Dooccttoorr ooff PPhhiilloossoopphhyy -...

ISOLATION AND CHARCTERIZATION OF ANTIBIOTIC PRODUCTION FROM SOIL

ISOLATES BY FERMENTATION

TThheessiiss ssuubbmmiitttteedd iinn

PPaarrttiiaall FFuullffiillllmmeenntt ffoorr tthhee aawwaarrdd ooff

Degree of

DDooccttoorr ooff PPhhiilloossoopphhyy IInn

PHARMACEUTICAL SCIENCE (PHARMACEUTICS)

BByy

CHANDRASHEKHARA.S

MM.. PPhhaarrmm

UUnnddeerr tthhee GGuuiiddaannccee ooff

DDrr.. BASAVARAJ K. NANJWADE MM.. PPhhaarrmm..,, PPhh..DD

VINAYAKA MISSIONS UNIVERSITY

SALEM, TAMILNADU, INDIA.

FEBRUARY - 2010

VINAYAKA MISSIONS UNIVERSITY, SALEM

Declaration By The Candidate

I declare that the thesis entitled “ISOLATION AND

CHARCTERIZATION OF ANTIBIOTIC PRODUCTION FROM SOIL

ISOLATES BY FERMENTATION” submitted by me for the Degree

of Doctor of Philosophy is the record of work carried out by

me during the period from OCTOBER 2005 to NOVEMBER 2009

under the guidance of DR. BASAVARAJ K. NANJWADE M.PHARM.,Ph.D

and has not formed the basis for the award of any degree,

diploma, associate-ship, fellowship, titles in this or any other

University or other similar institutions of higher learning.

Place: Belgaum Mr. CHANDRASHEKHARA .S M.PHARM Date: Dept. of Pharmaceutics

KLE College of Pharmacy Belgaum, Karnataka– 590 010

?

? ?

? ? ?

?

?

?

? ?

?

VINAYAKA MISSIONS UNIVERSITY, SALEM

CERTIFICATE BY THE GUIDE

I certify that the thesis entitled “ISOLATION AND

CHARCTERIZATION OF ANTIBIOTIC PRODUCTION FROM SOIL

ISOLATES BY FERMENTATION” submitted for the degree of

Doctor of Philosophy by MR. CHANDRASHEKHARA .S is the

record of research work carried out by him during the period

from OCTOBER 2005 to NOVEMBER 2009 under my guidance and

supervision and that this work has not formed the basis for

the award of any degree, diploma, associate-ship, fellowship or

other titles in this University or any other University or

Institution of higher learning.

Place: Belgaum DR. BASAVARAJ K. NANJWADE,

Date: M.PHARM., Ph.D AASSSSOOCCIIAATTEE PPRROOFFEESSSSOORR

DDeepptt.. ooff PPhhaarrmmaacceeuuttiiccss KK LL EE CCoolllleeggee ooff PPhhaarrmmaaccyy BBeellggaauumm,, KKaarrnnaattaakkaa–– 559900 001100.. ?

? ?

? ? ?

?

?

?

? ?

?

Affectionately Dedicated to

My beloved Father and Brother

Shri. Shivanna.B.and Lokeshappa.S

ACKNOWLEDGEMENT “GRATITUDE IS THE MEMORY OF THE HEART”

- J. B. Massiew.

I take this opportunity to sincerely thank all the people involved either

directly orindirectly in my research work.

It is my pleasure to express deep sense of gratitude and thankfulness to my

beloved guide Dr. Basavaraj K. Nanjwade Associate Professor, Department of

Pharmaceutics, K.L.E.S’s College of Pharmacy, Belgaum, for his excellent guidance

and co-operation during this study. His simplicity, caring attitude and provision of

fearless work environment will be cherished in all walks of my life. I express my

cordial thanks to Dr. F. V. Manavi, Principal, K.L.E.S's College of Pharmacy,

Belgaum, for providing me all the necessary facilities for the research work. I also

express my sincere thanks to all the staff members of K.L.E.S’s College of Pharmacy,

Belgaum, for their generous and kind help. I express my heartiest gratitude to

Dr. P. B. Kore, Chairman, Board of Management, K.L.E. Society and chancellor, K

L E University, Belgaum and also Members of Board of Management, K.L.E.

Society, Belgaum for their visionary towards their staff.

I offer my sincere and humble gratitude to Prof.J.K. Saboji Principal,

K.L.E.S’s College of Pharmacy, Nippani, Prof. A. D. Taranahalli, Vice-Principal

and staff of K.L.E.S's College of Pharmacy, Belgaum for their constant inspiration.

I take this opportunity to express my sincere thanks to Mr. Prakash.

Goudanavar staff of K.L.E.S's College of Pharmacy, Nipani for his intense

support throughout my course of study.

I greatfully acknowledge Prof. R. V. Kardi, Dr. E.N. Gaviraj, D.N. Shastri,

Satish Kavatgimat, V. S. Mannur, Dr. S. R. Pattan, Prof. B. C. Koti, B. N.

Ingalgi, Bharathi, Anjana Adyapak, Ramesh Kinal, Nayan Katib, V. S. Nagoor, C.

