Dissertation52.172.27.147:8080/jspui/bitstream/123456789/2239/1/... · 2011. 1. 18. ·...

"INCREASED PRODUCTION OF ANTIBIOTIC BYMUTAGENESIS FROM SOIL ISOLATED

ACTINOMYCETE"

ByPRAKASH B. MOTE.

B.PHARM.

DissertationDissertationDissertationDissertationDissertationSubmitted to Rajiv Gandhi University of Health

Sciences, Karnataka, Bangalore,in partial fulfilment of the requirement for

the award of the degree of

Master of Pharmacyin

Pharmaceutical Biotechnology

Under The Guidance OfSHRI. R. V. KARADI.

M.Pharm.

DEPARTMENT OF PHARMACEUTICAL BIOTECHNOLOGY,K. L. E. SOCIETY's COLLEGE OF PHARMACY,

BELGAUM- 590 010, KARNATAKA, INDIA.

MAY - 2006

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,KARNATAKA, BANGALORE.

Endorsement by the HOD, Principal/Endorsement by the HOD, Principal/Endorsement by the HOD, Principal/Endorsement by the HOD, Principal/Endorsement by the HOD, Principal/Head Of the InstitutionHead Of the InstitutionHead Of the InstitutionHead Of the InstitutionHead Of the Institution

This is to certify that the dissertation entitled"INCREASED PRODUCTION OF ANTIBIOTIC BY MUTAGENESIS FROM SOIL

ISOLATED ACTINOMYCETE" is a bonafide research work

done by PRAKASH B. MOTE under the guidance of Shri.R. V. KARADI, Assistant Professor and Head,Department of Pharmaceutical Biotechnology,K.L.E.S's College of Pharmacy, Belgaum.

Dr. F.V. MANVI.M.Pharm., Ph.D.

Principal,K.L.E.S's College of Pharmacy,Belgaum - 590 010.

Date :Place : Belgaum.

Shri. R. V. KARADI.M.Pharm.

Assistant Professor & Head,Department of PharmaceuticalBiotechnology,K.L.E.S's College of Pharmacy,Belgaum - 590 010.Date :Place : Belgaum.


Certificate By GuideCertificate By GuideCertificate By GuideCertificate By GuideCertificate By Guide

This is to certify that the dissertation entitled "INCREASED

PRODUCTION OF ANTIBIOTIC BY MUTAGENESIS FROM SOIL

ISOLATED ACTINOMYCETE" is the bonafide research work

done by PRAKASH B. MOTE in partial fulfillment of the

requirement for the Degree of MASTER OF PHARMACY IN

PHARMACEUTICAL BIOTECHNOLOGY.

Shri. R. V. KARADI. M.Pharm.

Assistant Professor and Head,Dept. of Pharmaceutical Biotechnology,K.L.E.S's College of Pharmacy,Belgaum - 590 010.Karnataka.



Declaration by the CandidateDeclaration by the CandidateDeclaration by the CandidateDeclaration by the CandidateDeclaration by the Candidate

I hereby declare that this dissertation entitled "INCREASED

PRODUCTION OF ANTIBIOTIC BY MUTAGENESIS FROM SOIL

ISOLATED ACTINOMYCETE" is a bonafide and genuine

research work carried out by me under the guidance of Shri. R. V.

KARADI, Assistant Professor and Head, Department of

Pharmaceutical Biotechnology, K.L.E.S's College of Pharmacy,

Belgaum.

PRAKASH B. MOTE. B.Pharm.

Dept. of Pharmaceutical Biotechnology,K.L.E.S's College of Pharmacy,Belgaum - 590 010.Karnataka.



Copyright DeclarationCopyright DeclarationCopyright DeclarationCopyright DeclarationCopyright Declaration

I hereby declare that the Rajiv GandhiUniversity of Health Sciences, Karnataka, Bangalore shall havethe rights to preserve, use and disseminate thisdissertation / thesis in print or electronic formatfor academic / research purpose.

PRAKASH B. MOTE. B.Pharm.

Dept. of Pharmaceutical Biotechnology,K.L.E.S's College of Pharmacy,Belgaum - 590 010.Karnataka.


(C) Rajiv Gandhi University of Health Sciences, Karnataka.

Acknowledgements

“Thus do your duty without attachment Perform the work that has to be done Since the person attains supreme form By doing duty in a spirit of sacrifice”

(The Bhagvatgita Chp. III – Verse 19)

The completion of this dissertation is not only fulfillment of my dreams but also the dreams of my Parents, who have taken lots of pain for me in completion of my higher studies.

I am indebted to the rich source deep inspirer and my esteemed guide and teacher SHRI R. V. KARADI, Assistant Professor and Head, Department of Pharmaceutical Biotechnology. His words of advice have been etched in my heart and I will always endeavour to hold up his ideas. His simplicity, untiring and meticulous guidance, caring attitude and provision of fearless work environment will be cherished in all walks of my life. I thank you sir for bringing out the best in me.

It’s a great pleasure to utilize this unique opportunity to express my deep sense of gratitude and offer my most sincere and humble regards to Dr.B.Jaiprakash, Professor, for his continuous encouragement and support in completion of my Course and Dissertation successfully.

I am immensely thankful to Dr. F.V.Manvi, Principal, and Prof. A. D. Taranalli, Vice- principal, K.L.E.S’s College of Pharmacy, Belgaum, for providing me the necessary facilities and help in carrying out my dissertation work.

I owe my warmest and humble thanks to Shri. E.N.Gaviranj Assistant Professor, Shri. Chandrashekhara S., and Shri.D.N.Sastry. Department of Pharmaceutical Biotechnology. K.L.E.S’s College of Pharmacy, Belgaum, for their timely help, encouragement, boosting my confidence in the progress of my academics.

It is with deep sense of gratitude and humbleness I express my heartfelt thanks to Dr.A.R.Bhat, Dr. K. R. Alagawadi, Dr. M. B. Patil, Shri. R. V. Savadi, Shri. B. C. Koti, Shri. V. S. Mannur, Shri. Kalyani, Shri. C.R. Patil, Shri. G. S. Neeli, Dr. (smt.) K. S. Patil, Shri. S. S. Jalalpure, Shri. M. B. Palkar and Shri. Vinod. for their valuable guidance.

I am oweful to Shri. P. V. Karadi for his kind co-operation and timely help throughout the study. I am also thankful to non-teaching staff Shri. Muddapur, Shri. Vasant, Shri. Mahadev and Mr. Bijay Pujari.

I extend my special thanks to CENTRAL BANK OF INDIA, KASABKHEDA, A’BAD. for providing me the financial help for the completion of my studies successfully.

I take this opportunity to thank my seniors Vijay Pratap, Sanjiv Heroor, Jayprakash, Navneet, Sailaju, Mathur, Manish, Ambar, Keyur, Kapil, Gulshan, Basavraj, Nagesh, Mamatha.

I extend my thanks to my colleagues Shri. Satish Kavatagimath, Om, Shete, Kedar, Siddharth,Shri. Raut, Shri Kumbhar, Rupesh, Vikesh, Ashok, Jiten , Mahajan, Vijay Singh, Bonde, Javesh, Vishal, Badgujar, Asif, Rajanna,Rajkumar, Pinkal, Venkey, Alok , Santosh, Mrs Suralkar, Nagraj, Sunil and Shanthkumar.

I shall also thanks my juniors Nagre, Gupta, Yogesh, Sunil, Lokesh, Mrs. Renuka, Rajesh, Kiran, Pandey.

I would like to express my love and gratitude to my PAPPA AND AAI and my grandparents from depth of my heart for giving me more than what I deserved. Their blessings always inspire me to work hard and to overcome all the difficulties in between way. It gives me an immense pleasure to dedicate my research work at their feet without whose blessings and vision I would not have been able to achieve this task.

The acknowledgement is not complete until I render my gratitude to my Little Sister, Ms. Manisha for consistent emotional support during my studies.

My personal regards to my friends Naim, Nilesh, Vaibhav and all my B. Pharm friends, who have contributed directly or indirectly to my studies.

I am thankful to Mr.Subhash of Saitech Computers, Belgaum, for his neat typographical work and friendly co-operation.

I am also thankful to Mr. Ram of Balaji Photo Studio for giving nice photographs of my dissertation.

Last but not the least I thank ‘God’ the Almighty, for the blessings and courage to ladder the success.

Thankful I ever remain…..

Date: Place: Belgaum Prakash Mote.

CONTENTS

SL. NO.

TITLE PAGE NO.

ABSTRACT

1. INTRODUCTION 1 – 35

2. NEED AND OBJECTIVES 36 – 40

3. REVIEW OF LITERATURE 41 – 55

4. MATERIALS AND METHODS 56 – 77

5. RESULTS AND DISCUSSION 78 – 123

6. SUMMARY AND CONCLUSION 124 – 127

7. BIBLIOGRAPHY 128-135

8. ANNEXURE 136-140

LIST OF TABLES

TABLE NO.

