site of an

operon, initiating coding for antibiotic inducible enzymes, which would ultimately

go on to stimulate transcription. As more NAD+ was used up during

phosphorylation of cAMP to ATP, this would have led to a removal of the NAD+

feedback inhibition and with more ATP produced (high E.C.),cell growth and a

tremendous increase in antibiotic production would have ensued.

Further increases in r.p.m. would not only have increased NAD+ and cAMP levels

~-~~---- on account of excessive oxygenation but would have physically stressed the cells.

Altogether· this would have lead to progressively stronger and earlier occurring

NAD+ feedback inhibition, resulting in a limitation in both growth and antibiotic

production. A similar effect was obtained by Grossowicz, et al (1957) who

observed that pyocyanine production from Ps. aeruginosa was stemmed upon

vigourous shaking during incubation. Cultures incubated at progressively lower

r.p.ms. would have experienced corresponding decreases in oxygenation, resulting

in poor growth and antibiotic production.

Variation of the temperature of incubation for different cultures, shaken at 120

r.p.m., gave 37°C as being optimal for both growth and antibiotic production in

this medium (Figs. 3.34 and 3.36). Cell aggregation or clumping in this minimal

medium, increased as the temperature decreased, clearly illustrating clumping to

be a nutritional/temperature dependant phenomenon (Calleja, et aI, 1979; Calleja

199

I

I

~ NADH is first oxidized to NAD+ by the electron transfer chain (ETC;

Fig. 4.2) involving the reduction of oxygen. The resulting 2H+ are then used in

a phosphorylation process which gives rise to an increased cellular concentration

of ATP (Fig. 4.3) and " decrease in cAMP and NAD' level,. A high energy

charge (EC) situation at the beginning: of log phase is thus obtained.

The high levels of ATP may at first depress antibiotic production (feedback

inhibition) (Janglova, et a1. 1969j Martin, 1976; Manin, et al. 1976) and allow

utilization of ATP for cell metabolism only but, after a threshold value (ATP

concentration), feed·back inhibition is lifted and antibiotic production proceeds.

~ Using ATP cataJ"zed by the enzyme adenyI.ate cyclase, glucose is

phosphorylated to glucose-I-phosphate and this goes on to be used for cen,

metabolism alone, or cell metabolism and antibiotic production. This results in

an increase in cellular cAMP and a decrease in ATP leading to a low E.C .. In

time, end~product inhibition/repression would lead to an increase in the level of

NAD+, resulting in feedback inhibition and subsequent entry into stationary/mid~

lag phase.

~ Cyciic AMP combines, sequentially, with various activator proteins (AP)

including firstly. AP (2nd carbon source) which results in the production of second

carbon source metabolic enzymes (constituting a pathway); secondly, AP (AB1)

which results in the production of antibacterial biosynthetic enzymes and thirdly,

AP (Nn) which results in the prOduction of antifungal biosynthetic enzymes

(Alper, tlAL 1978).

Stage 4 After completion of the metabolic and biosynthctic pathways, the cAMP

is then phosphorylated to ATP and this ultimately leads to the removal of the

NAD+ feedback inhibition and a high E.C. It is possible that the twO antibiotic _

pathways may have different ATP concentration thresholds thus enabling the

feedback inlubition of one to be lifted before that of the other.

~ The ATP thus accumulated here is used for growth alone or growth and

sequential antibiotic production thus giving rise to biphasic growth. In time,

depletion of essential nutrients and end·product inhibition/repression would curtail

production of ATP and hence lead to a low ac and increased NAD+ levels.

This would then effect feedback inhibition resulting in a stationary phase.

"

N o o

PROBABLE SCHEMATIC DIAGRAM FOR REGULATION OF ANTIBIOTIC PRODUCTION AND GROWTH ETC (See FIg 4.2) c ~ ~ ~ AMP ~ ATP ~ NAD + ~ NADH "OJ- ,.- NADH)

(Existing in dynamic equilibria) 2 2

_( NAD + __ (S~ FIg 4.3) Depression of H20 -- r----------------------,

! I ~ . ~ ATP m :. cAMP m : I High E.C. AGE I cAMP A ST

I t Glucose-I-phosphate (ete) ~ Cel! metaboUsm alone or cell metabolism fIrst then antibiotic production

AdenYlate-{ p A- I (strict sequence) cyclase I I L _______________ ~

"-CAMP

Glucose End-product inhibition (NAD +)

AGE 2 ~ :. cAMP m :. ATP ill :. NAD + m:. Feedback Inhibition I Low E.C. ST

nd -V .

genes AP(2 carbon source) I cAMP I AP I

u l operon AP(AbI>

u u

u~u <D 2 nd carbon source metabolic enzymes AP(Af D) chromosomes to

inducer enzymes encoded +- initiate production of :-CD Ab I biosynthetic enzymes

AGE 3 cAMP m . CD Af n biosynthetic enzymes st

£: \\ - Depression of

ATP .. :. ATP +H --r-----------------~-----------------1 I

----I - --- .

I I I I

NAD+ :. NAD + ill :.: Removal of Feedback. Inhibition I . I • I : I AGE 4 NADH ~ :. NADH m I High E.C. ST

.lO~-- --f I , 2 2 L. To be used for growth alone or growth fIrst then antibiotic production

(strict sequence)

<D Depletion of essential nutrients CD End-product inhibition I repression

ST

cAMP

* ~ ATP

.

HV- ~ :. NAD + m:. Feedback Inhibition I AGE 5 --ETC'" NAD+ • Low E.C. I I .. L ____ +----------

F~dback Inhibition

GlROwnI PHASES

LAG PHASE

LOG PHASE

STATIONAR y I MID-LAG

PHASE

BIPHASIC GROWTH

y STATIONAR PHASE

Fig. 4.2:

(After van Demark and Batting, 1987)

The electron transfer chain fETC) in a bacterium. The first carrier, a flavoprotein

(Fp) accepts hydrogen from NADH and passes electrons along to a non·haeme

iron carrier [nhFe]. Thereafter the electrons are passed sequentialIy to coenzyme

A(CoQ), cytochrome·b (Cytb), and finally to cytochrome.O(Cyto 0). The latter

functions as the tenninal member of the respiratory chain to reduce oxygen.' The

flow of one pair of eiectron leads to the extrusion of 2 pairs of protons (H+).

Merr:crcne s:o!enllOt

••••••• Memcrcr .•

~------~-~-~-~-:-:-:-NN~.O;H~+~H~.:'\\=;::::;~-+:2H. NAQ.t;

iE:=:r-+: 2H' Icw proton coneentrotlon

Fig. 4.3:

(After van Demark and Batzing, 1987)

ATPa!ie mediated phosphorylation within a bacterial cel!. As protons arc

extruded they accumulate outside the membrane of the cell. This creates a

proton gradient across the membrane. Protons 3rc then returned to the inside of

the cell through channels provided by the enzyme ATPase which spans the

diameter of the membrane. For each protan pair passing 'through, the ATPase

enzyme generates one ATP molecule from ADP and H}PO,. Thus the potential

energy of the proton gradient is converted into the chemical energy of the 'high

energy' bond ATP (Mitchel, 1968).

201

•

1984). Increasing the temperature of incubation for different flasks, above the

optimal growth temperature, may have progressively increased the rate of

denaturation of certain essential enzymes and certain proteins of the cells,

resulting in death and cell lysis, especially at the longer time periods (van Demark

and Batzing 1987) .. Additionally, high temperatures may have activated the

autolytic enzymes of the cell, causing cell lysis. From Fig. 3.36, the narrow·

temperature range of 32°C - 42°C suggests that the biosynthesis of this antibiotic

may be highly thermoregulatory.

The various initial O.Ds. in different flasks, during incubation, showed that the

optimum antibiotic production occurred at the low initial O.D. of 0.06 (Figs. 3.37 -

3.39) and as the initial O.D. increased along the series, so the antibiotic

production fell to a cut off point at initial O.D., 0.1 (Fig. 3.39). Though the

critical factors here are not yet well understood (Smith and Calam, 1980; Calam,

1986), this phenomenon of high initial O.D. stemming antibiotic production may

have been associated with a low Ozfcell packed volume ratio and to toxic end

products. giving rise te partial NAD± feedback inhibition. Consequently, a low.,.