K. Sompur, S. K. Nimbal, S. M. Patil, R. S. Bagli, R. D. Hiremath, Konnur, Sakan

Gowda, Miss. Madhuri, Rajshree Patil, Sunanda Chavan, Amrutha, Ravi Raj for

their guidance and invaluable help.

My sincere thanks to Dr. K. S. Patil, Professor & Head, Department of

Pharmacognocy, K.L.E.S's College of Pharmacy, Belgaum for valuable suggestions.

I am immensely grateful to my beloved mother Shivamma and Brother

Veerbhadra Swamy, Yediurappa B. S, Shivakumar N and my daughters Tanmyie,

Ramya, Chethan, Sister Geetha who always covered me under shade of their love,

affection and blessing.

I thanks to my friends Kalogi rao, Maltesh, Vishwa, Venkatesh, Raju R,

Dhanpal, Jagadish, Raju, Savithri Sonad, Anil Motekar, Vijaylakshmi S.G, R. B.

Patil, Shamriz Ali whose constant inspiration and support made my work easy.

My thanks to Miss. Veena and Mr. Deepak of Sai DTP & Xerox centre, for

the help in designing and printing the dissertation copy.

Lastly I thanks to God, the Almighty to show the path to the ladder of

success.

Thankfully I ever remain ……………………….

Chanrashekhara.S

CONTENTS

SL. NO. TITLE PAGE

NO.

1. INTRODUCTION 1-53

2. NEED AND OBJECTIVES 54-59

3. REVIEW OF LITERATURE 60-79

4. MATERIALS AND METHODS 80-109

5. RESULTS AND DISCUSSION 110-172

6. SUMMARY AND CONCLUSION 173-177

7. BIBLIOGRAPHY 178-188

8. ANNEXURE 189-195

LIST OF TABLES

TABLE NO.

TITLE PAGE NO.

1 Microorganisms producing the various antibiotics 11-12

2 Factors affecting antibiotic production 31

3 Inhibition of antibiotic production by readily

utilizable nitrogen sources

33

4 Influence of metals on antibiotic production 37

5 Improvement of antibiotic yields during the first 20

years of antibiotic development (Riviere, 1977)

48

6 Modification and improvement of the strain by

various mutagens

51-53

7 Collection of Soil Samples and Number of

Actinomycetes in Isolation Plates

112

8 Sensitivity of different microorganisms towards the

soil isolates by agar streak method

115

9 Taxonomical Characterization of Soil Isolates 116

10 Morphological and cultural characterization of the

strain A-4

118-119

11 Antibiotic Productivity by UV Mutant Strains 123

12 Comparison of UV mutant strains for the antibiotic

production

124-125

TABLE NO.

TITLE PAGE NO.

13 Effect of Streptomycin and Rifampin on UV

mutants and antibiotic productivity of UV-drug

resistant mutants

127

14 Morphological and cultural characterization of the

strain A-4 mutant

129

15 Optimization of Carbon Source 132

16 Optimization of Nitrogen Source 136

17 Optimization of Temperature 139

18 Optimization of pH for optimum growth and

antibiotic productivity

143

19 Optimization of Dissolved Oxygen Concentration

(DO2)

147

20 Optimization of duration of fermentation for the

maximum growth and antibiotic production

151

21 Composition of Fermentation Medium 154

22 Thin layer chromatography of purified

antimicrobial compound

156

23 Determination of MIC for an isolated antimicrobial

compound against bacteria

157

24 Determination of MIC for an isolated antimicrobial

compound against fungi

158

25 Physical properties of purified antimicrobial

compound

158

TABLE NO.

TITLE PAGE NO.

26 IR spectroscopical data and their functional groups 162

27 NMR Spectroscopical data and their functional

groups

162

LIST OF PLATES

PLATE NO.

TITLE PAGE NO.

1 Photograph Showing Crowded Plate Method 164

2 Test for Microbial Sensitivity 164

3 Test for Melanoid Pigment Formation 165

4 Test for Nitrate Reduction 165

5 Test for Milk Coagulation and Peptonization 165

6 Test for Gelatin Liquefication 166

7 Test for Amylolytic Activity by Starch Hydrolysis 166

8 Carbohydrate Assimilation Test 166

9 Morphology of A-4 Strain on ISP-1 and ISP-7 167

10 Morphology of A-4 Strain on ISP-3 & ISP-5 167

11 Morphology of A-4 Strain on ISP-4 167

12 Morphology of A-4 Strain on ISP-6 168

13 Microscopy of A-4 Strain under 10X 168

14 Microscopy of A-4 Strain under 100X 168

15 Microscopy of A-4 Mutant Strain under 10X 168

16 Microscopy of A-4 Mutant Strain under 100X 168

17 Morphology of A-4 Mutant Strain on ISP-3 169


PLATE NO.

TITLE PAGE NO.




22 Photograph showing Laboratory Fermenter of 3L

Capacity of Sartorius B-Lite Company 171

23 Photograph showing Antimicrobial Activity of

Broth Collected at an interval of 24 hr during

bioprocess

171



bioprocess

172



bioprocess

172



bioprocess

172

LIST OF FIGURES

FIGURE NO.