TITLE PAGE NO.

1 Microorganisms producing the various antibiotics 8

2 Factors affecting antibiotic production 21

3 Inhibition of antibiotic production by readily utilizable nitrogen

sources

22

4 Influence of metals on antibiotic production 24

5 Improvement of antibiotic yields during the first 20 years of

antibiotic development (Riviere, 1977)

32

6 Modification and improvement of the strain by various mutagens 34

7 Collection of Soil Samples and Number of Actinomycetes in

Isolation Plates

79

8 Sensitivity of different microorganisms towards the soil isolates

by agar streak method

81

9 Taxonomical Characterization of Soil Isolates 82

10 Morphological and cultural characterization of the strain A-4 84

11 Antibiotic Productivity by UV Mutant Strains 87

12 Comparison of UV mutant strains for the antibiotic production 87

13 Effect of Streptomycin and Rifampin on UV mutants and

antibiotic productivity of UV-drug resistant mutants

89

14 Morphological and cultural characterization of the strain A-4

mutant

90

TABLE NO.

TITLE PAGE NO.

15 Optimization of Carbon Source 91

16 Optimization of Nitrogen Source 94

17 Optimization of Temperature 97

18 Optimization of pH for optimum growth and antibiotic

productivity

100

19 Optimization of Dissolved Oxygen Concentration (DO2) 103

20 Optimization of duration of fermentation for the maximum

growth and antibiotic production

106

21 Composition of Fermentation Medium 109

22 Thin layer chromatography of purified antimicrobial compound 110

23 Determination of MIC for an isolated antimicrobial compound

against bacteria

110

24 Determination of MIC for an isolated antimicrobial compound

against fungi

111

25 Physical properties of purified antimicrobial compound 111

26 IR spectroscopical data and their functional groups 114

27 NMR Spectroscopical data and their functional groups 114

LIST OF FIGURES

FIGURE NO.

TITLE PAGE NO.

1 Antibiotic gene clusters encoding doxorubicin. 15

2 (A) A-factor for S.griseus, (B) Genetics of streptomycin production

in S.griseus

17

3 The spread in productivity of chlortetracycline of natural variants of

Streptomyces viridifaciens

31

4 The spread in chlortetra productivity of a UV-treated population of


31

5. Solvent Extraction Tree 72

6. Comparison of Packed Cell Volume (%) at different Concentrations

of Lactose for A-4 and A-4 Mutant Strains

92

7. Comparison of Zone of Inhibition at different Concentrations of

Lactose for A-4 and A-4 Mutant Strains

93

8. Comparison of Packed Cell Volume at Different Nitrogen Sources

for A-4 and A-4 Mutant Strains

95

9. Comparison of Zone of Inhibition at Different Nitrogen Sources for

A-4 and A-4 Mutant Strains

96

10 Comparison of Packed Cell Volume at Different Temperatures for

A-4 and A-4 Mutant Strains

98

11 Comparison of Zone of Inhibition at Different Temperature for A-4

and A-4 Mutant Strains

99

12 Comparison of Packed Cell Volume (%) at specified pH for A-4


101

FIGURE NO.

TITLE PAGE NO.

13 Comparison of Zone of Inhibition at different pH for A-4 and A-4

Mutant Strains

102

14 Comparison of Packed Cell Volume (%) at Different DO2 Levels

for A-4 and A-4 Mutant Strains

104

15 Comparison of Zone of Inhibition at Different DO2 Levels for A-4


105

16 Comparison of PCV at Different Fermentation Durations for A-4


107

17 Comparison of Zone of Inhibition at Different Fermentation

Durations for A-4 and A-4 Mutant Strains

108

18 IR Spectrum for an Isolated Antibiotic 112

19 NMR Spectrum for an Isolated Antibiotic 113

LIST OF PLATES

PLATE NO.

TITLE PAGE NO.

1 Photograph Showing Crowded Plate Method 115

2 Test for Microbial Sensitivity 115

3 Test for Melanoid Pigment Formation 115

4 Test for Nitrate Reduction 116

5 Test for Milk Coagulation and Peptonization 116

6 Test for Gelatin Liquefication 116

7 Test for Amylolytic Activity by Starch Hydrolysis 117

8 Carbohydrate Assimilation Test 117

9 Morphology of A-4 Strain on ISP-1 and ISP-7 117

10 Morphology of A-4 Strain on ISP-3 & ISP-5 118

11 Morphology of A-4 Strain on ISP-4 118

12 Morphology of A-4 Strain on ISP-6 118

13 Microscopy of A-4 Strain under 10X 119

14 Microscopy of A-4 Strain under 100X 119

15 Microscopy of A-4 Mutant Strain under 10X 119

16 Microscopy of A-4 Mutant Strain under 100X 119

17 Morphology of A-4 Mutant Strain on ISP-3 120


PLATE NO.

TITLE PAGE NO.




22 Photograph showing Laboratory Fermenter of 3L Capacity of

Sartorius B-Lite Company 122

23 Photograph showing Antimicrobial Activity of Broth Collected

at an interval of 24 hr during bioprocess 122







ABSTRACT

Introduction:

In the present study of increased production of antibiotic from actinomycetes,

several actinomycetes were isolated from soil samples collected from various places in

Belgaum, India. About 18 actinomycetes showed the capability of producing antibiotics.

Out of 18 actinomycetes strains, 6 strains showed promising antimicrobial scores against

different strains of bacteria and fungi. From the six strains selected, one strain designated

as A-4 showed maximum antimicrobial property against gram positive and gram negative

strains as well as various fungi and the same strain is taken for the present research.

Antibiotics play a crucial role in the development of various tissue culture

techniques and also in various fields like biochemistry, molecular biology, microbiology

and genetics etc. The continuing success of biotechnology in the search of microbial

metabolites as antimicrobial compounds is useful in combating human, animal and plant

diseases. As antibiotics are secondary metabolites, they do not play any role in microbial

metabolism so their biosynthesis is in very less amounts. Therefore, it is always desirable

to try to improve the yield of an antibiotic during fermentation and subsequent processing

steps.

Antibiotics are the best known products of actinomycete. The genus

streptomycete is responsible for the formation of more than 60% of known antibiotics.

Materials and methods:

Crowded plate technique is used for the isolation of actinomycetes using media

like soybean – casein digest medium actinomycetes isolation agar.

From the isolated actinomycetes, A-4 strain is selected, on the basis of higher

scores against various microbial strains, for further research study.

The taxonomical characterization as well as morphological and cultural

characterization of A-4 strain is performed as per International Streptomycete Project

described by Shirling and Gottlib.

Strain improvement and medium formulation for specific strains helps to improve

the antibiotic production. Therefore strain improvement of A-4 was performed by UV

irradiation and drug resistance methods. In drug resistance method, streptomycin and

rifampin antibiotics in the concentration of 5-7 mg/ml were used for mutation.

In medium formulation study for A-4 and A-4 mutant, various carbon and

nitrogen sources were tested for maximum antibiotic production using zone of inhibition

and packed cell volume (%) as parameters. Various fermentation conditions like pH,

temperature and DO2 were also optimized for the maximal production of antibiotic from

both A-4 and A-4 mutant. All medium formulation as well as bioprocess parameters for

A-4 and A-4 mutant strains were compared.

The antibiotic was extracted using ethyl acetate and it is then purified by cold

methanol and thin layer chromatography.

The spectroscopical studies including UV, IR and NMR were performed to obtain

the type and structure of the compound.

The MIC of the compound against various bacteria and fungi were determined.

Result and discussion:

Taxonomical as well as morphological and cultural studies showed that A-4 is

belongs to actinomycete genus. The strain improvement study by UV irradiation and

drug resistance showed the improved production of antibiotic by A-4 mutant, strains.

The morphological and cultural characteristics of A-4 mutant showed cellular and

aerial growth as well as soluble pigment formation in various ISP media.

In medium formulation study, lactose as a carbon source and peptone as a

nitrogen source were selected and optimized as 4% lactose and 1% peptone for both A-4

and A-4 mutant strains. In fermentation parameters optimization, pH was set at 7.0,

agitation at 200 rpm and temperature at 28°C.

In the study of ‘duration of fermentation’ 96 hr duration was found to be ideal for

antibiotic bioprocess.

In solvent extraction, ethyl acetate was found to be ideal solvent and used for

extraction. The purification of antibiotic compound was done by cold methanol and TLC.

In the determination of structure and type of antibiotic, UV, IR and NMR were

performed and from data obtained by spectroscopical study, antibiotic is categorized as

aminoglycoside type and its structure ellucidation is in progress.