E.C. situation which favoured growth to the exclusion of antibiotic production

(strict sequence) may have occurred. (AJper et aI, 1978, Fig. 4.1) .. The reason

why no biphasicity occurred as the initial O.D. decreased along the series from

0.06, may have been due to a relatively higher Oz/cell pack volume ratio and a

relatively higher end-product dilution, both cases occurring on account of the

smaller numbers of cells present, especially at the earlier growth stages.

Subsequently, this must have given rise to better growth with no biphasicity and

to low level antibiotic production. In support of the existence of a sensitive role

. for nutritional end-products on growth and subsequently, on antibiotic production,

Martin and Demain (1980) pave revealed the existence of a mechanism, mediated

by cAMP and a specific activator protein, which is highly responsive to catabolite

concentrations. The mechanism, when activated,initiates the production of

substances which can depress carbon source metabolism. It is to be noted that

the fact that not much antibiotic production occurred in the first 24 hrs for initial

O.Ds 0.03 and 0.015 (Fig. 3.38), was probably directly due to the overall number

of antibiotic producing cells present during this time (Fig. 3.37).

202

All the above optimization studies seem to lend particular weight to the group of

workers who support the contention (as discussed at sectionl'3) that stress factors

are important initiators of antibiotic production. (Bruehl, et ai, 1969; Katz and

Demain, 1977; Martin and Demain, 1980; Nisbet and Porter, 1989).

The final optimized medium and conditions were as follows:

NH4CI, 0.6 gmlL; Nai50., 0.2 gm/L; KH2P04, 11.3 gm/L; KzHP04, 2.9 gmlL;

glucose, 3.0 gmlL; pH 6.0; 120 r.p.m. (orbital incubator); 37°C; 72 hrs and this

gave an inferred (log dose-response curve; Fig. 3.44) yield of 0.372 mg/ml. This

yield compares very favourably with other wild-type (non-mutated) optimization

studies (Takeda, 1958; White, et ai, 1986). A factorial increase of 9.3 starting

from the controlfcomplete' medium was achieved (Table 3.6). Variation of r.p.m.

(aeration/agitation) gave the greatest single increase in antibiotic production.

Stochastic errors of variation, inherent in the whole optimization procedure, gave

an O.D. coefficient of variation (C.V.) of 12.3%, a pH C.V. of 1.2%, a zone of

inhibition C.V. of 9.7% and a dose-response calibration curve assessment of

antibiotic concentrations in broth culture C.V. of 21.9%. Altogether these show

good significance (Sussman, 1964). The pH C.V. was particularly low because of .

the strong buffer used .

. A simple scale-up procedure from the orbital incubator. (50 ml in 250 ml conical

flask) to a '6 litre laboratory scale batch fermentor was adopted (Table 3.7 a & b)

because of the need to produce enough quantities of the antibiotic for chemical

analysis. ,This resulted in the optimum conditions of 150 tr/min (Rush ton

impeller), 4Umin (aeration), 72 hrs (incubation time) at 37°C and gave an

inferred yield of 0.038 mg/ml. This yield was poor when compared to the

optimized 'conical flask/orbital incubator. fermentation inferred yield of 0.372

mg/ml and thus reflects directly. on the simplicity of the scale-up procedure. It

- must be stressed, however, that because of the many critical factors that govern

antibiotic production (Fig. 1.5), even slight differences in fermentor design would

inevitably result in some loss of antibiotic production when moving from an

already optimized system (Trilli, 1986).

203

During the fermentation, a black plastic bag was secured around the glass vessel

of the fermenter to cut out sunlight in case the antibiotic was significantly

susceptible to photo-degradation (deleterious range = 290 to 450 nm; U.S.

Pharmacopoeia XIX 1975).

Downstream processing (Fig. 3.40 3.43, Table 3.8), involving

methanol/chloroform (1/9) for broth extraction, ethylacetate for TLC extraction

and hexane for trituation, gave rise" to a yellow crystalline product the purity of

which was confirmed by HPLC (retention time (Rt) 10.4 mins). The solventtrLC

. extraction route (Fig. 3.42) was the preferred extraction route as it gave rise to far

better percentage efficiency of extraction than the solvent/solvent extraction route

(Fig. 3.41): 62% against 13%. Furthermore it was far easier to manipulate.

During the quest for a suitable mobile and stationary phase to isolate the

antibiotic from the broth .culture, the Aszalos and Issaq flow chart (Aszalos and

Issaq, "1980), originally devised as an aid for the identification of established

antitumour agents, was used. The rationale behind using this was centered on the

premise that a solvent, capable of eluting the active substance up a. TLC plate, is

one that was also likely to extract the antibiotic from the broth. The ideal solvent

system to aim for would be one that was immiscible with the aqueous broth and

one that would extract the antibiotic with as least a number of co-extractives as

"" possible. Methanol/chloroform fulfilled these objectives quite well.

During the extraction experiments, a yellow/green, non-active fluorescent material

was often seen on the TLC plates when examined under U.V. light (Table 3.8,

Fig. 3.43). This may well have been a pseudobactin (siderophore) produced by

the cell to chelate trace quantities of Fe2+ from the basal medium. (Wendenbaum

et aI, 1983; Palleroni, 1986). It is interesting to note that in literature (Attafuah

and Bradbury, 1989), strain A is cited as belonging to a Pseudomonas group which

does not produce fluorescent pigments. To account for this anomaly, it is possible

that under these unusual/stressful conditions, a metabolic pathway which can

produce fluorescent pigments may have been triggered. This would suggest that

the existing biochemical test (King, E. O. et aI, 1954) for the ability of an

organism to produce fluorescent pigments, may not be accurate enough to account

for all cases. A similar case was recorded when Jamieson (1942) accidentally

204

triggered the production of blue/green pigments from organisms previously

considered to be Achromabacter spp. He later had to recommend a modification

in the classification of the microorganisms, by suggesting they be placed under

Pseudomonas spp. (Hellinger E., 1951).

Overall, TLC has traditionally been the preferred initial, small scale, extraction

route for this type of antibiotic research as it allows for quick and easy

solid/mobile phase manipulations and can give rise to very good product yields

(Calam et aI, 1986; Chan arid Aszalos, 1986). The final extraction yield of the

antibacterial substance obtained from the lab-scale fermenter was 236 mg/10L and

this compares very favourably with some wild-type bacteria lab-scale fermentation

extractions (Takeda R, 1958; Primrose S. B., 1987) .

. The calculation of percentage efficiency of extraction, (only made possible by the

log dose-response calibration curve) from the lab-scale fermentation, was 62%.

It is interesting to note that although the initial analytical TLC test elution with

solvent A, only eluted 1 spot (Table 3.8), this spot was further split into 5

components after ethylacetate elution (Fig. 3.42). The resulting antibacterial

antibiotic fraction from this, was again split into two further fractions with hexane.

All this clearly illustrates the renowned difficulties associated with isolation and

. purification which are often encountered by investigators working on unknown

compounds derived from novel (particularly natural) sources. Such difficulties

occur because the active broth can often present an extraordinarily complex

mixture of primary and secondary metabolites (some quite closely related),

occurring at concentrations ranging from nanograms to milligrams per millilitre

and in which only one of the compounds may be of interest (White, et aI, 1986) .

. Retention factor (Ri) determinations of the antibiotic in 9 different solvents,

covering an even cross-section of solvent polarities, gave a value of 0.67 for

methanol which was the best eluent, followed by 0.64 for solvent A

(methanol/chloroform) (Table 3.9). However, at the other end of the polarity

scale, hexane did not move the antibiotic spot thus showing why it was such a

good trituating agent. From all the above it was quite evident that the compound

possessed some polar characteristics. It shOlild be noted here that since only one

205 ..