TITLE PAGE NO.

1 Antibiotic gene clusters encoding doxorubicin. 22

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

streptomycin production in S.griseus

25

3 The spread in productivity of chlortetracycline of

natural variants of Streptomyces viridifaciens

46

4 The spread in chlortetra productivity of a UV-treated

population of Streptomyces viridifaciens

47

5. Solvent Extraction Tree 104

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

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

Strains

133

7. Comparison of Zone of Inhibition at different

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

Strains

134

8. Comparison of Packed Cell Volume at Different

Nitrogen Sources for A-4 and A-4 Mutant Strains

137

9. Comparison of Zone of Inhibition at Different

Nitrogen Sources for A-4 and A-4 Mutant Strains

138

10 Comparison of Packed Cell Volume at Different

Temperatures for A-4 and A-4 Mutant Strains

140

Vinayaka Mission University, Salem

FIGURE NO.

TITLE PAGE NO.

11 Comparison of Zone of Inhibition at Different

Temperature for A-4 and A-4 Mutant Strains

141

12 Comparison of Packed Cell Volume (%) at specified

pH for A-4 and A-4 Mutant Strains

144

13 Comparison of Zone of Inhibition at different pH for

A-4 and A-4 Mutant Strains

145

14 Comparison of Packed Cell Volume (%) at Different

DO2 Levels for A-4 and A-4 Mutant Strains

148

15 Comparison of Zone of Inhibition at Different DO2

Levels for A-4 and A-4 Mutant Strains

149

16 Comparison of PCV at Different Fermentation

Durations for A-4 and A-4 Mutant Strains

152

17 Comparison of Zone of Inhibition at Different

Fermentation Durations for A-4 and A-4 Mutant

Strains

153

18 IR Spectrum for an Isolated Antibiotic 160

19 NMR Spectrum for an Isolated Antibiotic 161


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 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.

? 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 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.

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

? m 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

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.

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.


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


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 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), ? -lactum (cephalosporins), macrolides (erythromycin),

and tetracyclines.


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.

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


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.

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.


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. A-factor [?-

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. (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.

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.

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.

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.


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 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

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 (<10 mM), similar to the

carbon or nitrogen source.

As described above, the amount of inorganic phosphate

strongly influences the production of antibiotics, but the precise

mechanism has not been clarified. However, it is presumed that when

the concentration of inorganic phosphate in the culture is high, the

intracellular concentration of ATP is increased and the primary

metabolism is accelerated. When the amount of inorganic phosphate

is lowered, the ATP concentration decreases; this decrease

depresses metabolic conversions which are required for the

production of antibiotics.

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


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 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 ? -

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 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 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.

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 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).

0

5

10

15

20

25

30

20 40 60 80 100 120 140 160 180

Productivity (%)

% o

f Cu

ltu

re

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

variants of Streptomyces viridifaciens


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 Streptomyces viridifaciens


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 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).


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

Low

Low


AT ? GC,

GC ? AT

transition

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

Frame shift,

loss of

Low


pairs plasmids and

microdeletions

Biologicals:

11. Phage, plasmid, DNA

transposing

Base substitution

and breakage

Deletions,

duplication,

insertion

High

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

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.

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.

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.

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) Optimization of fermentation media for maximum antibiotic

productivity.

a. Medium formulation

b. Optimization of fermentation parameters

i. pH

ii. Temperature

iii. Dissolved oxygen concentration

4) Down stream processing

a. Solvent extraction

b. Thin layer chromatography

5) Determination of spectrum of activity against different strains of

microorganisms.

6) Structural identification of an antibiotic

a. UV spectroscopy

b. IR spectroscopy

c. NMR spectroscopy


REVIEW OF LITERATURE

By far the most extensively studies of the actinomycete

suborders is Streptomycineae, mostly because the genus

Streptomyces has been the source of vast numbers of useful

antibiotics. Streptomyces are widespread in soil and are the most

frequently isolated actinomycetes. Hundreds of Streptomyces spp.

have been described, and more than 70 have phylogenetically

sorted.

The spore-to-spore streptomycete life cycle progresses through

several stages of differentiation: germ tubes emerge from spores and

grow into filamentous hyphae that branch to form a dense “substrate

mycelium”; certain hyphae grow away from the substrate to form an

“aerial mycelium”, and finally, the aerial hyphae septate into

compartments that become spores. The various Streptomyces

species aerial hyphae differ in their spore chain morphologies (e.g.,

smooth, spiny, hairy or warty).1

EO Stapley et al. (1972)45 isolated a number of actinomycetes from

soil which were found to produce one or more number of a new family

of antibiotics, the cephamycins, which are structurally related to

cephalosporin C. The cephamycin were produced in submerged

fermentation in a wide variety of medium by one of more or eight


different species of Streptomyces, including a newly described

species, S.lactamdurang. These antibiotics exhibit antibacterial

activity against a broad spectrum of bacteria which includes many

that are resistant to the cephalosporins and penicillins.