The MIC against various bacteria and fungi were found to be the range of 100-

125 mg/ml.

Keywords: Actinomycete; Antibiotic; MIC; Fermentation; Spectroscopical data;

Aminoglycoside; Solvent extraction; PCV; Zone of Inhibition.

Introduction

INTRODUCTION

The term antibiotic appeared as early as 1928 in the French microbiological

literature as antibiosis. The phenomenon of antagonism between living organisms was

frequently observed even since 1877, when Pasteur and Joubert noticed that aerobic

bacteria antagonized the growth of Bacillus anthracis.1

However, the world in its present restrictive meaning, “a chemical substance

derived from microorganisms, which has the capacity of inhibiting growth and even

destroying other organisms in dilute solutions” was introduced by Selman and Waksman

in 1942.1

In 1940 Waksman had forecasted, “We are finally approaching a new field of

domestication of microorganisms for combating the microbial enemies of man of his

domesticated plants and animals. Surely microbiology is entering a new phase of

development.1

During the 1960s, the phase of discovery of antibacterials slackened, but efforts

were then made to research also for antifungal, antimycoplasmal, antispirochetal,

antiprotozoal, antitumor, antivirual and antiphage compounds, as well as for antibiotics

for non-medical uses such as antioxidants.1

The problem of the bacterial resistance to antibiotics had evolved and new

compounds or derived from the known antibiotics had to be found to replace existing

ones.1

Progress of trends in Antibiotic Search: 2,3,4

The continuing success of a biotechnologist in the search of microbial metabolites

as antimicrobial compounds (antibiotics) is useful in combating human, animal and plant

Dept. of Pharmaceutical Biotechnology, KLES CP, Belgaum. 1

Introduction

diseases for stimulating the belief that microorganisms constitute an inexhaustible

reservoir of compounds with pharmacological, physiological, medical or agricultural

applications.

Antibiotics continue to play a crucial role in the development of tissue culture

techniques and basic screenings, primarily in biochemistry, molecular biology,

microbiology and genetics including genetic engineering and to a lesser extent,

pharmacology and organic chemistry.

In the research for new antibiotics, the leading position of Japan, United States

and England remains unchanged. Recently, the marketing products have been in number

of analogs, minor modifications of earlier known antibiotics. Mainly as a result of novel

strain isolation and selection methods, refined compound isolation and characterization

procedures and in vivo assay systems, completely new compounds still emerge at a

slower pace. Though chemical derivatives or bioconversions of antibiotic offer potentials

to yield more useful compounds, finding new antibiotics remains the most desirable

objective.

Goals of Antibiotic Research:5

The study and development of antibiotics certainly share some of the same aims

as other areas of biotechnology. For example, it is always desirable to try to improve the

yield of an antibiotic during fermentation and subsequent processing steps.

A very large fraction of antibiotic research is directed towards development of

new agents, because –

• Many microorganisms, including most fungi and viruses, do not have truly

effective and safe antibiotics.

• Some bacteria, such as Pseudomonas aeruginosa, also are intrinsically resistant to

most antibiotics.


Introduction

• Pathogenic bacteria are acquiring or developing resistance to existing antibiotics

in correlation with the level of use of these antibiotics to treat them.

• Many potentially important antibiotics have associated nephrotoxicity and

ototoxicity.

• Most of the existing antibiotics are more costlier.

Because of the above reasons, research in newer antibiotics and optimization of

yield / productivity of antibiotics is still important.

Actinomycetes: [A brief guide to generic groups] 6,7

The actinomycetes are gram positive, high G+C (>55%) organisms that tend to

grow slowly as branching filaments. Many actinomycetes will grow on the common

bacteriological media used in the laboratory, such as nutrient agar, trypticase soy agar,

blood agar, and even brain-heart infusion agar. Actinomycetes encompass a wide range

of bacteria. They have universal occurrence and play an active part in the cycle of nature.

Sporoactinomycetes require special media to allow differentiation and

development of characteristics spores and pigments. For example, the pale, shiny, hard

colonies of a Streptomyces species on nutrient agar can be transformed into bright yellow

colonies with a powdery white aerial mycelium and spirals of arthrospores when the

organism is subcultured onto a more suitable growth medium, such as oatmeal or

inorganic salts starch agars.

Actinomycetes show outgrowths from a spore or fragments of mycelium (Colony-

forming units, CFUs) develop into hyphae that penetrate the agar (substrate mycelium)

and hyphae that branch repeatedly and become cemented together on the surface of the

agar to form a tough, leathery colony. The density and consistency of the colony will

depend on the composition of the medium. Nocardioform actinomycetes exhibit


Introduction

fragmentation; the hyphae break up into rods and cocci, thus leading to soft or friable

colonies. In strains of certain genera (e.g. Streptomyces), the colony becomes covered

with aerial mycelium: free, erect hyphae surrounded by a hydrophobic sheath that grow

into the air away from the colony.

Actinomycetes grow slowly. A branching mycelium growing at the surface of

transparent agar can be seen with the aid of a microscope after 24 hours, and visible

colonies may appear in 3-4 days, but mature aerial mycelium with spores may take 7-14

days to develop, and some very slow growing strains may require up to 1 month’s

incubation.

The saprophytic actinomycetes are oxidative and may grow poorly when the air

supply is restricted. Actinomycetes can grow in broth but need to be cultivated under

specialized conditions. The growth of Streptomycete in a stationary broth tube is usually

restricted to a surface pellicle and perhaps a cottony sediment, leaving the broth quite

clear. Liquid cultures require considerable aeration and agitation to give the suspended

growth. Tubes and flasks must be incubated on a shaker at high speeds (e.g. 200 – 250

rpm) to give the supply of oxygen and mixing necessary for maximum growth. Even,

diffuse mycelial growth may require the higher agitation and mixing rates achieved by

baffles or springs.

The morphology of an actinomycete growing on agar can provide useful and rapid

clues to its identity, but viewing isolated colonies can give little worthwhile information.

Morphological characters are still widely used for characterizing genera, for example, the

presence or absence of spores on the substrate mycelium or the formation of zoospores in

specialized spore vesicles or sporangia. The ability to produce motile spores is more

widespread in the actinomycetes.


Introduction

Preservation of both sporing and non-sporulating actinomycetes can be achieved

by freeze drying or storage in liquid nitrogen. Freezing suspensions in 20% (v/v) glycerol

at -20°C to -40°C has proved to be a very useful method in a busy laboratory.

Filament may fragment into irregular sized elements (0.5 – 1.0 μm in diameter) or

remain stable and produce arthrospores. Spores are produced singly, in chains of various

lengths, or in sporangia. Spores are usually non-motile, but some genera produce

flagellate spores. Some genera that do not produce branching filaments are

phylogenetically related to this group. Genera of actinomycetes are distinguished on the

basis of their morphology and marker chemical constituents of the cell wall, membranes

and whole cell hydrolysates. Actinomycetes are mainly aerobic, but some genera are

facultative or obligately anaerobic. Actinomycetes are ‘chemoheterotrophic, using a wide

variety of energy sources, including complex polymers. Mainly free living in a wide

range of habitats (water and soil). Some are pathogens for human, animals or plants.

Generic groups of Actinomycetes: 6,7

1) Nocardioform actinomycetes:-

This is a heterogenous group which forms filaments and fragment into shorter

elements. Aerial growth is formed by some genera and may produce chains of spores.

Genera are distinguished primarily by well chemotypes, presence or absence of mycolic

acids and other chemical characters.

Subgroup 1: Mycolic acid-containing bacteria (Genus – Gordona, Nocardia,

Rhodococcus and Tsukamurella)

Subgroup 2: Pseudonocardia and related genera (Genus – Actinobispora,

Actinokineospora, Actinopolyspora, Amycolata, Pseudoamycolata,

kibdelosporangium, Saccharomonospora and Saccharopolyspora).

Subgroup 3: Nocardioides and Terrabacter


Introduction

Subgroup 4: Puomicromonospora and related genera (Genus – Jonesia,

Oerskovia)

2) Genera with multilocular sporangia:

These genera form filaments that divide by longitudinal and transverse septa. This

produces large number of coccoid-like elements, which may be motile (Dermatophilus,

Geodermatophilus) or non-motile (Frankia).

3) Actinoplanes:

Stable filaments are formed, with little or no aerial growth. Motile spores are

produced in sporangia. (Actinoplanes, Ampullariella, Dactylosporangium, Pilimelia) or

non-motile spores are produced singly (Micromonospora) or in chains (Catellatospora).

Cell wall contains meso-DAP and glycine, arabinose and xylose are found in whole cell

hydrolysates.

4) Streptomycetes and related genera:

A heterogenous group, all of which have cell wall containing L-DAP and glycine.