. spot was seen eluted each time, in all the solvents used, this was a further

indication that the isolated antibiotic was pure.

pH/thermal stability tests (Table 3.10) revealed the antibiotic to be thermostable

at low pHs (pH 2,0) but unstable at high pHs (pH 9.0). This again suggests some

polar characteristics for the antibiotic and if later found to be non-toxic, would be

excellent for the preferred oral route, drug administration, mainly because it is

likely to be stable at the lowstomach pHs. Ultra-violet light stability tests (Table

3.11) showed comparable stability with the established drug, tetracycline and

therefore suggests acceptable U.V. stability for the antibiotic as a potential drug.

Minimum inhibitory concentration (MIC) tests (Table 3.12), against a number of

microorganisms, using an agar plate diffusion assay, indicted activity to be in the

pg/ml range for sensitive microorganisms (E. coli 1.3 pg/ml and ~. abony 1.0

pg/ml). Antibiotic challenge against the growth profiles of E. coli (Fig. 3.45) and

'in situ' subculturing, indicated a rapid onset, non-lytic, antibacterial activity. (Fig.

3.45). This therefore, suggests that the antibiotic is bacteriostatic.

During structure determination, qualitative chemical analytical tests, done on the

antibiotic, showed it did not contain a pyrrol ring (Kovac's test); it was not a

carbohydrate (anthrone test) but did contain a ketone (Feigl test); it did not

co~tain aldehyde· (SchiffsJFuschsin . test) nor did it contain an amino·· acid

(ninhydrin test); finally, it did not contain alkyl/aryl halides (formalin test).

It is possible from the uvMs spectrum (Fig. 3.47) that the antibiotic may contain

2-3 conjugated bonds in its structure as exemplified by two peaks below 300 nm

(Dudley and Fleming, 1987).

It was also found from KBr disc I.R. scans (Fig. 3.48) that the structure may

contain saturated C-H groups, 298OCm.l; secondary amide groups of unknown

origin, 312OCm.l; >C = N (imines) groups, 1690-1640,m.1 and conjugated cyclic

systems, 1660-1480,m-l. (Steele, 1971; Dudley and Fleming, 1987). Next, a local

search/comparison of the finger print region, with those of established antibiotics

. was made but this failed to reveal the identity of the compound. (British Pharm.

2116

I

I

I

I

I

I

I. R. Ref. Spectrar., 1980; Keller, R. J. (Sigma I. R. Library) 1986; Pouchart, C ..

J. (Aldrich I. R. Library), 1981).

Mass spectral studies suggested that the first electron/mass (e/m) ratio is 193

(measured accurate mass, 193.0599; Fig. 3.49) and it is therefore highly likely that

ihis, in actuality, is the mass number or atomic mass unit (a.m.u.) of the antibiotic ..

Also, from the list of the arithmetically possible atomic compositions of the

compound (Table 3.13), the ratio which had the least percentage deviation (-0.1

millimass unit (1 millimass unit =. 0.5 p.p.m.)) from the theoretical mass was C

(7), 13C(0), H(7), N(5), 0(2). From all the above considerations this elemental

ratio must strongly suggest the empirical formular (C,H7Ns0 2) of the compound

(Dudley and Fieming, 1987).

Bond lengths and angles (Tables 3.15 and 3.16) of the antibiotic crystal, which had

been very carefully grown in the most suitable solvent (chloroform) to avoid

imperfections such as twinning, were obtained from x-ray diffraction. The results

were used, together with an atomic numbering scheme, to generate the molecular

structure (Fig. 3.50). This final structure was obtained with a R factor of 7.3'(and

is therefore highly likely to be correct.

A structure search for the identity of the antibiotic revealed it to be xanthothricin

(1-6-dimethylpyrimido[5,4-e ]-1,2,4-triazine-5, 7(lH 6H)-dione), also known as

toxoflavin (Fig. 4.2), a powerful but toxic antibiotic (I.V. LDso = 1.7 mglkg and

oral LDso = 8.4 mg/lg against mice), only previously known to be produced by Ps.

cocovenenans and" a streptomyces spp. (van Veen and Mertens, 1934; van

Damme, et ai, 1960;" Bycroft, 1988). Ps. cocovenemins also produces a toxic

antifungal substance known as bonkrekic acid (Nugteren and Berends, 1957).

Although a Cambridge databank search in 1988 revealed that the structure had

not been confirmed by x-ray crystallography, a more recent search (1990) from the

same source, revealed that the structure was confirmed by x-ray crystallography

by the Russian, Aleksandrov et ai, (1986).

It is thought that xanthothricin's mode of action may involve it acting as a highly

efficient electron-carrier which provides a by-pass of the ETC, giving rise to lethal

207

concentrations of toxic end-products of Oz metabolism, such as HzOz (Latuassan

and Berands, 1961; Wolff, et aI, 1986; Kako, 1987). This· may explain why

anearobic ceIIs such as Cl. sporogenes and catalase-abundant ceIIs such as C.

albicans remained unaffected even at high concentrations (> 2000 /,g/ml)."

Fig. 4.2: Xanthothricin

o

[1,6-dim·ethyl pyrimido[ 5,4-e ]·1,2,4,.triazine·5, 7 (lH, 6H)·dione].

Similar structures of interest include the antibiotic, Fervenulin (a), obtained from

Streptomyces fervens (Daves et aI, 1961) and the chemotherapeutic agent

Xanthinol nicotinate (b). AIl 3 have similar methylalloxan rings (KeIIer, R. J.,

(Sigma I. R. Library), 1986).

A VCOOH

(a) (b)

208

Following the confirmation that apart from Ps. cocovenenans, strain A also

produced xanthothricin, it became important to ensure that these two

Pseudo monads are indeed different species and comparison tests between the two

organisms strongly suggested that they are indeed different (see section 4.1).

4.3 The effect of magnesium salt omission from the medium

The cell propagation experiments (Fig. 3.52) showed a visually assessed increase

in yellow pigmentation when moving from the SLR 'complete' basal medium to

. Med A2. Thereafter, subsequent propagations in SLR Med A2, resulted in

further pigment production at this increased level. Repeating the above

experiments with AR 'complete' basal medium, yellow pigmentation was found to

be of the same order as its counterpart in the SLR medium (,complete') but here,

a single propagation into AR Med A2 did not result in pigmentation though

similar growth was observed. Further propagation in AR Med A2 did not result

in growth. Clearly, since the latter medium's (AR Med A2) constituents were of

a higher quality, it was highly likely that there would be less Mg2+ from impurities

present than in its SLR counterpart (see appendix). From all this, it appears that

there may be, for this organism, a critical mass of Mg2+ necessary for propagation

of growth and increased pigmentation, and a critical mass for growth alone,

immediately below which growth stops. Such a point may have been reached ..

when the Mg2+ concentration was so depleted such that basic metabolic functions

would have been adversely affected.

From the atomic absorption measurements of Mg2+, in the SLR and AR media

and from Fig. 3.53,a summary of Mg2+ levels and its effects on strain A, at start

of first and second generation propagations in the two media types (SLR and

AR)' were tabulated. (Table 3.17). From all the above, it becomes clear that a

Mg2+ concentration of 0.140 ppm was needed to perpetuate peak antibiotic

production and a concentration of 0.102 ppm was needed to support growth

without detectable antibiotic production.· With reference to the latter

concentration, it is possible that this phenomenon was caused by the organism

being further stressed, on account of the reduced Mg2+ concentration and

consequently, priority of Mg2+ usage would have gone to growth and not antibiotic

209

production. These results are similar to those obtained by Grossowicz et al (1957)

in their work on the effect of MgS04 on pyocynine production in Ps. aeruginosa.

Here, it was obselVed that a critical mass of Mg2+ was necessary. for peak

pyocynine production. Furthermore, Young and Kempe (1984) showed whilst

investigating the effect of lincomycin production in Streptomyces lincolnensis that

as initial concentrations of Mg2+ was reduced along a series of shake flasks,

lincomicin production fell but this fall colorated with increases in the biomass.