Yasuji et al. (1975) demonstrated a new antibiotic, fumaramidmycin,

has been isolated from a Streptomycete NR-7GG1 which was

characterized and named Streptomyces kurssanovii. The strain

produced the antibiotic only when grown on agar plates but not in the

submerged culture broth, where the contact with the vegetative

mycelia appears to cause the inactivation of the antibiotic. The

antibiotic shows an antimicrobial activity against both gram-positive

and gram-negative bacteria.

Satoshi Omera et al. (1982)14 reported that 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 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 methods, selection of producing

microorganisms, and cultivation methods. These are closely related

to each other, and their efficient combination is indispensable for


successful screening. Since Waksman intentionally employed

Mycobacterium tuberclosis 607 as a test organism in seeking anti-

tuberculosis drugs and found streptomycin, many antibiotics with anti-

bacterial, anti-fungal, anti-protozoal, anti-parasitic, anti-viral and anti-

cancer activities have been discovered by employing various

detection methods. Though the detection method in screening is

important, new substances can be found by combining the detection

method with the improved methods of culture selection and

cultivation.

Richard A. Nelson et al. (1986)48 isolated a microorganism

designated as Rv-79-9-101 and now identified as Micromonospora

purpurea chromogenes subspecies halotolerans, from a mud sample

in the Philippines, has shown to produce a complex of antibiotics

called crisamicins. Thin layer chromatography and bioautography,

employing solvent extracts of whole fermentation broths, revealed a

minimum of five antimicrobial components. The major biologically-

active component of the antibiotic complex-crisamicin A, was

obtained in pure from after preparative silica gel column

chromatography followed by crystallization. Based on

physicochemical data crisamicin A has been identified as a novel

member of the isochromanequinone group of antibiotic. It exhibits in


vitro activity against gram positive bacteria but little or no activity

towards gram-negative bacteria or fungi.

Juan Soliveri et al (1988)24 described the effect of different nutrients

on the production of the macrolide polyene antibiotics. PA-5 and PA-

7, produced by Streptoverticillium sp. 43/16. Optimal production

yields of PA-5 and P-7 have been achieved with L-proline and glycine

as nitrogen sources, respectively. The presence of manganese as a

metallic ion stimulated the antibiotic production significantly. The

requirement of phosphate for optimal yields of both antibiotics (50

mM) is much higher than those descried for strains of Streptomyces,

which produce other macrolide polyene antibiotics. Magnesium is

essential for this production. The specific production of PA-5 and PA-

7 increases with concentrations of glucose up to 40 mM, but is

inhibited at higher concentrations. It was found that the presence of

ammonium salts and the amino acids L-cysteine and/or L-

valine as nitrogen sources has negative effects on the production of

both antibiotics.

James A. Matson et al. (1989)28 isolated Actinomadura melliaura

culture, SCC 1655 from a soil sample collected in Bristol Cove,

California by plating soil suspensions on water agar containing 75

? g/ml Cephalosporin C. The compounds AT2433-Al (A1), AT2433-A2


(A2), AT2433-B1 (B1) and AT2433 B2 (B2) were isolated from the

culture broth of A.melliaura, SSC 1655. Structurally these compounds

are closely related to rebeccamycin (L), an indolocarbazole antitumor

antibiotic. A1, A2, B1 and B2 were active against Staphylococcus

aureus A9537, Streptococcus faecalis A20688, Streptococcus

faecium (ATCC 9790), Micrococcus lutea (ACC 9341), Bacillus

subtilis (ATCC 6633). A1 and B1 were active against p388 leukemia

in mice.

Keuichi Suzuki et al. (1989)9 isolated a strain SCM-127 identified as

Streptomyces hygroscopicus subspecies luteolus subspecies nov,

was found to produce a new antitumor antibiotic, phospholine. They

described the taxonomy of the producing strain, isolation,

physicochemical and biological properties of phospholine.

MP Lechevalier et al. (1989)50 was isolated a novel family of

antitumor antibiotics, the calicheamicins from the fermentation broth

of Micromonospora echinospora subspecies Calichensis. These

antibiotics exhibited significant activity against gram-positive and

gram-negative bacteria in vitro . Calicheamicin ?1I demonstrated

antitumor activity against P388 leukemia and B16 melanoma in vivo.

PDA James et al. (1991)22 reported that Streptomyces

thermoviolaceus was grown in a chemostat under conditions of


glutamate limitation. The effects of growth rate on production of the

antibiotic granaticin, extra cellular protein and protease activity as

components of secondary metabolism were studied a 37, 45 and

50?C. The amount of each secondary metabolite synthesized was

highly dependent on growth rate and temperature. Granaticin yields

were highest at growth rates of 0.1 to 0.15 h-1 at 37?C, 0.175 h-1 at

45?C and 0.045 h-1 at 50?C. Protease activity of culture supernatants

responded to low nutrient concentration and/or low growth rate.

Measurement of extracellular protein revealed complex changes in

amount which were dependent on growth rate and temperature. At

45?C and a growth rate of 0.15 h-1, biomass yeld was highest

between pH 5.5 to 6.5 whereas granaticin synthesis was low at pH

5.5 and rose to highest values at between pH 6.5 and 7.5.