Stable filaments are formed and may produce extensive aerial growth with long spore

chains (Streptomyces, Streptoverticillium). Other genera (Intrasporangium, Kineospora,

Sporichthya) produce little or no aerial growth and have a variety of spore forms.

5) Maduromycetes:-

Stable filaments are formed and produce spore bearing aerial growth. Short chains

of non-motile arthrospores are produced by Microbispora (two spores), Microtetraspora

(four spores), and Actinomadura (varying number). Other genera produce spores in

sporangia which are motile (Planobispora, Planomonospora, Spinillospora) or non-

motile (Streptosporangium). The cell walls contain meso-DAP, and cell hydrolysates

contain madurose.


Introduction

Subgroup 1: Streptosporangium and related group (Genus – Microbispora,

Microtetraspora, Planobispora, Planomonospora, Spirillospora,

Streptosporangium).

Subgroup 2: Actinomadura

6) Thermomonospora and related genera:

Stable filaments are formed and produce aerial growth bearing spores that are

single (Thermomonospora), in chains (Actinosynnema, Nocardiopsis), or in sporangia-

like structures (Streptoalloteichus). The cell wall contains meso-DAP, but not

characteristic amino acids or sugars in whole cell hydrolysates.

7) Thermoactinomycetes:

This comprises only one genus, Thermoactinomycetes. The stable filaments

produce aerial growth. Single spores (which are endospores) are formed on both aerial

and vegetative filaments. All species are thermophilic. The cell wall contains meso-DAP

but not characteristic amino acids or sugars.

8) Other genera:-

This group comprises three genera (Glycomyces, Kitasatosporia and

Saccharothrix) that cannot at present be assigned to other groups. They all produce aerial

growth bearing chains of spores.

Source of Antibiotic Producing Microorganisms: 8,9

Antibiotics are produced by many microorganisms in various ecological

conditions. Producers of antibiotic can be found in rivers, lakes, decaying plants and

animal remains etc. but majority of microorganisms that produce antibiotic inhabits soil.


Introduction

Table 1: Microorganisms producing the various antibiotics

Sl. No. Microorganisms Antibiotics

1. Bacillus licheniformic Bacitracin

2. Cephalosorium acremonium Cephalosporin C

3. Penicillium chrysogenum Penicillins

4. Streptomyces antibiotics Actinomycin, Oleandomycin

5. Streptomyces griseus Indolmycin, Streptomycin, Candicidin

6. Streptomyces kanamyceticus Kanamycin

7. Streptomyces fradiae Neomycin

8. Streptomyces albinogen Puromycin

9. Streptomyces sioyaensis Siomycin

10. Streptomyces lavendulae Streptothricin

11. Bacillus subtillis Bacillin, subtillin

12. Streptomyces cinnamonesis Monensin

13. Streptomyces veneguelae Chloramphenicol

14. Streptomyces verticillatus Mitomycin

15. Penicillin griseofulvin Griseofulvin

16. Penicillin urticae Patulin

17. Pseudomonas aureofaciens Pyrrolnitrin

18. Streptomyces caelestis Celesticetin

19. Streptomyces sp. X-53 Echinomycin

20. Streptomyces cacaoi Polyonins L & M

21. Streptomyces sp. P-8648 Viridogrisein

22. Stretpomyces sp. Novobiocin

23. Micromonospora sp. Micromonosorin

24. Thermophilic actinomycetes Thermomycin, Thermocyridin, Refcin (anthracin)

25. Streptomyces spinosus Spinosad

26. Streptomyces hygnoscopicus Rapamycin

27. Streptomyces pencetius Avermectin

28. Streptomyces erythrea Erythromycin


Introduction

Role of Actinomycete in the field of Research: 10

Antibiotics are the best known products of actinomycete. Over 5000 antibiotics

have been identified from the culture of gram positive, gram negative organisms and

filamentous fungi, but only 100 antibiotics have been commercially used to treat human,

animal and plant disease. The genus Streptomycete is responsible for the formation of

more than 60% of known antibiotics. While further 15% are made by number of related

Actinomycete, Micromonospora, Actinomadura, Streptoverticillium and

Thermoactinomycetes.

Many of the microbial products including antibiotics are considered to be

‘Secondary metabolites’ because they seem to have no direct role in those aspects of

metabolism which support necessary functions in the cell namely energy production,

growth and reproduction. There is a great structural variety among the secondary

metabolites but organisms also have the ability to produce closely related metabolites.

Some are antimicrobially active and some are not.

Antibiotics, because of their industrial importance, are the best known products

of actinomyctes. Streptomycete is responsible for the formation of more than 60% of

known antibiotic while further 15% are made by number of related actinomycete –

Micromonospora, Actinomadura, Stretoverticillium and Thermoactinomycetes.

The selection of superior producing microorganisms was earnestly pursued by

Weinstein et al. They thoroughly screened microorganisms of the genus Micromonospora

which had rarely been studied and found gentamicin and several other antibiotics. With

this work as a turning point, studies shifted to methods for effectively isolating

actinomycetes other than Streptomyces which are less frequent in soil. Rare

actinomycetes were found to produce many new antibiotics. However, since rare


Introduction

actinomycetes do not usually produce antibiotics abundantly and grow slowly, research

on and development of them are difficult.

Antibiotic Diversity:11

The actinomycetes produce an enormous variety of bioactive molecules e.g.

antimicrobial compounds. One of the first antibiotics used was Streptomycin, produced b

S.griseus. The last 55 years have seen the discovery of more than 12,000 antibiotics. The

actinomycetes produce about 70% of these, and the remaining 30% are products of

filamentous fungi and non-actinomycete bacteria.

Of the plethora of known bioactive compounds, approximately 160 are currently

used in clinical practice. Streptomyces species produce about 55% of these. The

disproportionate representation of Streptomycetes among the producing strains may have

more to do with the relative ease of isolating and screening them than with a lack of

biosynthetic capability in other actinomycetes. For example, Micromonospora and

Aeromicrobiuim produce structurally and genetically related macrolide-type antibiotics.

More over, many other evolutionary distant groups of bacteria, including the

mycobacteria are known to produce diverse bioactive compounds, but these organisms

are relatively difficult to culture for screening programs.

Most of the bioactive compounds from actinomycete sort into several major

structural classes: aminoglycosides (e.g. Streptomycin and Kanamycin), ansamycins

(Rifampin), anthracyclines (doxorubicin), β-lactum (cephalosporins), macrolides

(erythromycin), and tetracyclines.


Introduction

Concepts of Industrial Research in Antibiotic Production: 2,3,4

The industrial fermentation industry received its greatest impetus for expansion

and profits with the advent and exploitation of antibiotics as chemotherapeutic agents.

The demand for penicillin during World War II, and later for Streptomycin and other

antibiotics, brought on the undertaking of intensive research programs designed to find

organisms capable of producing good antibiotics, and oriented toward the development of

means for producing antibiotics on a large scale. New cultural procedures were devised,

and the technique of submerged agitated-aerated fermentations in deep-tank fermentors

came into action. As a result much of the knowledge gained during the development of

antibiotic fermentation processes then became available for the commercial development

of other new antibiotic fermentation processes on a large scale production.

Screening of antibiotics has been widely performed for about 30 years, and new

antibiotics are still being found. However, the possibility of discovering new antibiotics

merely by random screening is reduced now a days, and new approaches are required for

finding new antibiotics efficiently.

In screening of new antibiotics, three major factors must be considered i.e.,

detection method, selection methods. These are closely related to each other, and their

efficient combination is indispensable for successful screening.

These days, new strain development for antibiotic production has been essential

prerequisite for scale up of antibiotic production and also for search of new antibiotics.

Current industrial practices involve natural selection, mutation and protoplast fusion for

Streptomyces and other related genera. A major feature of industrial antibiotic production

is directed to the screening programmes to generate new potent antibiotics producer

microorganisms either from natural sources or from established culture.


Introduction

Screening for antibiotic producing microorganisms can be considered as the use

of highly selective procedures to allow the detection and isolation of only those

microorganisms of interest from a large population. Screening can be direct or indirect.

Direct screening involves assay of product either by bioassay or by chemical means, in

contrast indirect screening do not rely on assay of product but rather on some other

characteristics of strains which is correlated with antibiotic production.

Factors considered in selection of antibiotic:2,3,4

Only a comparatively few of the many hundreds of antibiotics produced by

microorganisms have been selected for manufacture on an industrial scale. Antibiotics for

use in the field of medicine should be relatively non-toxic. They should not precipitate

the serum proteins, cause hemolysis, or adversely affect the phagocytes. They should be

soluble in water, reasonably stable, and effective against pathogens under the conditions

of use. They should be well tolerated by the individual in the doses required and should

bring as undesirable side responses as possible. They should be free of pyrogenicity and

should not cause histamine-like responses.