Using tM. atomic absorption spectometer, the Mg2+ content/mg of washed cells,

previously grown in basal medium was compared to those grown in Med Al. It

was found that for the former, the cells had accumulated a Mg2+ concentration

of 0.31 pprn/mg of cells and the latter,0.11 ppm/mg. These results suggest that

the cells may have an active uptake mechanism for Mg2+.

From all the studies done above it becomes clear that strain A seems to be a far

more efficient scavenger of Mg2+ then either Ps. aeruginosa (control in the SLR

propagation experiments) or Ps. cocovenenans (Fig. 3.51 a?d 3.52 respectively).

In view of all this, it is possible that strain A may have an industrial or scientific

research application where powerful microbial scavengers of Mg2+ are required ,

but further research may be needed to confirm this.

The cell viablity growth cUlVe was constructed to determine how the drop in Mg2+

was affecting the true growth situation. From Fig. 3.54, the control/complete basal

medium shows a short declining stationary phase before a steep decline phase.

The shortness of this stationary phase is probably due to the poverty of the

complete basal medium. However, with Med Al, no stationary phase was

obselVed but excellent growth occurred followed by a sharp fall. This

phenomenon where excellent growth occurs as Mg2+ concentration is decreased

to a cut off point has also been obselVed by Young and Kempe (1984). It may

be argued here that the inevitable reduction in already low Mg2+ with time, thus

bringing the microorganism nearer crisis point may have resulted in a stimulation

in some metabolic processes, giving rise to increased antibiotic production. This

stimulation in metabolism may have been due to the low Mg2+ concentrations

which, as has been reported, enhances the permeability of the cell (Brown, 1975;

210 .

Benz and Hancock, 1981; Yoshimura and Nikaido, 1982; Nicas and Hancock,

1983), and as such, may have resulted in an increase in uptake of nutrients

(Leive, 1965). Consequently, by 36 hrs. (Fig. 3.54), the remaining Mg2+

concentration in Med A2 may have been too low to sustain the microorganism

into stationary phase, thus leading to rapid death. This may have been principally

because Mg2+ are important for ribosomal stability, are critical as co-factors and

are involved in all phosphorylating enzymes, including the enzyme that catalyses .

the conversion of the energy rich molecules, ADP to ATP, which are essential for

metabolism (Lipman, 1941; Alberty, 1968; van Demark and Batzing, 1987).

4.4 Antifungal activity studies

Although cultures grown in PDLM made from Oxoid PDA batch No. 156 21497

gave rise to very large zones of inhibition (Fig. 3.55), Med q was preferred

because it is chemically defined and therefore permited medium component study.

Furthermore, PDLM, per se, proved inconsistent: PDLM made from Oxoid PDA

lot No. 070 40305, and from Leicestershire grown King Edwards' potatoes, both

failed to elicit antibiotic production (Table 3.5). This inconsistency may have been

due to the fact that the production of some antibiotics is acutely sensitive to the

presence/concentration of trace elements. Thus potatoes grown in different

. localities, .. therefore different soil types, .. may accumulate different

. arrays/concentrations of trace elements and thereby affect production .. Many

examples of this erratic elicitation phenomenon exist (Foster, 1943; Chesters and

Rolinson,1951; Katz, 1958; Kurachi,1958; Weinberg and Tonnis, 1966) and can

have serious implications to do with loss of revenue in industry, where complex

media fermentations are the norm. (TriIIi, 1986).

It should be noted that when Ps. aeruginosa was grown in PDLM batch No. 156

21497, it did not, unlike strain A, give rise to zones of inhibition but it did

however, inhibit A. niger invasive growth, on PDA of the same batch,to the same

degree (Table. 3.4). Here, it could well be that the biosynthetic pathways

responsible for producing strain A's particular antifungal compound does not exist

in Ps. aeruginosa, or that being a different organism, different factors were needed

211

------------ ------ ------

to trigger prOduction, or that some antifungal production may have occurred but

the assay method used was not sensitive enough to detect it.

A preliminary heat stability test (20 mins at 100°C) on the antifungal activity of

the brOth (section 3.5.2) resulted in a 5.6% loss in activity. Like the antibacterial

stability test (section 3.2.2a), this low value again suggested that the active

principal was prObably not an enzyme, toxin or similar prOteinacious substance but

rather may be a substance of some interest and therefore worthy of pursuit.

The pigmented inoculum orbital incubator experiment, using Med q, much like its

basal medium counterpart, gave rise to greater zones of inhibition than the

'washed' inoculum situation. The probable reasons for this phenomenon, which

includes reduction of pigment and consequent enhanced uptake of O2 frOm

agitation, as already discussed above (section 4.2), would also be applicable here.

However, unlike the static incubation experiment of the basal medium where no

detectable antibiotic production occurred,' _ Med q gave both antifungal and

antibacterial zones of inhibition after 10 days and further static incubation

increased pigmentation. The probable explanation for the pigmented inoculum

basal medium experiment as already discussed above, may have been in part,

applicable here. However, in addition to this, a respiriltory prOcess, known as

dissimilatory nitrate reduction, involving -conversion of nitrate to nitrite and-·

involving electrOn and hydrogen respiratory carriers, may have ultimately led to

relatively more ATP prOduced to initiate detectable levels of antibiotic production

(Palleroni, 1986; van Demark and Batzing, 1987). (Refer Table 3.2 for nitrate

reduction biochemical test). Static incubation in Med q with 'washed' cells

however, did not give rise to zones of inhibition and this must therefore have been

solely due to the lack of pigmented in the inoculum, as already discussed.

Optimization studies based on the same rationale as previously (medium

component single del ... tion and sequential variation of essential nutrients for

antibiotic prOduction; section 4.2) but this time using Med q, gave results which

were similar to ones achieved with the basal medium study, in that, trace

quantities of Mg2+ and low nitrogen concentrations, both significantly increased

prOduction (Figs. 3.58 and 3.59;', Fig. 3.61 and 3.62).

212

The medium component single deletion (MCSD) experiments, revealed that Med

B3 enabled fairly good relative growth (O.D. 0.49 in 24 hrs) of strain A (Fig.

3.57). Like Mgl+' Fe2+ is traditionally termed a macronutrient and is normally

needed in significant quantities (10.3 - lO-4M) being a key component of heame

containing respiratory enzymes (Mandelstam et aI, 1973; van Demark and

Batzing, 1987). This growth may have therefore been due to the fact that certain

Pseudomonas of which strain A could be one, are capable of producing low

molecular weight Fe2+ chelates known as pseudobactins (siderophores). They are

secreted into the medium in times of low Fe concentrations, chelate trace

quantities of Fe present and transport the resultant complex back into the cell via

specific receptors (Wendenbaum, et aI, 1983; Moores, et aI, 1984; Palleroni,

1986; . van Demark and Batzing 1987). Furthermore, comparing the. curves of

Med Band B3 at Fig. 3.57 and MedB and B3 at Figs. 3.58 and 3.59, where

growth alone occurs in the latter (Med B3), suggests that Fe may be important in

the biosynthesis of the antifungal substance.

Omission of K+ from the medium recipe, (Med B4), also permitted growth of

strain A (Curve of Med B4 at Fig. 3.57). Since potassium is also termed a

macronutrient activating enzymes involved in the formation of peptide bonds

during protein synthesis (Mandelstam and McQuillen, 1973; van Demark and

.. Batzing, 1987) this' may indicate like' for Mgl+ omission; a receptor mediated .

active uptake mechanism for strain A. Here again, like for Mgl+ omission, the

probable stress imposed upon the organism as a consequence, may have resulted

in the production of detectable amounts of both antibiotic types. (Med B4 at

Figs. 3.58 and 3.59).

Analysis of Fig. 3.57, showing growth of strain A in Med BS and Med B6 which

was relatively r pid in the former, indicates that in the complete medium, the

organism would metabolise sucrose first (rapid) before synthesizing new enzymes

to metabolise glycerol (slow). This observation where one carbon source is first

metabolised before the other is well documented (Drew and Demain, 1977;

Martin and Demain, 1980). Also, a pattern seems to be emerging where

antibiotic production occurs at rise in the secondary log phase of biphasic curves.