Masayuki Hayakawa et al. (1991)37 described the two methods for

the isolation of the rare actinomycetes Streptosporangium and

Dactylosporangium from soil. The methods use the ability of both the

sporangiospores of Streptosporangium and the globose bodies

(aleuriospore) of Dactylosporangium to withstand drug heating and

treatment with benzethonium chloride (BC). In addition, the

differential antibiotic resistance of these actinomyetes is also utilized

(i) to isolate Streptosporangia, an air-dried soils ample is first


subjected to dry heat treatment (120?C, 1 hr). A water suspension of

the heated sample is then treated with 0.01% BC, diluted and

cultured on HV agar supplemented with nalidixic acid and

leucomycin. (ii) To isolate Dactylosporangia, a water suspension of a

soil sample given dry heat and 0.03% BC treatment is cultured on HV

agar supplemented with nalidixic acid and tunicamycin. The dry heat

and BC treatment drastically eliminated bacteria and unwanted

actinomycetes contaminants, including Streptomyces, from the

isolation, plates, thereby facilitating the selective isolation of

Streptosporangia and Dacrylosporangia.

Masayuki Hayakawa et al. (1991)36 described a simplified

enrichment method for the highly selective isolation of the zoosporic

actinomycetes Actinoplanes spp. from soil. The method consists of

baiting the species with Pinus pollen grains, desiccating (30?C, 2 h)

the baits bearing sporangia in dried soil particles with the aid of silica

gel and following the spore liberation upon immersion in water.

Portions of the liquid enriched with zoospores are plated out on humic

acid-vitamin (HV) agar supplemented with nalidixic acid at a

concentration of 10 g/ml. The desiccation stage has enabled the

almost complete elimination of associated bacteria from colonized

baits while allowing the Actinoplanes sporangia to survive and still


possess the ability to release many spores. A total of four different

soil samples from fields of corn, peach, vegetable and paddy rice

were examined. The pollen-baiting and drying method consistently

resulted in the highly selective isolation of Actinoplanes spp. which

accounted for over 83% of the total number of microorganisms

recovered on HV agar containing nalidixic acid.

Ahmed Lebrihi et al. (1992)32 reported the effect of ammonium on

growth and spiramycin biosynthesis in Streptomyces ambofaciens on

a chemically defined medium. Spiramycin biosyntheis was better in

the presence of valine and isoleucine than in the presence of

ammonium. This production was reduced in the presence of excess

ammonium (100 mM). The addition of catabolic intermediates of

valine and isolencine reserved the negative effect of ammonium.

Valine dehydrogenase (VDH), the enzyme responsible for valine,

bencine and isobencine catabolism, was repressed when excess

ammonium was present in the medium. This repression was

approximately 25% when the ammonium concentration was

increased from 50 to 100 mM.

Li H, et al. (1992)51 mutagenized natural non-antibiotic producing

Streptomyces sp. 1254 by UV irradiation and two active mutants were

isolated. Mutant 113 produced novel anthracycline compounds


designed mutactimycins. Mutactimycin A was active against the

bacteriophage of B.subtilis and some viruses in tissue culture. The

mutant 2-6 synthesized a basic water-soluble antimicrobial antibiotic.

Chemical analysis of the whole cell hydrolysate and the

morphological characterization showed that the strain 1254 and its

mutant 2-6 were of chemotype I, belonging to the genus of

Streptomyces, and the mutant 113 was of chemotype IV without

mycolic acid. Co-synthesis test of strain 1254 and a blocked mutant

of strain 113 gave the active compounds identical with mutactimycins.

Using the act I gene as a probe, the southern hybridization revealed

homology between the actinorhodin polyketide biosynthase gene and

the total DNA of the strain 1254, based upon above data, they

concluded that stretpomyces sp. 1254 should have a biosynthesis

pathway for mutactimycin, but some of its genes might fail in

expression and mutagenesis would make the silent gene(s) active.

ST Williams et al. (1993)11 described new methods for the detection

and identification of novel actinomycetes from a range of environment

and also approached to the detection of actinomycetes ranged from

investigation of neglected habitats and extreme environment (e.g.

alkali soils and oil drills) to the analysis of DNA extracted from the

environment and use of specific phages. The continuing problems of

the identification of actinomycete were also considered.


JC Ensing et al. (1993)16 described the physiology of some

actinomycete genera. Actinomycetes are widespread in the

environment and are mainly organotrophic. Studies of their ecology

have been primarily focused on their detection and isolation, with

comparatively little attention to the control mechanisms that

determine their occurrence and behavior in their natural

environments.

Several actinomycete genera produce motile spores. The

significance of flagella proteins and factors influencing spore motility

and germination are considered. The genus Frankia forms nitrogen

fixing associations with non-leguminous plants. Molecular techniques

have been used to clarify the endophyte-host relationship.

Micromonospora species are common in the environment. The

growth and physiology of a gentamicin-producing strains are

described. Thermophilic actinomycetes in the genus

Thermoactinomyces are common in composts and other self-heating

environments.