Selective search strategies for new antibiotic:-

1) Target directed screening:

a. Use of antibiotic resistant test organisms

b. Use of antibiotic supersensitive test organisms

2) Non-target directed screening:

a. Inhibition of antibiotic inactivation enzymes

b. Inhibition of target enzymes or receptor type


Introduction

Antibiotics and its Classification:

The term “antibiotic” has been defined by Selman Waksman as being an organic

compound, produced by one microorganism, that, at great dilution, inhibits the growth of

or kill another microorganism or microorganisms. Obviously, this definition does not

include materials extracted from green plants or from other non-microbial sources, nor

does it include organic acids or amines that may inhibit microbial growth, but which are

not active at high dilution.

For any one antibiotic there is a specific group of microorganisms, comprising its

“inhibition spectrum”, which is sensitive to the antibiotic at therapeutically possible

dosage levels.

The organisms susceptible to the inhibitory or lethal effect of an antibiotic

constitute its spectrum. According to their spectra, antibiotics may be classified as:-

1) Antibiotics mainly effective against Gram-positive bacteria

a. Those employed for systemic infections e.g. Penicillins, Erythromycin,

Lincomycin, Oleandomycin, Vancomycin, Novobiocin and Fucidin.

b. Those employed topically e.g. Bacitracin.

2) Antibiotics mainly effective against Gram-negative bacteria

a. Those used mainly for systemic infections e.g. Streptomycin, Kanamycin,

Gentamicin, Colistin, Polymyxin B and Cycloserine.

b. Those used locally in the intestines e.g. paramomycin.

3) Antibiotics effective against both Gram-positive and Gram-negative bacteria:

a. Those employed for systemic infections e.g. Ampicillin, Amoxycillin,

Carbanecillin, Cephalosporins, Rifamycins.

b. Those employed topically e.g. Neomycin, Tyrothricin and Framycetin.


Introduction

4) Antibiotics effective against both Gram-positive and Gram-negative bacteria,

rickettsiae and Chlamydia – Tetracyclines and Chloramphenicol.

5) Antibiotics effective against acid-fast bacilli (M.tuberclosis):- Streptomycin,

Cycloserine, Viomycin, Caprieomycin, Kanamycin and Rifampicin.

6) Antibiotics effective against protozoa:- Paramomycin, Tetracyclines, Fumagillin.

7) Antibiotics effective against fungi:- Nystatin, Amphotericin B, Griseofulvin,

Hamycin and Pimaricin.

8) Anti-inflammatory antibiotics e.g. Actinomycin D, Mitomycin and Azaserin.

Physiology of Antibiotic Production:12,16

Antibiotics are small molecules whose synthesis often requires dozens of

enzymes. Enzyme activities are of necessity closely regulated in such complex pathways.

It is therefore important to understand the physiology of the producing organisms in order

to maximize the fermentative production of antibiotics.

As antibiotics are secondary metabolites, they do not seem to play a central role in

growth and metabolism of the organisms. Antibiotics have complex, unusual structures.

Antibiotic genes occur in clusters:12

The examples of antibiotics that have been especially well characterized

genetically are streptomycin and the antitumor drug doxorubicin.

The genes encoding the doxorubicin, polyketide synthase, hydroxylase, and other

tailoring enzymes are all clustered in the producing organism’s genome. In fact, the entire

set of 37 open reading frames (ORFs) that are required for doxorubicin synthase is linked

in a contiguous gene cluster (Fig. 1). Indeed, it is a general observation that the genes for

synthesis of a given antibiotic are clustered.


Introduction

Fig. 1: Antibiotic gene clusters encoding doxorubicin.

Usually, antibiotic genes are chromosomal, although in one case –

methylenomycin – the genes are on plasmid.

The organization of transcripts expressed from the doxorubicin gene cluster is

complex. Transcriptional regulators specific to the doxorubicin genes are also associated

with the gene cluster, as are genes that encode doxorubicin resistance functions. Linkage

of dedicated regulatory and resistance genes to an antibiotic biosynthetic gene is another

typical feature of antibiotic genes clusters.

The gene cluster for streptomycin synthesis (Pipersberg, 1977) that is found in

S.griseus repeats the theme established by the doxorubicin cluster linked biosynthetic,

regulatory, and resistance genes arranged in a complex pattern of transcript.

Autoregulator signaling system:12

An actinomycete signaling system that regulates an antibiotic pathway-specific

regulator and also morphogenesis has been relatively well characterized in S.griseus.


Introduction

A-factor [γ-butynolactone], a hormone-link autoregulator with a structure that permits

diffusion through membrane, was dissolved more than 30 years ago on the basis of its

ability to restore sporulation, as well as, streptomycin biosynthesis and resistance, to an

S.griseus Bld-mutant.

The A-factor triggered regulatory cascade regulating antibiotic synthesis in

S.griseus (Fig. 2) starts with A-factor binding to its receptor protein, ArpA, which is a

cytoplasmic DNA-binding protein (Onaka and Horinouchi, 1997). A-factor binding

relieves ArpA repression of the gene AdpA (for A-factor dependent protein). AdpA is a

regulator of StrR, the pathway-specific activator for streptomycin biosynthesis.

The role of A-factor in linking regulation of morphogenesis to Streptomycin

biosynthesis is due to its effects on the expression of AmfR (for aerial mycelium

formation). AmfR is a response regulator-like protein required for normal aerial-

Mycelium development. Similarly to its role in depressing antibiotic synthesis, A-factor

also depresses AmfR transcription Fig 2. It does this by relieving repression of AmfR by

the protein AdpB, which binds just upstream of the AmfR promoter is regulated by ArpA

or if not, how its repressor-like function is otherwise regulated by A-factor is not yet

clear.

Molecular genetic studies of antibiotic-producing prokaryotics, such as

Streptomyces, are finally focused on the complex regulatory mechanisms that operate in

these organisms. One striking fact is that the classic producers of antibiotics are soil

microorganisms that go through sporulation processes such as Streptomyces, Bacillus,

fruiting Mycobacteria, and eukaryotic fungi. It has been established in Bacillus that the

sporulation process, at the end of the growth phase, require the differential transcription

of various types of promoters by several different sigma subunits of RNA polymerase.


Introduction

(The sigma subunit is one of the subunits of this giant enzyme; it plays a decisive role in

recognizing a specific DNA sequence as a promoter). A similar situation apparently

exists in Streptomyces. Recently, a sigma subunit called WhiG was shown to be

necessary for the formation of spores in Streptomyces. Streptomyces is thought to contain

at least seven species of sigma subunits, each of which presumably recognizes a different

promoter sequence. The sequence of DNA upstream from coding regions appears to be

very complex in Streptomyces. It has not been proved that the genes of antibiotic

biosynthesis are regulated in exactly the same way as the genes involved in sporulation,

and some differences are likely to exist. Nevertheless, similar principles probably operate

in both classes.

Fig 2: (A) A-factor for S.griseus, (B) Genetics of streptomycin production in S.griseus

Fermentation:13

The term ‘fermentation’ is derived from the Latin verb fervere, to boil, thus

describing the appearance of the action of yeast on extracts of fruit or malted grain. The

boiling appearance is due to the production of carbon dioxide bubbles caused by the

anaerobic catabolism of the sugars present in the extract.


Introduction

The catabolism of sugars is an oxidative process which results in the production

of reduced pyridine nucleotides which must be reoxidized for the process to continue.

Under aerobic conditions, reoxidation of reduced pyridine nucleotides occurs by electron

transfer, via cytochrome system, with oxygen acting as the terminal electron acceptor.

However, under anaerobic conditions, reduced pyridine nucleotide oxidation is coupled

with the reduction of an organic compound, which is often a subsequent product of the

catabolic pathway. Thus, the term fermentation has been used in a strict biochemical

sense to mean an energy-generation process in which organic compound acts as both

electron donors and terminal electron acceptors.

The production of alcohol by the action of yeast on malt or fruit extract has been

carried out on a large scale for many years and was the first industrial process for the

production of a microbial metabolite. Thus industrial microbiologists have extended the

term fermentation to describe any process for the production of product by the mass

culture of microorganism.

The range of fermentation processes:13

There are five major groups of commercially important fermentations:-

i) Those that produce microbial cells (or biomass) as the product e.g. single cell

proteins.

ii) Those that produce microbial enzymes.

iii) Those that produce microbial metabolites

iv) Those that produce recombinant metabolites

v) Those that modify a compound which is added to the fermentation – the

transformation process.