This phenomenon is supported by the findings of Aharonwitz and Demain 1978

213

From the nitrogen (NaN03) variation experiment (Fig. 3.60), the 0.2% curve

appeared to have the best overall growth, rising to 0.0. 1.68 in 96 hrs and ending

at 0.0. 1.44 in 192 hrs. As the concentration was increased from here, from flask

to flask, overall growth was slightly reduced and this may have been due to

substrate inhibition. As the concentration was decreased, there was a tendency

towards biphasicity with its best expression at 0.05%. In the flask where NaN03

had been omitted from the medium recipe (0.0%), the 0.0. gradually rose to

0.147 in 96 hrs, reducing thereafter to 0.0. 0.08 in 144 hrs. (NB initial OD was

0.9). This small rise in 0.0. may have been due to trace nitrogenous compounds

. from impurities in the SLR constituted medium causing some growth.

All the above may be explained by postulating that initially, as the first carbon

source was being metabolised, this may have resulted in an increase in

intracellular concentrations of ATP, (Fig. 4.1). After giving up its energy to the

cell, ATP eventually turns to cAMP and therefore, a stage would arrive when with

a depleting first carbon source, the cAMP level would have risen to quite high

levels. However, this stage may not have been solely brought about by a depleting

first carbon source (c.f. Fig. 3.57. Med B6 curve, where 0.0. rose to 0.838 in 12

hrs): end-product inhibition may also have played a part here, sending the curve

into a mid-Iag phase at 0.0. 0.2. Here, stages 3 and 4 of Fig. 4.1 might have then

been followed With particular reference to second carbon source metabolism. The

period of time accounting for the mid-Iag phase may represent the time taken for

the initiation and synthesis of enzymes necessary for the new metabolic pathway

(Fig. 3.65).

The fact that the lower nitrate concentration of 0.05% gave rise to optimal

antibacterial and antifungal production strongly suggests that after the two step

nitrate reduction to ammonia during assimilatory nitrate reduction (Hartingsvelt

et aI, 1971; Sias et aI, 1980) there occurs a predominance of the GS-GOGAT

enzyme system over the GDH enzyme system. As already mentioned, the 0.2%

nitrate concentration was optimal for growth and thus, from Figs. 3.63 and 3.64,

one gets very clear evidence in support of nutritional stress encouraging antibiotic

production. This is because on moving away from 0.2%, antifungal activity

become more evident outside the range, 0.1 - 0.3% and this was also true for

214

antibacterial activity but to a lesser degree, outside the range, 0.15 - 0.25%.

Clearly this property of low nitrate concentration, stimulating the antibiotic

production of the microorganism, would, under appropriate conditions in the wild,

be of significant survival advantage.

FoIloWing the gradual trend in loss of biphasity from the 3% to 2% to 1% flasks

. in the sucrose concentration variation experiments, it is possible that at the latter

concentration, an early, smaIl and unfeatured biphasic depression may have

triggered, as previously described, antifungal production which was eventuaIly

detected at the start of stationary phase (Figs. 3.66 and 3.68). It is also possible

that as the organism entered stationary phase, production was triggered by stress,

having gone through stages 1-3 of Fig. 4.1. Such a confluence of log and mid-

. stationary phase has already been reported in 2 carbon source antibiotic

production studies (Pirt et aI, 1967; Lurie, et aI, 1975; Froyshov, 1978).

From the 0.5% and 0.0% sucrose (NB glycerol also present at 5%) media curves

(Fig. 3.66 - 3.68) it can be said that in general, during biphasic growth, antifungal

production seems to occur before antibacterial production. However, during

conventional growth, the reverse is true. These observations contradict the

popularily held view that there is always a strict sequence governing the

production of differeIlt antibiotics by an organism and clearly, this sequence would

not work simply· via set differences in binding affinities of different antibiotic

activator proteins for cAMP (AJper and Ames, 1978; Aharonowitz, 1980).

During the glycerol variation experiment (under orbital incubation conditions)

antibacterial activity was not detected in media containing concentrations of

glycerol below 4% (Fig. 3.71 and 3.73). However, for antifungal activity, there was

no detection of zones below 2%. As the concentration of glycerol was

progressively increased from 0%, stress caused by substrate inhibition, end-product

inhibition and catabolite repression, with increasing time, may have promoted

progressively earlier glycerol metabolism 'switch over' (stage 1-3 of Fig. 4.1) which,

as previously discussed, always seemed to concur with initiation of antibiotic

production. As a result, this may have given rise to the step by step appearance

of first, antifungal activity and then antibacterial activity. (Figs. 3.73 & 3.74).

215

The pH variation gave optimum growth and antibiotic production at the initial

medium pH of 8. As Med q was being sequentially optimized from the NaN03

variation experiment, a clear biphasic trend produced by strain A in successive"

'carry forward' media was seen and this is again evident, albeit not so pronounced,

in the medium of initial pH 8 (Figs. 3.65, 3.69 and 3.75). Another biphasic trerid

is also clearly evident when varying the pH from 6 - 8 (Fig. 3.76). This latter,

apparent biphasicity (120 - 192 hrs) may have signified a 'switch over' to glycerol

metabolism with its customary 'switch on' of antibiotic production and this is

supported by the late occurrence of a zone of inhibition of 25.4 mm at 192 hrs.

(Fig. 3.80). The pH profile here (initially pH8) dropped sharply to levels below

5 before rising and levelling above 5, with time. This drop in pH was a clear

indication of rapid sucrose (first carbon source) metabolism, resulting in excess

acid production (MacFaddin, 1983) which may have consequently caused the weak

buffer to collapse. The rapid growth may have been due to an initially adverse

pH environment, resulting in an influx into the cells, of just enough undissociated

bases to stimulate general metabolism (van Oemark and Batzing 1987). This is

in stark contrast to the basal medium situation which gave excellent growth at pH

6 (0.0. of 2.0 at 24 hrs) but poor overall growth at pH 8 (Fig. 3.27). Here, it is

possible that differences in media co~position may have been solely responsible

for bringing about this disparity in growth and antibiotic production. Further

" increases in the initial pH in the different flasks resulted in a suppression in both

growth and antibiotic production and this must have been due to the toxic effects

of increased levels of undissociated bases entering the cells.

Unlike the basal medium r.p.m. variation experiment, static incubation in this

"medium gave fairly good growth (0.0 0.603 in 72 hrs, Fig. 3.81) with detectable

antifungal production (17.8 mm in 168 hrs, Fig. 3.82». This may have been due

to additional ATP production from dissimilatory nitrate reduction of the NaN03

present in the medium and probably to undissociated bases entering the cell,

causing stimulation of general metabolism. The former postulation is supported

by the fact that the biochemical test study (Table 3.7), indicates the organism to

be capable of reducing nitrate to nitrite, giving rise to free N2 with extra ATP

obtained via dissilimatory respiration (MacFaddin, 1983; Palleroni, 1986; van

Oemark and Batzing, 1987).

216

Like Fig. 3.63 and Fig. 3.64 for nitrogen variation, Fig. 3.83 for aeration/agitation

may also support stress factor triggering of antibiotic production with 90 r.p.m.

being least stressful. Here, with relatively more oxygenation than the 60 r. p.m.

situation, the dynamic equilibrium of NAD+ (feed back inhibition) to ATP

formation/use would have tipped the scale in favour of the latter, thus initiating

a late mid-stationary phase, (Fig. 3.81; Fig.'4.1). As a result, more cAMP may

have been available to initiate a 'switch over' to glycerol metabolism and then

trigger the creation of antibiotic biosynthetic pathways. The 120 r.p.m. situation

(Fig. 82) showed better antibiotic production and to explain this, it is possible that

. at the lag phase, a higher stress may have triggered more production of antifungal

biosynthetic enzymes and this may have given rise to a higher rate of pre-second

log phase antifungal production. During this production, more A TP would have

been used, leading to more cAMP available to mop up any excess NAD+. This

would have prevented NAD+ feedback inhibition and thereby maintain, virtually

. unhindered, growth. NAD+ feedback inhibition of growth may however have

occurred later, between 168 and 192 hrs (Fig. 3.82), triggering a 'switch over' to

glycerol metabolism and thus initiating events which would have ultimately led to

a significant increase in antifungal production.