Masayuki Takizawa et al. (1993)52 isolated actinomycetes from

sediment samples collected in Chesapeake Bay with an isolation

medium containing nalidixic acid, which proved to be more effective

than heat treatment of samples. Actinomycete counts ranged from a


high of 1.4 x 105 to a low of 1.8 x 102 CFU/ml of sediment.

Antimicrobial activity profiles indicated that diverse populations of

Actinoplanets were present at each station. Actinomycetes

constituted 0.15 to 8.63% of the culturable microbial community. The

majority of isolates from the eight stations studied were

Actinoplanetes.

Yuka Momose et al. (1995)35 isolated the rare actinomycete

Actinomadura viridis from a variety of field soils by a new plate culture

method in which a water suspension of dry heated (110?C, 1 h) soil

was treated with 1% phenol and cultured on Humic acid vitamin agar

supplemented with Kamamycin, josamycin, lysozyme and nalidixic

acid. The methods significantly eliminated unicellular bacteria and

undesirable actinomycetes contaminants from the isolation plates,

thereby achieving the highly selective isolation of A.viridis.

Rong Yang Wu et al. (1995)27 isolated that an actinomycete,

designed strain KS3-5, was from a soil sample collected from

Kaohsiung, Taiwan, ROC. This organism is capable of producing a

series of antibiotics that strongly inhibit the growth of gram-positive

and gram-negative bacteria and yeast like fungi. The spore

morphology and cell wall chemotype suggest that strain KS3-5 is a

Streptomycete . Further cultural and physiological characterization


and the DNA homology suggest that strain KS3-5 is identical to

Stretomyces toxytricini.

Shigetoshi Okada et al. (1997)33 described the scale-up of

Streptomyces hygroscopicus subspecies aureolacrimosus in

submerged culture and the production of milbemycin, a 16-membered

macrolide pesticidal antibiotic. The primary scale-up of a dissolved

oxygen (DO) stat culture to 12,000 dm3 failed for milbemycin

production, when only agitation was used for DO control. Whenever

production deteriorated in a plant-scale fermenter, several symptoms

(such as morphological changes) were observed; termed SUDS

(scale up deteriorated production syndrome). A series of experiments

generating SUDS intentionally, replacing agitation, and examining

internal pressure or temperature effects indicated alternative DO

control procedures to prevent SUDS. Among these procedures

investigated, a sole procedure resulted in successful scale-up to

plant-scale (6000 and 1200 dm3). In the control procedure, these

control variables were employed in the order of internal pressure,

aeration, agitation and culture temperature.

Hiromitsu Iino et al. (1997)36 described a simple isolation method for

rare actinomycetes belonging to the family Streptosporangiaceae was

developed, in which a water suspension of air-dried soil was first


treated with chloramine-T (1.0%), a chlorine releasing biocide, diluted

in water, and subsequently cultured on plates of humic acid - vitamin

medium. The chlorinated treatment significantly inhibited the growth

of non-filamentous bacteria and undesirable actinomycete

contaminants on the plates, thereby facilitating the selective isolation

of bacteria from the Herbidospora, Microbispora, Microtetraspora and

Streptosporangium genera.

Koichi Ogata et al. (1997)53 reported a brownish-orange crystalline

antibiotic having acid-base indicating property has been isolated

from the culture filtrate of a strain of Streptomyces grown on MgSO4-

rich medium. The mature aerial mycelium of this strain was gray and

bore long straight chains of spores with smooth surface. Several

taxonomic characteristics were investigated. The antibiotic, named A-

60 had strong activity against gram-positive bacteria and weak

activity against gram-negative bacteria. The effect of MgSO4 and

other metal salts on production of the antibiotic was investigated. The

productivity was accelerated by the addition of MgSO4, MgCl2, MnCl2,

FeSO4, FeCl2 or BaCl2.

Yoshiko Hosoya et al. (1998)20 reported that physiological

differentiation (including antibiotic production) in microorganisms

usually starts when cells encounter adverse environmental conditions


and is frequently accompanied by an increase in the accumulation of

intracellular ppGp. They have found that the acquisition of certain

Streptomycin-resistant (Str) mutations enables cells to overproduce

antibiotics, demonstrating an increase in productivity 5- to 50-fold

greater than that of wild-type strains. The frequency of such

antibiotic-over producing strains among the Str mutants was shown to

range from 3 to 46%, as examined with several strains of the genera

Streptomyces, Bacillus, and Pseudomonas. Analysis of Str mutants

from Bacillus subtilis Marburg 168 revealed that a point mutation

occurred within the rpsL gene, which encodes the ribosomal protein

S12, changing Lys-56 (corresponding to Lys-G3 in E.coli) to Asn, Arg,

Thr, or Gln.

Penka Moncheva et al. (2000-2002)55 reported that forty-seven

actinomyces strains were isolated from antartic soils-nineteen of them

showed antagonistic activity against gram-positive and Gram-

negative bacteria. Six of the strains possessed a broad spectrum of

antibacterial activity. Results obtained from the physiological and

biochemical analyses including determination of 39 characteristics

proved that two of the strains (23 and 29) were similar whereas all the

rest differed among each other. Morpholgoical studies indicated that

the strains belonged to the genera Streptomyces, Actinomadura and

Kitasatosporia.