Introduction

Trophophase and Idiophase:14

Though the production of antibiotics is sometimes evident during growth of the

microorganisms, usually the production is actively carried out after growth reaches the

stationary phase. Doskoil et al. studied growth, synthesis of DNA and RNA, respiration,

morphology, utilization of carbon and nitrogen sources, accumulation of pyruvate, and

production of oxytetracycline by Streptomyces rimosus, and divided the process into five

stages. They indicated that the morphological and physiological properties of the strains

are greatly changed before and after antibiotic accumulation begins. Particularly stage 2,

in which respiration is high and vegetative growth is accelerating by utilizing constituents

of the medium, is well constructed to stage 5, in which growth stops and the production

of antibiotic reaches at maximum. The fermentation period including stage 2 is known

“Trophophase” and that including stage 5 “idiophase”. Conditions for antibiotic

production are more restricted than the growth conditions, and thus the efficient

conversion from the trophophase to the idiophase is important for the production of

antibiotics. The termination of the trophophase does not always lead to the idiophase and

thus events that occur during trophophase are important for the production of antibiotics.

Therefore, it is necessary that precursors and other factors required for antibiotic

production should be prepared during the trophophase when primary metabolism occurs

vigorously.

Medium Formation:13

Medium formulation is an essential stage in the design of successful laboratory

experiments, pilot scale development and manufacturing processes. The constituents of a

medium must satisfy the elemental requirements for cell biomass and metabolite

production and there must be an adequate supply of energy for biosynthesis and cell

maintenance. The first step to consider is an equation based on the stoichiometry for

growth and product formation of an anaerobic fermentation.


Introduction

Carbon and

energy source

+ Nitrogensource

+ O2 + Other requirements Biomass + Products + CO2 + H2O + Heat

This equation should be expressed in quantitative terms, which is important in the

economical design of media.

Medium formulation is very important criteria in antibiotic fermentation, so

detailed investigation is needed to establish the most suitable medium for an individual

fermentation process. Thus medium formulation should be done to meet as many as

possible of the following criteria.

1. It should produce the maximum yield/ concentration of the product or biomass per

gram of substrate used.

2. There should be minimum yield of undesired product formation.

3. It should be of a consistent quality and easily available.

4. It should cause minimal problems during media making and sterilization.

5. It should cause minimal problems in other aspects of the production process

particularly aeration and agitation, extraction, purification and waste treatment.

6. Foaming may aid in contaminating the fermentation medium so the deformers

used in termination should not affect the product formation.

Numerous studies on the nutritional requirement for production of antibiotics and

other non-essential metabolites have demonstrated that there is a link between nutrient

limitation and biosynthesis of secondary product.

Medium formulation consists of two factors as

1. Medium composition and

2. Fermentation conditions which may affect the antibiotic production.


Introduction

Table 2: Factors affecting antibiotic production

Medium Composition Fermentation Conditions

Carbon source

Nitrogen source

Inorganic phosphates

Inorganic salts

Trace metals

Precursors

Inhibitors

Inducers

pH

Temperature

Oxygen

1. Medium Composition:13,14,15,23,24

Carbon Source:-

It is now recognized that the rate at which the carbon source is metabolized can

often influence the formation of biomass or production of primary or secondary

metabolites. Fast growth due to high concentrations of rapidly metabolized sugars is

often associated with low productivity of secondary metabolites.

Readily metabolized carbon sources, such as glucose, can suppress antibiotic

production by preventing the synthesis of a key enzyme in the biosynthesis pathway. The

phenomenon has been referred to as “catabolic repression”.

As in penicillin (PC) fermentation, PC production is better in the presence of

lactose which is slowly utilized than of glucose.

The commonly used carbon sources in fermentations are glucose, maltose,

sucrose, lactose, glycerol, corn steep liqor, molasses etc.

The method of media preparation particularly sterilization, may affect the

suitability of carbohydrates for individual fermentation processes. It is often best to


Introduction

sterilize sugars separately because they may react with ammonium ions and amino acids

to form black nitrogen containing compound which will partially inhibit the growth of

many microorganisms.

Nitrogen Source:-

It is well known that changes in the kind and concentration of nitrogen source

influence greatly antibiotic production. For example, as shown in table, antibiotic

production is inhibited by a rapidly utilized nitrogen source (NH4+, NO3-, certain amino

acids, etc.).

Table 3: Inhibition of antibiotic production by readily utilizable nitrogen sources

Microorganisms Antibiotics

Streptomyces antibioticus

Streptomyces erythreus

Streptomyces kitasatoensis

Streptomyces clavuligerus

Streptomyces viridoflavum

Fusidium coccineum

Oleandomycin

Erythromycin

Leucomycin

Cephamycin

Candihexin

Fisidin

Antibiotic accumulation begins to increase in many cases only after the nitrogen

source in the culture broth has been almost entirely consumed. In candihexin production,

addition of a nitrogen source in the idiophase, returns the fermentation to the

trophosphase and production is reduced. Recently, the presence of nitrogen regulation

was revealed to the enzymatic level in fermentation of cephamycin and patulin.

The inhibition of production by a nitrogen source can be usually avoided by

selecting an adequate production medium with the proper kind of nitrogen source. The

quantity of nitrogen source is chosen keeping in mind the quantity of carbon source

present and this reflect C/N ratio. The use of complex nitrogen sources for antibiotic


Introduction

production has been common practice. They are thought to help create physiological

conditions in the trophophase which favour antibiotic production in the idiophase. For

e.g. in the production of polyene antibiotics, soyabean meal has been considered a good

nitrogen source because of the balance of nutrients, the low phosphorous content and

slow hydrolysis.

Inorganic Phosphate:-

When adding a large amount of inorganic phosphate, consumption of carbon and

nitrogen sources and respiration are accelerated resulting in good growth, but the

production of antibiotics is usually reduced.

Aharonowitz and Demain studied the relation between the concentration of

inorganic phosphate in a medium and the production of antibiotics in the fermentation of

cephalosporin by Streptomyces clavuligerus; the production of cephalosporin was

increased with increase in phosphate until the concentration reached 25 mM. Further

addition of phosphate progressively decreased the production.

Microorganisms have their optimal phosphate concentration for growth in a range

of 0.3 to 300 mM, but the amount of inorganic phosphate adequate for the production of

antibiotics is usually much lower than the amount required for growth (

Introduction

Inorganic salts:-

It is well known for rather long time that addition of inorganic salts such as NaCl

to antibiotic production media increases the production. In 1946, Rake and Domovick

reported that a marked increase in the production of streptomycin was observed by

adding 0.5% NaCl, but addition of larger amount of NaCl usually inhibits the production.

Usually the amount of NaCl added in antibiotic fermentation medium is 0.5% or

less but Okami et al. found that apalasmomycin is produced best in the presence of NaCl

as high as 1.0 ~ 3.0%.

Trace Metals:-

It is obvious that fermentation processes are based on the reaction of enzymes.

Not only enzymes and substrate but co-factors such as metals are needed for the reactions

to proceed smoothly. Therefore, one can presume that some specific metals will be

related to the production of individual antibiotics, and in fact various metals affect the

production of antibiotics.

Table 4: Influence of metals on antibiotic production

Metal concentration (x 10-5 M) Antibiotic Producing organism

Positive effect Negative effect

1. Actinomycin Streptomyces antibioticus Fe (10*) --

2. Monensin Streptomyces cinnamonesis Fe (100) --

3. Neomycin Streptomyces fradiae Fe (1.0), Zn (0.1)

Fe (15), Zn (1.0), Mn (10)

4. Bacitracin Bacillus licheniformis Mn (0.07) Mn (9)

5. Griseofulvin Penicillium griseofulvum -- Zn (20*)

Without * mark : 100% effective with concentration

With * mark: 50% effective with concentration


Introduction

The amount of trace metals required for the antibiotic production is larger than

that required for growth: the concentrations added for growth are usually 10 to 100 fold

greater. Addition of further amounts inhibits production.

Precursors:-

It is well known that in penicillin fermentation by Penicillium chrysogenum a

high amount of penicillin-G is selectively produced by adding phenylacetate as a

precursor. Although the addition of precursors has been tried with many antibiotics, it is

rather have for the antibiotic production to increase merely by addition of precursors.

Inhibitors:-

Upon adding ethionine, an analog of methionine, and sulfa drugs affecting carbon

transfer reactions to antibiotic fermentations, demethylated derivatives of the original

antibiotics are produced. These include 7-chloro-6-demethyltetracycline in tetracycline

fermentation and N-demethyllincomycin in the lincomycin fermentation.

It is known that chloramphenicol inhibits protein biosynthesis, but does not inhibit

biosynthesis of peptide antibiotics. The production of actinomycin and polymyxin, which

are peptide antibiotics, is reported to increase by chloramphenicol addition.

The relationship between antibiotics and producing microorganisms present many

interesting problems for e.g. the autotoxic antibiotics which inhibit the producing

microorganisms themselves and xenotoxic antibiotics such as antimycin, penicillin,

polyene macrolides and polyomines which have no target site in the producing

microorganisms.