A similar postulation may be attributed to growth at 150 and 180 r.p.m. but here,

. unlike 120 r.p.m., no sign of biphasicity was obseIVed and this was supported by·

the absence of a steep rise in antifungal activity between 168 to 192 hrs (Fig.

3.82). Consequently, it is possible that stage 3 of Fig. 4.1 may not have been

activated. It is interesting to note that at 180 r.p.m., antifungal activity was only

first detected at 72 hrs. This may have been due to a prolonged but unfeatured

primary lag phase, probably caused by NAD+ feedback inhibition (stress) which

may have staggered pre-second log phase antifungal production. The 210 r.p.m.

CUIVe shows a 24 hrs lag phase before good growth 0.997 in 72 hrs (Fig. 3.81).

This may have been due to stress caused by excess aeration/agitation, leading to

strong or prolonged NAD+ feedback inhibition. The excess NAD+ would have

been eventually moped up by the cAMP, resulting in a high E.C. situation and

subsequently, rapid growth would have ensued. However, because of the high

oxygenation, producing high NAD+ levels, any available cAMP from the resulting

217

growth metabolism would have been phosphorylated by NAD+ thus preventing the

ceIls metabolism entering stage 3 of Fig. 4.1 to initiate antibiotic production.

Although temperature variation gave optimum growth of strain A in medium 6 at

32°C., overaIl, antibiotic production was best at 37°C. (Figs. 3.85 &3.86). This

may have been due to stress, on account of the non-optimum growth conditions.

The 42°C O.D;curve rose to 0.733 in 24 hrs. then feIl steeply to 0.281 in 72 hrs,

before levelling out to 216 hrs (Fig. 3.85). This faIl would have been principaIly

due to the high temperature, which may have caused induction of autolytic

enzymes within the ceIl, although stress caused by nutrient limitation, end-product

inhibition and the high agitation may also have played a part. After 72 hrs at

42°C, a peculiar malodour was emitted from the flask and this may weIl have

resulted from ceIl lysis. It is interesting to note that the organism was able to

. grow fairly weIl at this temperature in the modified Cruickshank's medium (Fig.

3.34). CeIl aggregation was seen at 22°C and 27°C, from 72 hrs onwards and as

previously discussed (pagel'~, this may have been due to nutritional and or low

temperature stress. It is also interesting to note that in contrast to the modified

Cruickshank's medium, no ceIl aggregation occurred at 32°C and this may have

been due to differences in media composition.

Again, all the optimization studies above seem to lend particular weight to the

group of workers, who support the contentionthilt stress factors~a.re important

initiators of antibiotic production. (Bruehl, et al, 1969; Katz and Demain, 1977;

Martin and Demain, 1980; Nisbet and Porter, 1989).

The final optimal medium and conditions for antifungal activity were as foIlows: . . NaN03, 0.5 gm/L; KCl, 0.5 gm/L; NaHzPO •. 2HzO 0.5 gm/L; FeSO., 0.01 gm/L; .

sucrose, 30 gm/L; glycerol 20 mIlL; pH 8; 120 r.p.m. (orbital incubator); 37°C;

216 hrs.

A percentage increase of 109 in antifungal activity was achieved during the orbital

incubator optimization (Table 3.18). Increase in antibiotic production here, was

measured by percentage increase in zone of inhibition instead of the factorial

computation used in the basal medium fermentation (Table 3.6). This was done

because it was impossible to obtain an antibiotic concentration value via log dose-

218

response curve: .it was later found out that more than one antifungal compound .. .

was present (Fig. 3.92). Like the basal medium fermentation, variation of r.p.m.

(aeration/agitation) here, gave the greatest single increase in zone size.

Stochastic errors inherent in the whole orbital incubator optimization procedure,

gave an O.D. C.V. of 33%, a pH C.V. of 7.3%, an antibacterial zone of inhibition

C.V. of 11:2% and an antifungal zone of inhibition C.V. of 12.4%.· The high C.V.

value of the O.Ds., indicating inconsistent growth, may have been due to the fact

that the prototype medium was based on C~e.k Dox I..iquid Medium which was

primarily designed for good fungal growth (Smith and Onions, 1983) and not for

good bacterial growth. From this prototype, the medium was developed with a

view to increasing antifungal activity, however good consistent growth also

eventually resulted. Stochastic errors for the final optimized medium gave an

O.D. C.V. of 6.9%, a pH C.V. of 2.8% and an antifungal zone of inhibition C.V.

of 4%, grown at pH 8 and at 120 r.p.m.

A simple scale-up procedure, from the orbital incubator to the laboratory scale

fermentor, resulted in the optimum conditions of 100 tr/min (Rushton impeller),

4Umin (aeration), at 37°C in 144 hrs. (Table 3:19 a and b). Again, the

differences between the 6-litre fermentor conditions and the 250 ml conical

flask/orbital incubator condition, highlighted the problems associate~with scale-up

and how sensitive antibiotic production can be when subjected to changing

physical parameters.

-Downstream processing, based on the same rationale used previously, (section

4.2), gave acetone/chloroform· (1/9) for broth extraction and ethanol,

acetone/chloroform (1/9) and acetone/chloroform (1/9) again for TLC extraction

(Fig. 3.89 - 3.92, Table 3.20). This resulted in one yellow crystalline antibacterial

substance, AB1 and 3, light yellow, amorphous antifungal substances, AF1, AF2,

and AF3, whose purity were subsequently confirmed by G:C. (Rt 12.8 mins, 16.3

mins and 21.8 mins respectively).

The yields of the antifungal antibiotics, obtained from the lab-scale fermentor

after brothtrLC extractions, were 50 mg/lOL for AF1, 19 mg/10L for AF2 and 19

219

mg/l0L for AF3. AFl's final yield compares favourably with yields obtained from

some wild-type bacteria lab-scale fermentations. (Takeda, 1958; White, et ai,

1986). Antibiotic AF2 and AF3 yields were however poor and this was probably

due to the medium optimization path inadvertently taken which was selectively

optimal for AFl. The extraction procedure may also have adversely affected final

yields. Much higher antibiotic concentrations must have been obtained in the

orbital incubator optimization flasks as shown by the disparity between maximum

zones of inhibition, arising from the 2 fermentation systems: 35.5 mm (orbital

incubator) and 20.5 mi'n (lab-scale fermentor).

AFl was tested for MIC against a range of microorganisms (Table 3.21) and this

generally revealed fairly strong antifungal activity but also revealed some potent

activity against the Gram positive bacterium, s.. aureus, 'a peculiar characteristic

shared with the imidazole drugs (Edwards, 1980). The antibiotic was clearly most

potent against T. mentagrophytes, the causative organism for athletes foot (Fig.

3.93). Further incubation of the agar diffusion MIC assay plates (5 days), reduced

the initial zone of inhibition sizes and this suggests that the antibiotic may well be

unstable and or fungistatic (Fig. 3.93 - 96). Though the solvent extraction had

revealed possible polar characteristics of the antibiotic (on account of its

dissolution in the more polar solvents), it was not easily miscible with water which,

in relation to other solvents, is highly polar.- This immiscibility with water is

characteristic of many antifungal compounds and can make their MIC

determinations particularly difficult especially with the tube dilution technique

(Conner, 1986).

The original zones of all the 3 antifungal antibiotics, against Co albicans, after 24

hrs, were 21.3 mm, 24.6 mm and 23.3 mm for AFl, AF2 and AF3 respectively.