The three strains showed antibacterial activity against

phytopathogenic bacteria Xanthomonas axonopodis pv. Glycines, X.

vesicatoria, X.axonopodis pv. Phaseoli, Pseudomonas syringae pv.

Biological means for control had been developed yet. The

antibacterial compounds produced by these strains probably

possessed non-polar structure and consisted of several active

components.

Judith Schimana et al. (2000)54 reported two novel angucyclinone-

type antibiotics, simocyclinones D4 and D8, in the mycelium extract

of Streptomyces antibioticus Tii 6040 by HPLC-diode-array and

HPLC-electrospray-mass-spectrometry screening. The compounds

show antibiotic activities against gram-positive bacteria and cytostatic

effects on various tumor cell lines.

Tamotsu Furumai et al. (2000)58 showed Aristostatins A and B; new

members of tellocarcin class of antibiotics were isolated from the

culture broth of an actinomycete strain. The producing strain, TP-A

0316 was identified as Micromonospora sp. Aristostatins were

obtained from the culture fluid by solvent extraction and

chromatographic purification. They showed antibiotic activity against

gram-positive bacteria and antitumor activity.


Kozo Ochi et al. (2001)19 developed a new approach for improving

the production of antibiotic from Streptomyces coelicolor A3(2) by

inducing combined drug resistant mutations. Mutants with enhanced

(1.6 to 3-fold higher) actinorhodin production were detected at a high

frequency (5 to 10%) among isolates resistant to Streptomycin (Str),

gentamycin (Genr) or Rifampin (Rifr), which developed spontaneously

on agar plates which contained one of the three drugs. Construction

of double mutants (str gen and str rif) by introducing gentamicin

or rifampin resistance into an str mutant resulted in further increased

(1.7 to 2.5 fold higher) actinorhodin productivity. Likewise, triple

mutants (Str. Gen Rif) thus constructed were found to have an ever

greater ability for producing the antibiotic, eventually generating a

mutant able to produce 48 times more actinorhodin than the wild-type

strain. Analysis of Str mutants revealed that a point mutation occurred

within the rpsL gene, which encodes the ribosomal protein S12. Rif

mutants were found to have a point mutation in the rpoB gene, which

encodes the ? -subunit of RNA polymerase. Mutation point in gen

mutants still remains unknown. These single, double and triple

mutants displayed in hierarchial order a remarkable increase in the

production of Act II – ORF4, a pathway-specific regulatory protein, as

determined by Western Blotting analysis. This reflects the same

hierarchial order observed for the increase in actinorhodin production.


The superior ability of the triple mutants was demonstrated by

physiological analyses under various cultural conditions. They

conclude that by inducing combined drug-resistant mutations can

cause continuously increase the production of antibiotic in a stepwise

manner. This new breeding approach could be especially effective for

initially improving the production of antibiotics from wild-type strains.

Klaus Gebhardt et al. (2001)56 detected four novel cyclic

homodecapetide antibiotics, stretocidin A ~ D in the mycelium extract

of Stretomyces Sp. Tii 6071 by HPLC-diode-array and HPLC-

electrospray mass-spectrometry screening. The peptides which were

closely related in structure to the tyrocidines and gramicidins of

Bacillus brevis show antibiotic activities against gram-positive

bacteria.

Marcelo Beutasso et al. (2003)57 isolated strain Tii 6239 from a soil

sample collected in Brazil and determined as a new species of the

genus Stretomyces. In the course of HPLC-diode assay screening

program three metabolites were detected in the culture filtrate and

mycelium extract of strain Tii 6239. They were characterized as

members of macrolactum group, the new compound ripromycin,

previously described ikarugamycin and a new derivative of it,

ikarugamycin epoxide. They show antibiotic activity against gram-


positive bacteria and cytostatic effects to various human tumor cell

lines.

Norimasa Tamehiro et al. (2003) reported that the efficacy of

introducing drug resistance to Streptomyces albus producing

Salinomycin (10 mg/ml) produces mutations for further strain

improvement. Mutants with enhanced salinomycin production were

detected at a high incidence (7 to 12%) among spontaneous isolates

resistant to streptomycin, gentamicin, or rifampin. Finally they

successfully demonstrate improvement of the salinomycin

productivity of the industrial strain by 2.3-fold by introducing a triple

mutation.

Kozo Ochi et al. (2003)18 showed certain rpsL (which encodes the

ribosomal protein S12) mutations that confer resistance to

Streptomycin markedly activate the production of antibiotics in

Streptomyces spp. These rpsL mutations are known to be located in

the two conserved regions within the S12 protein. To understand the

roles of these two regions in the activation of silent genes, they used

site-directed mutagenesis to generate eight novel mutations in

addition to an already known (K88E) mutations that is capable of

activating antibiotic production in Streptomyces lividans. Of these

mutants, two (L90K and R9GG) activated antibiotic production much


more than the K88E mutant. Neither the L90K nor the R94G mutation

conferred an increase in the level of resistance to Streptomycin and

Paromomycin. Their results demonstrate the efficacy of the site

directed mutagenesis technique for strain improvement.