Inducers:-

Inducers and inhibitors, similar to their roles in primary metabolism, influence the

biosynthesis of antibiotics, and work together to control the production of substances. To


Introduction

reveal the presence of inducer is more difficult experimentally than inhibitors, and this is

the reason that understanding of inducers is meager with inhibitors.

For example, a small amount of A-factor induces streptomycin production and

lactose with the formula C12H22O4 inducers staphylomycin production.

One unusual application of an inducer is the use of yeast mannan in streptomycin

production (Inamine et al., 1969). During the fermentation varying amounts of

Streptomycin and mannosido streptomycin has only 20% of the biological activity of

streptomycin, the former is undesirable product. The production organism Streptomyces

griseus can be induced by yeast mannan to produce β-mannosidase which will convert

mannosidostreptomycin to streptomycin.

2. Fermentation Conditions:13,14,15,22

pH:-

The pH of fermentation affects not only the growth but the production as well as

does the medium constituents and the temperature. The inhibition of antibiotic production

by glucose or K2HPO4, is not only to the above described regulatory controls but also to

the effect on pH during fermentation. When keeping the pH of the culture broth at about

6.0 by addition of CaCO3, K2HPO4 or NaHCO3 in the fermentation of helvolic acid and

cerulenin by Cephalosporium caerulens, production of the former increased, but that of

the latter was little affected.

The control of pH may be extremely important if optimal productivity is to be

achieved. A compound may be added to the medium to serve specifically as a buffer, or

may also be used as a nutrient source. Many media are buffered at about pH 7.0 by the

incorporation of calcium carbonate. The balanced use of the carbon and nitrogen sources

will also form a basis for pH control as buffering capacity can be provided by the


Introduction

proteins, peptides and amino acids, such as in corn steep liquor. The pH may also be

controlled externally by addition of ammonia or sodium hydroxide and sulfuric acid.

Although pH of a medium is often noted after sterilization, but since the medium has

been subjected to severe conditions of high pressure sterilization before use, the pH

before sterilization is also important.

Temperature:-

It is known that thermophilic actinomycetes such as Thermoactinomycetes

produces new antibiotics at temperature higher than 40°C, but Streptomyces usually

produces antibiotics at temperatures near 27°C. Generally the range of a temperature

supporting good growth is as wide as 25 degrees, but the temperature range adequate for

good production of secondary metabolites is narrow i.e. 5 ~ 10 degrees.

Usually, cultivation for antibiotic production is performed under one constant

temperature from the beginning to the end, but the temperature adequate for growth is not

always the same as that for production. When a penicillin producing strain was grown at

30°C and then shifted to 20°C for production a highly effective process was obtained.

Streptomyces species no.81 strain, which produces antibiotic M-81 at 27°C, forms

cryomycin at the low cultivating temperature of 12°C. Thus temperature must be

considered separately for growth and for production. It would be of interest in antibiotic

screening to use temperature shifts.

Oxygen:

Many antibiotic producing microorganisms require oxygen for growth. The water

solubility of oxygen is very low and scale-up of antibiotic fermentation is based on

dissolved oxygen in a cultivation medium. For determining the conditions for large scale


Introduction

cultivation of individual antibiotics, aeration and agitation conditions are selected

depending on the optimal concentration of dissolved oxygen.

In the fermentation of Cephalosporium acremonium, penicillin-N production

decreases and cephalosporin C increases as dissolved oxygen is increased; it is caused by

the oxidative conversion of penicillin-N to cephalosporin.

The optimal level of dissolved oxygen may be different between growth and

production. Therefore, it is necessary to cultivate the fermentation not under one aeration

or agitation condition, but with shifts in this parameter.

Others:

Besides the fermentation parameters described above, there are other factors that

affect antibiotic production such as pressure, oxidation-reduction potential and light.

Although agitation is usually considered only from the viewpoint of oxygen, it

may have other effects. Cellular damage by agitation affects the production of

maridomycin by Streptomyces hygroscopicus. In the bicyclomycin fermentation by

Streptomyces sappronensis, the producing microorganism suffers a reduction in antibiotic

production by increased agitation, concomitant with an absence of aerial mycelium.

The Isolation and Purification of Antibiotics:9,17

The objective of isolation is to separate the comparatively minute amount of

antibiotic from the producing cells and the large volume of spent medium.

The first step in the recovery process is the removal of mycelium and /or cells by

filtration or centrifuging. Continuous vacuum filters, Bird-Young filters, basket

centrifuges or other equipment may be used depending on the nature of the cells.


Introduction

The second important step is to remove the antibiotic from the spent production

medium by solvent extraction, adsorption or precipitation, or a combination of methods.

Solvent extraction may be accomplished in countercurrent extractions such as

Podbielniak, in towers, in the Craig distribution system or otherwise, depending on the

volume of material and the objective. Amyl acetate, ethyl acetate, ether, chloroform,

acetone, and other solvents may be used for the extraction, according to the solubilties of

the antibiotic. The compatibility of solvents for antibiotics should be checked by

biological assay methods. Temperature and pH control are extremely important factors in

securing high yields of the antibiotics.

Adsorption may be carried out on active carbon, ion-exchange resins or other

materials. Purification is accomplished by additional solvent extraction, by distillation, by

sublimation, by column chromatography or by other means, depending on the nature of

the antibiotic, cost, etc. The product may be sterilized and rendered free of pyroens

(fever-producing substances) by filtration through seitz, molecular or other bacterial

filters of a suitable nature.

Benefits of Strain Improvement:12,14,18,19,20,21

Improvement of microbial strains for overproduction of industrial product has

been the hallmark of all commercial fermentation processes. Fermentation economics are

driven by the profitability of a marketed product. A key component of this value is based

on manufacturing cost per unit of product (Parekh 1999).

Lower fermentation, manufacturing and capital cost can be gained from

improvements in fermenter design engineering (Doran 1995). However, improvement of

the microbial production strain offers the greatest opportunity for cost reduction without

significant capital outlay. Natural isolates usually produce commercially important


Introduction

products (e.g. antibiotics) in very low concentrations and therefore every attempt is made

to increase the productivity of the chosen organism. Increased yields may be achieved by

optimizing the culture medium and growth conditions, but this approach will be limited

by the organisms’ maximum ability to synthesize the product. The potential productivity

of the organism is controlled by its genome and, therefore, the genome must be modified

to increase the potential yield. The cultural requirements of the modified organisms

would then be examined to provide conditions that would fully exploit the increased

potential of the culture. Thus, the process of strain improvement involves the continual

genetic modification of the culture, followed by reappraisal of its cultural requirements.

Dulaney and Dulaney (1967) compared the spread in the productivity of

chlortetracycline of natural variants of Streptomyces viridifaciens with the spread in

productivity of the survivors of an ultraviolet treatment. The results of their comparison

are shown in figure 3 and 4, and from which it may be seen that although there are more

infectious producers amongst the survivors of the ultra violet treatment, there are also

strains producing more than twice the parental level, far greater than the best of the

natural variants.

The use of ultraviolet light is only one of a large number of physical or chemical

agents which increase the mutation rate – such agents are termed mutagens.

Thus, strain improvement programmes had to be developed which meant that they

depended on the random selection of the survivors of mutagen exposure. Elander and

Vournakis (1987) described these techniques at “hit or miss” methods that require brute

force, persistence and skill in the art of microbiology”. However, despite the limited

knowledge underlying these approaches they were extremely effective in increasing the

yields of antibiotics (Table 5).


Introduction

0

5

10

15

20

25

30

20 40 60 80 100 120 140 160 180

Productivity (%)

% o

f Cul

ture

Fig.3: The spread in productivity of chlortetracycline of natural variants of


0

5

10

15

20

25

30

20 40 60 80 100 120 140 160 180

Fig.4: The spread in chlortetra productivity of a UV-treated population of



Introduction

Table 5: Improvement of antibiotic yields during the first 20 years of antibiotic

development (Riviere, 1977)

Antibiotic Initial yield at time of discovery (units cm-3)

Improve yield in France 1972 (Units cm-3)

Penicillin

Streptomycin

Chlortetracycline

Erythromycin

20 (1943)

50 (1945)

200 (1948)

100 (1955)

12,000 – 15,000

12,000 – 15,000

12,000 – 15,000

300

The benefits of strain improvement is achieved when a strain is selected that can

synthesize a higher proportion of the product using the same amount of raw materials i.e.

the productivity of the culture and process is enhanced. Alternatively, strains that utilize

either low-cost materials such as starch or corn syrup, or spent products like molasses

(instead of refined glucose) can significantly reduce fermentation costs. In addition

fermentation capacity can be freed (or new capital outlay avoided) via strain

improvement to facilitate the launch of other fermentation products at a multi-product

fermentation plant. Regardless of the methods or strategy, strain improvement relies on

the iteration of three operations: genetic alterations, fermentation, and assay.