However, after 4 days incubation, at 30°C, only AF2's zone more or less

maintained its size with a clear cut circumference, whilst the other 2 zones had

virtually been overgrown. (Fig. 3.97). This strongly suggests that AF2 may be

fungicidal, and AFl and AF2, fungistatic.

Preliminary structural analysis, including IR/UV spectroscopy and low resolution

mass spectrometry (elm = 193), suggests that the antibacterial substance, ABl,

220

was xanthothricin (toxoflavin). Preliminary structural analysis of the 3 antifungal

compounds, including IRJUV spectroscopy and !H-NMR (AF1 only), were done.

The uv/vis spectra of the 3 antifungal compounds, dissolved in UV ethanol and

shown at Figs 3.98 ~ 100, suggests some conjugation in their structures and it is

known that compounds having one or only a few bands below about 300 nm

probably contain only 2 or3 conjugated units (Dudley and Fleming, 1987). This

seems to apply to antibiotic AF1 and AF3. It is also known that a spectrum with

many peaks stretching into the visible region i.e. from 200 to 400 nm, shows the (hoJl\

presence of a long conjugatedLor polycyclic aromatic chromophore (Edwards,

1980; Dudley and Fleming, 1987) and this seems to apply to antibiotic AF2.

Furthermore, this type of spectrum (AF2) appears to be typical of polyene

antibiotics which may either be of the linearly conjugated acyclic type or macrolide

type (Dinya and Sztaricskai, 1986). It also appears to have some polyene

antifungal characteristics, namely, it is light yellow, amorphous, fungicidal,

sparingly soluble to insoluble in H20 and has a polar/apolar quality (Edwards,

1980).

An I.R. spectrum of AF1, shown at Fig. 3.101, with bands at 752.6 cm-! and 698.5

cm-I, suggests that the structure may contain a mono-substituted ring system.

Further evidence of this is given by bands between 2000 - 1600 cm-! which

although weak, are typical of mono-substituted ring systems. The bands at 3540

cm-! to 3428 cm-! suggest OH groups. The band at 1457.2 cm-! may indicate CH2

or CH) groups and the bands at 2924 cm-! and 2852 cm-! suggest the presence of

saturated aliphatic C-H groups. The band at 752:6 cm-! may be due to a chain of

CH2 units. It is to be noted that there is no absorption in the CH) umbrella

region (1400 - 1350 cm-I) and this'suggests absence of CH) terminal groups

(Steele, 1971; Dudley and Fleming, 1987) Fig. 3.102 shows the NMR spectrum

of AFl. The chemical shifts at 7.4c5(d) and 7.2 c5(d) indicate aromaticity (Ar - H).

Since the doublets do not 'lean' towards each other it may not be correct to

assume that the ring is disubstituted. From the I.R., it appears mono-substituted.

The chemical shifts between 3.8 • 4.3 c5(q) may be Ar-CH2-O-, 0-CH2-O-, Ar-OH,

C-OH; at 1.9 c5(d) may be R-CH2-Ar; at 1.25 c5(m) may be R~CH2-CO, -CH2; at 0.6

c5(d) may be C-OH, C-NH. The integration curves gave a proton ratio of 2:1:1:2

and from this if one is to proceed with mono-substitution, a more realistic proton

221

distribution may be 5:2:2:5. ((Dudley and F1eming, 1987; Sorrell, 1988). From

all the above it is possible that AFl may contain a mono-substituted benzene with

CHi chain and terminal hydroxyl groups. It is to be stressed however that these

findings are tentative and that further work need to be done before· a more ,

comprehensive idea of the structure is known.

The I.R. spectrum of AF2, shown at Fig. 3.103, suggests that the compound may

be non-aromatic, with no clear bands between 1600 - 1500 cm-I; may posses 1,2-

diketones, S trans open chains, 1732 cm-I; may possess CH .. groups,1454 cm-I and

. terminal CH3 groups,1376 cm-I; may possess hydroxyl groups, 3364 cm-I and or

alcohol (C - OH) groups, 1066 cm-I (Steele, 1971; Dudley and F1eming, 1987). All

the above suggests that the antibiotic may be a polyene antifungal compound

(characteristic uvMs scan) of the linearly conjugated acyclic type but again these

postulations are tentative and further work needs to be done on structure

elucidation.

The I.R. spectrum of AF3, shown at Fig. 3.104, suggests that the compound may

possess hydroxyl groups, 3300 cm-I; may possess CH3 and CHz groups, 2920 -

2850 cm-l, supported by 1462 cm-I; but with 1880 cm-I not present, suggests no

terminal CH3 group; may be an aliphatic aldehyde, 2920 cm-I and 2852 cm-I

--- -- ·(ferriiidoublt~t), 1420 cm-I and overlap of the CH stretching bands and may

suggest carboxyl groups, 1732 cm-I. No bands at 750 - 720 cm-I suggests absence

of a chain of 3 or more CHz units (Steele, 1971; Dudley and F1eming, 1987;

Lambert, 1987). Again postulations here are tentative and further work needs to

be done on structure determination.

The present study has established that Ps. antimicrobica (strain A) is an unusual

microorganism. With its ability to· sustain growth under limited Mg2+

concentrations and now being identified with a select group of acidophilic

pseudomonads, it is of potential interest to researchers. Furthermore, with its

array of four antibiotics, Ps. antimicrobica is a microorganism of commercial

interest.

222

4.5 Conclusion/futuretrends

Morphological, biochemical/tolerance and antibiotic producing properties obtained

from the present study tend to support the contention that the two strains A and

B are members of a new species of Pseudomonas. Both strains seem to be slow

growers in basal medium (mgt of 170 ± 6 mins for strain A and mgt of 150 ± 5

mins for strain B) but growth can be stimulated with the addition of phenylalanine

and histidine for strain A and methionine, ornithine, arginine and lysine for strain A ~J

B (mgt reduction of 77 ± 6 mins for strainlmgt reduction of 53 ± 5 mins for

strain B). Acidophibtests for strain A has revealed for the first time that it should

now become the fifth member of a select group of Pseudomonas, all of which

share an ability to grow at pH 4.0.

Four liquid media, able to elicit antibiotic production from strain A were

developed: a chemically defined medium (CDM) for antibacterial activity, a CDM

and a complex medium for antifungal activity, and a CDM for both antibacterial

and antifungal activity~

Optimization of antibacterial and antifungal activities increased yield, by a factor

of x 9 and by 109%, respectively. Nitrogen and magnesium limitation increased

yields and from these optitnization studies, there is clear evidence to support the

contention that stress factors are important initiators of antibiotic production. A

probable scheme for the regulation of antibiotic production is proposed and this

has been constructed in light of all the antibiotic studies done and from current

knowledge of the biochemistry/genetics of antibiotic regulation.

The medium component single deletion 'of MgSO. (MCSD-Mg+2) from the

medium recipe, w\Tue excellent growth" and antibiotic production occurred, was

investigated. From this, it is possible that strain A may be an efficient scavenger

of Mg2+ and may in addition, be able to subsist on low levels of Mg2+.

Three procedures for the isolation of the active substances have been developed:

two for an antibacterial substance, AB 1 and the other for the antifungal

substances, Afl, AF2 and AF3. With the· aid of a log-dose response curve, a

223 I

I

percentage efficiency of extraction of 62, from the broth to the crystals, was·

obtained for AB!.

Agar diffusion, M.I.C. assays for the compounds, indicate activity to be in the

pg/ml range for. sensitive microorganisms. Antibiotic challenge, against test

microorganisms suggest bacteriostatic activity for ABl, fungistatic activity for AFl

and AF3,and fungicidal activity for AF2.

Structure determination involving spectral analysis and X-ray diffraction indicated

ABl to be 1,6- dimethylpyrimido[S,4-ej-l,2,4-triazine-S,7 (1.H, 6H)-dione

(Xanthothricin) a toxic metabolite previously detected in foods contaminated with

&.. cocovenenans. Selective media developed for strains A and B did not support

growth of Ps. cocovenenans. Preliminary structural analysis suggests that AFl may

possess a monosubstituted ring system with CH2 chain and terminal hydroxyl

groups; that AF2 may belong to the polyene group of antifungal antibiotics and

that AF3 may be an aliphatic aldehyde with hydroxyl groups.

Further work needs to be carried out for complete structure elucidation of the 3

• antifungal compounds. A neat way to do this would be first to establish a

functional group on the antibiotic then to derivatise this group with a known

. reagent which upon combination would form a. crystalline product .. This crystal

should then be subjected to X-ray crystallography ... From the final structure

obtained here, the structure of the known reagent should be subtracted, leaving

behind the structure of the unknown.

If novelty is established, then toxicity tests (Tissue Culture MIC and LDso),

stability tests and efficacy against a whole range of microorganisms, including

. viruses and cancer cells, should be done.

The ability of strain A to grow in limited Mg2+ concentrations should be further

investigated· and this may involve continuous culture and electron-microscopic

examination of the ribosomes.

224

Strain B should also be investigated, as per strain A, to see if different antibiotics

are produced.

225

APPENDIX ,

TRACE ELEMENT COMPOSITION OF MEDIA CONSTITUENTS.

a) Medium for antibacterial substance production

Constituents Trace NH4Ci NazSO. K1H2PO. K2HPO. Glucose substances

Mg 5 ppm 50ppm 50ppm 5 ppm -Nitrogenous - 7 ppm 30ppm 10ppm -compounds

P04 . 5 ppm 10ppm - - -Pb 3 ppm 2ppm 5 ppm· 5 ppm 1 ppm

Fe 2ppm 2ppm 20ppm 5ppm 2ppm

Cu 1 ppm 2ppm 10ppm 5 ppm -Ca 10 ppm 50ppm 100 ppm 50ppm . -

b) Medium for antifungal substance/s production

Constituents Trace NaN03 KCl FeS04 NaH2P04 . Sucrose Glycerol substances

Mg 12ppm ~

150 ppm 35 ppm 10 ppm - -Nitrogenous - 10 ppm 10ppm - 20ppm -compounds

.

PO. 5 ppm - 5 ppm - - -Pb 5 ppm 2ppm 25 ppm 10ppm 1 ppm 1 ppm

Fe 3 ppm 3 ppm --- ...

5 ppm 1 ppm 2ppm

. Cu - 2ppm 10ppm - 1 ppm -Ca 12ppm 10 ppm 150 ppm 35 ppm - -Na - 500 ppm 250 ppm - - -K - - 50ppm 200 ppm· . - -

The trace element values of individual medium constituents were of AR quality and were obtained from their respective container labels.

226

EXTRACTED FROM THE PROCEEDINGS OFTHE 16.h INTERNATIONAL CONGRESS OFCHEMOTHERAPY,.JUNE 1989 ISRAEL

Production and characterisation of an antibacterial agent from a pseudomonad isol.ted from an insect vector.

E. Attafuah, W.G. Salt· and R.J. Stretton.

·couespondence

Microbiology Unit, Chemistry Department, University of Technology, Loughborough, -Leicestershire, UK. -

Antimicrobial activity has been demonstrated in cultures of a pseudomonad isolated from an insect vector. The antibacterial agent has been isolated and identified as xanthothricin (toxoflavin) and its production and isolation optimised.

Pseudomonas sp. (NCIB 9897) which produces yellow pIgments in a range of media and which was originally isolated from the gut of the insect virus vector Planococcoides njalensis has been reported to produce both antibacterial and antifungal compounds (1). The organism may warrant separate species status (2). This communication reports investigations of the production and nature of the antibacterial agent.

The organism was initially grown (370C; orbital incubator) in a defined medium, in which preliminary experiments had established growth with good yield. The medium (lOOml batches) contained (g/l): ammonium chloride, 3; magnesium chloride, 0:2; sodium sulphate, 2; glucose, 3; all in 0.2M (potassium) phosphate buffer (final pH 7.2). When necessary the medium was solidified by the addition of 2\w/v agar. Antimicrobial activity in culture supernatant fluid was assessed at various times

'by·the-plate diffusion method using a range of microorganisms to detect activity via growth inhibition zones. Medium composition was varied by single component deletion/addition to produce' a range of media, in each of which the production of an antibacterial compound was as~essed using Escherichia coli as test organism. The medium in which maximum agent yields were obtained was then used in studies ' intended to optimise pH, incubation temperature and shaking rate for best agent yields. During these procedures, batch size was increased to 41 (61 Batch Fermenter; Life Science-Laboratories, UK) and optimisation reconfirmed.

The antibacterial agent was isolated by a combination of solvent extraction and thin layer chromatography. The yellow crystalline product was subjected to analysis by IR/UV spectroscopy , Mass Spectrometry and X-ray Diffraction.

Minimum inhibitory concentrations (MICs) were determined for the pure product against a variety of organisms in routine laboratory media using the agar diffusion method.

RESULTS AND DISCUSSION

In both solid and liquid media the pseudomonad (NeIB 9897) produced diffusable,

227

antibacterial and antifunqal ceapounds. Opti ... production of the antibacterial coapound occurred in a maqnesium limited chemically defined aediu. containinq (q/l): a..oniu. chloride, 0.6; sodiu.sulphate, 0.2; potasaiu. dibydroqen phosphate, 11.3; dipotassium hydroqen phosphate, 2.9 and qlucose, 3.0; final' pH was 6.0. Despite the absence of added maqnesiua salts, the orqanis. qrev veIl ('iq. I). Adequate maqnesiu. levels for qrovth appear to be provided by other aediu. coaponents and atomic absorption spectroscopy shoved the presence, of aaqnesiua at 1.6 x 10-6H. Haxiaum product yield occurred after 30-40 hours. Haxi .... concentration was, hovever, typically less in 41 batches than in 50-l00a! •

Opti.u. conditions for the production of the antibacterial coapound in up to 41 batch cultures of this aediu. vere an air flow rate of 41/ain with paddle aqitation of 150 rpl at 37oC.

X-ray diffraction analysis indicated that the compound is 1,6 -dlaethylpyriaido 15,4-e) -1,2,4-triazine - 5,7 (lH,6H) - dione (Xanthothricin also known as

'Toxoflavin,' 'Iq. 2), a toxic aetabolite previously detected in foods conta.inated with Ps. cocovenenans (3). This latter'orqanis. would not, however, qrow in a selective _diu. developed for HCIB 9897. Hini .... inhibitory concentration values have been assessed by the aqar diffusion _thod are qiven in Table 1 and indicate activity in the uq/.l ranqe.

• 0 ....

e'.

Table 1.

rig. 2. strlctlre of lalthot.rlcla.

0_0--0--0

o 10 20 30 .0 !>Cl eo TIME (1'1)

Hiniaua'lnhibitory CoJlQefttrations for Xanthrothricin (aqar'diffusion).

Gr ... ba lie 1"/aI) fldtrlol, CllI i.) '110011, .Il0l, 1.1 IIdlln nr ... 25.' CI •• trl.l .. rptr.,..,. >2'" ,t,,.,I.r.cror 1If." 11.t It.p' •• ,I'mldls 1 •• ' '.rr.tl, IIretln.' 511.5 ' ..... rOlfttl Clr.,I.I •• m.) C1 •• lds .I.ICI •• )211' , .. Idlll .. orp.,m. m.l

Thouqh inherent toxicity (4) may 1i.it syste.ic use, Xanthothricin or its derivatives may form the basis of a qroup of relatively low .olecular 88SS

(Xanthothricin: 193) anti.icrobial aqents.,

RD'ERENC!!l

1. Attafl1ah, A. 1980. UK Patent Appllcation GB 2036792A. 2. Attafl1ah, A. and Bradbury, J.'., 1989, J. !pp!. Bact., in press. 3. Van D,,_, P.A., Johannes, A.G., Cox, H.C. and Berends, W., 1960, Rec. Trav.

Chi •• , 11: 255. 4. Glaso)" J.B. ,In Encyclopaedia of Antibiotics. P 345. WlleyP1Jbl. London, 1976.

228.

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