Mutation: (Strain Improvement) 19,21

Mutation is the permanent alteration of one or more nucleotides at a specific site

along the DNA strand. The strain that harbour the mutation is called a mutant strain.

Mutation may be associated with the change of a single nucleotide (point

mutation), through substitution, deletion, or rearrangement of one or more nucleotide

base pairs in the chromosome. Mutations may also arise due to faulty re-union of DNA.

Most mutations occur at low frequency at any point along the gene (10-5–10-10/

generation). In many cases mutations are harmful, but certain mutations occur that make


Introduction

the organism better adapted to its environment and improve its biocatalytic performance.

The challenge is to isolate those strains which are true mutants and which carry beneficial

mutations.

In nature, metabolism in microbes is carefully controlled to avoid wasteful

expenditure or the enzymes needed for their biosynthesis. This tight metabolic and

genetic regulation and synthesis of biologically active compound is ultimately controlled

by the nucleotide sequences that program the biological activities. To improve microbial

strains, the sequence of the genes must be altered and manipulated. It is assumed that the

manipulations result in subtle alteration and reprogramming of the DNA (on the genes) to

shift or bypass the regulatory controls and check points. Changes in the genetic

programming manifest variability in a population of mutated cells. Ultimately, through

genetic alteration, strains are anticipated to evolve which are likely to be relaxed in

regulation and capable of devoting their metabolic machinery towards producing key

biosynthetic enzymes, leading to overproduction of metabolites to the levels needed for

economical industrial use.

Modification and improvement of the strain through mutation are typically

achieved by subjecting the genetic material (in vivo or in vitro) to a variety of physical or

chemical agents are called mutagens (Table 6).


Introduction

Table 6: Modification and improvement of the strain by various mutagens

Mutagen Mutation induced Impart on DNA Relative effect

Radiations: UV radiations like x-rays, gamma rays

Single or double stranded breakage of DNA

Deletions structural changes

High

Short wavelengths: 2. Ultraviolet rays

Pyrimidines diamerizations and cross links in DNA

Transversion, deletion, frame shift, transitions from GC AT

Medium

Chemicals: Base analogs 3. 5-chlorouracil 5-Bromouracil

Results in faulty pairing

AT-GC, GC AT transition AT GC, GC

AT transition

Low

Low

4. 2-Aminopurine deaminating agents

Errors in DNA replication

GC AT transition

Low

5. Hydroxylamine (NH2OH) Deamination of cytosine

Bi-directional translation, deletion

Low

6. Nitrous and (HNO2) Deamination of A, C and G

AT GC and /or GC AT

Medium

Alkylating Agents: 7. N-methyl-N’-nitro N-Nitrosoguanidine

Methylation, high pH

GC AT transition

High

8. Ethyl methane – Sulfonate Alkylation of C and A

GC AT transition

High

9. Mustards di-(2-chloroethyl)-sulfide

Alkylation of bases C and A

GC A transitions

High

Intercalating agents: 10. Ethidium bromide, acridinedyes

Intercalation between two base pairs

Frame shift, loss of plasmids and microdeletions

Low

Biologicals: 11. Phage, plasmid, DNA transposing

Base substitution and breakage

Deletions, duplication, insertion

High


Introduction

The objective of mutagenesis for developing improved strain is to maximize the

frequency of desired mutations in a population (culture).

Screening of Mutants:19

After inducing a mutation, survivors from the population are randomly picked and

tested for their ability to produce the metabolite of interest. Screening a large number of

mutated organisms usually identifies improved mutants. Moreover, it offers a significant

advantage over the genetic engineering route by yielding gains with minimal start-up

time and sustaining such gains over years, despite lack of scientific knowledge of the

biosynthetic pathway or genetics of the producing microbe.

Random screening has been widely adopted by the fermentation industry

following its successful improvement in penicillin titers since World War II.


Need and Objectives

NEED AND OBJECTIVES

With the rapid biotechnological advances in infectious disease management threat

poised by the emergence of highly resistant infectious agents becomes the next challenge.

The antibiotic earlier shown to be effective in controlling a microorganism is no longer

able to do so. The strike-back of pathogens has revitalized the search for new antibiotics

to counter drug-resistant bacteria, fungi, and viruses. In this respect, the new antibiotics

obtained from actinomycetes and other bacteria, having inhibition spectra for gram-

positive and gram negative organisms, should not be toxic to human beings, plants and

animals.

The mass production of antibiotics began during World War II with the invention

of streptomycin and penicillin. Their specific action against particular group of organisms

made their use more important in medical, veterinary and agricultural practices. But more

vexing problem is the emergence of resistant strains among the microorganisms that were

sensitive to antibiotics before the drug became widely used. This phenomenon tends to

limit severely the useful life of any new antibiotics, requiring the pharmaceutical industry

to come up with new compounds continually. The need for new antibiotics is especially

acute because of the following unfortunate situation. In any modern hospital, huge

amount of antibiotics are used in the treatment as well as the prevention of infectious

disease. As a result, the hospital environment becomes highly enriched for

microorganisms that are resistant to those antibiotics. At the same time, the immune and

other defense mechanisms of the body are not functionally well in many hospitalized

patients, who are thus especially vulnerable to ‘nasocomial’ (hospital-acquired) infection

by these resistant bacteria. Scientists all over the world are constantly working to

discover newer effective antibiotics to combat resistant strains.


Need and Objectives

Screening of antibiotics has been widely performed for about 50 years, and new

antibiotics are still being found. However, the possibility of discovering new antibiotics

only by random screening of microorganisms (actinomycetes and other bacteria

producing antibiotics) is reduced now-a-days, and new approaches are required for

finding new antibiotics efficiently.

In the screening of new antibiotics, three major factors are considered as:-

1) Detection methods

2) Selection of producing microorganisms

3) Cultivation method or fermentation conditions

These are closely related to each other, and their efficient combination is useful

for successful screening of new antibiotics.

Modification of the fermentation process by changing the medium composition,

mutation and cultivation temperature has been proved successful in the screening of new

antibiotics.

By changing detection methods for microorganisms producing antibiotics from

soil such as varying the incubation temperatures can give rise rare actinomycetes from

which new antibiotics can be discovered. And it is possible to increase the production of

a trace antibiotic component by changing the constituents of the medium or the

cultivation conditions, resulting in the discovery of a new antibiotics.

Microbes also have proved to be an exceptionally rich source of other useful

products such as enzymes, proteins, organic acids and there is every indication that they

will continue to be so in the future.


Need and Objectives

During last 2-3 decades, focus has been centered on actinomycetes for their

ability to produce secondary metabolites mainly antibiotics. More than 75% of broad

spectrum antibiotics currently in use are derived from actinomycetes.

Literature based reports reveal that actinomycetes produce a large number of

antitumor antibiotics, potent antifungal antibiotics, anti-HIV proteins

(e.g., Actinorhodins) and anthelmintics either in pure form or in mixture of compounds.

Recent research works have proved that most of microorganisms develop

resistance to existing antibiotics, which provokes the need for a constant research on

production of newer antibiotics in order to overcome resistant microorganisms.

The biosynthesis of antibiotic involves very complex procedure as they are

secondary metabolite which does not play any role in the growth and culture conditions

of microorganisms. As the submerge fermentation condition does not support the

formation aerial growth of actinomycetes as a result the antibiotic yield would be low in

submerged condition. Therefore, it is important to increase the yield of antibiotic by

varying the medium composition and fermentation conditions.

The important objectives of the present research work are as follows:-

1. To isolate an actinomycete by various new detection methods, from soil sample

which may be capable of producing a newer antibiotic.

2. To increase antibiotic yield by optimizing different fermentation parameters and

by strain improvement techniques.

3. To make the process economical in pilot scale and to enhance the scale up

techniques.


Need and Objectives

Hence, the present research work emphasizes on isolation and strain improvement

of antibiotic producing actinomycetes species and optimization of fermentation process

for maximum production, purification and characterization of produced antibiotic.

Plan of Work:

The research work was planned as:-

1) Isolation and characterization of actinomycetes

a. Collection of soil samples from different places.

b. Screening of soil samples for actinomycetes, capable of producing

antibiotic.

c. Morphological and taxonomical characterization of isolates.

d. Preliminary screening for the effectiveness of crude antibiotic produced.

2) Strain improvement:

a. Enhancement of antibiotic productivity by strain improvement programme

using UV radiations.

b. Isolation of stable mutants

c. Comparison of antibiotic productivity of mutated strain with parent strain.

3) Optimizati

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