JIN Zhi-Hua et al. (2004)29 reported that strain improvement and

medium optimization to increase the productivity of spiramycin were

carried out. Of oil tolerant muant strains screened, one mutant,

Streptomyces ambofaciens XC2-37, produced 9% more spiramycin

than the parent strain S. ambofaciens XC-1-29. The effects of

soyabean oil and propyl alcohol on spiramycin production with

S.ambofaciens XC 2-37 were studied. The potency of S.ambofaciens

XC 2-37 was improved by 61.8% with addition of 2% soyabean oil in

the fermentation medium and 0.4% propyl alcohol at 24 hours after

incubation. The suitable time for feeding propyl alcohol is at 24 hours

after incubation in flask fermentation and at 20 hours after incubation

in fermenter fermentation. The new process with S. ambofaciens XC

2-37 was called up for industrial scale production of spiramycin in a

60m3 fermenter in Xinchang Pharmaceutical Factory.

BP Kapadnis et al. (2004) isolated about 312 actinomycetes from

soil samples on chitin agar. All the isolates were purified and

screened for their antifungal activity against pathogenic fungi. Out of


these, 22% of the isolates exhibited activity against fungi. One

promising isolate with strong antifungal activity against pathogenic

fungi was selected for further studies. This isolate was active against

both yeasts and molds. Various fermentation parameters were

optimized. Based on morphological and biochemical parameters, the

isolate was identified as Streptomyces. The correlation of antifungal

activity with growth dependent production of antimetabolite. Maximum

antifungal metabolite production (600 units/ml) was achieved in the

late log phase, which remained constant during stationary phase, and

it was extracellular in nature.

PC Jain et al. (2004)34 isolated a strain of Streptomyces

purpeofuscus CM 1261, from a sample of compost collected locally,

was found to possess strong antagonistic activity against 4 human

pathogenic fungi i.e. Candida albicans, Aspergillus niger,

Microsporum gypseum and Trichophoton sp. The active antifungal

compound produced by it found to be a heptaene group of polyene

antifungal antibiotic.

Liutskanova DG, et al. (2005) used conventional mutagenesis (UV

irradiation and exposure to nitrosoguanidine) to produce and

regenerate protoplasts, aiming at increasing the antibiotic activity of a

Streptomyces fradiae strain producing tylosin. Variants exceeding the


activity of the initial procedure strain by 0.5 – 28.3% were obtained.

The most active variants were produced by a combined exposure to

UV and nitrosoguanidine, as well as upon regeneration of protoplasts

formed from the cells of clones produced by UV irradiation. Unstable

inheritance of the trait of increased tylosin production was

demonstrated.

Shu-Wei Yang et al. (2005) reported a new microbial metabolite Sch

725424(1) was isolated from the culture of Kitasatospora sp. The

structure elucidation of 1 was accomplished based on NMR

spectroscopic analyses as well as extensive structure elucidation of

its dehydration product Sch 725428 (2). Compound 1 showed

inhibitory activity against Staphylococcus aureus with MIC values 1 ~

2 ? g/ml, and also displayed weak antifungal activity against

Saccharomyces cervisiae (PM 503) with an MIC 32 ? g/ml.

Noemi Antal et al. (2005)47 isolated a new xanthone compound

named retymicin together with galtamycin B and Saquayamycin Z,

new members of the galtamycin and Saquayamycin families

respectively, and the new lumichrome derivative 1-(? -ribofuranosyl)-

lumichrome from Micromonospora strain Tii 6368, isolated from a soil

sample collected in Romania. Retymicin, galtamycin B ad

Saquayamycin Z show cytotoxic effects to various human tumor cell


lines whereas saquayamycin Z is also active against gram-positive

bacteria.

V. Gesheva et al. (2005)23 reported the influence of different carbon

and nitrogen sources on the production of AK-111-81 nonpolyenic

macrolide antibiotic by Streptomyces hygroscopicus 111-81.

Substitution of glucose with lactose or glycerol significantly affected

maximal antibiotic AK-111-81 productivity as the growth rate was

close to that of the basal fermentation medium. Addition of

ammonium succinate to the fermentation medium markedly increased

the antibiotic productivity as the growth rate was low. Divalent ions as

Mn2+, Cu+2, Fe2+ stimulated AK-111-81 antibiotic biosynthesis. These

results allowed to develop a new fermentation medium showing 6-fold

increase of AK-111-81 antibiotic formation compared with the basal

fermentation medium.


MATERIALS AND METHODS

4.1. MATERIALS

1. Laboratory fermentor model LF2

2. Autoclave

3. Glassware-Borosil

4. pH meter

5. Electronic weighing balance

6. FT-IR spectrometer

7. UV spectrometer

8. Micropipettes

9. Incubator

10. Hot air oven

11. Vortex shaker

12. Laminar air flow bench-Klenzaids

CHEMICALS

1. Dextrose IP

2. NaCl

3. Soyabean meal

4. n-Butanol

5. Silicon oil

6. Dimethyl sulphoxide (DMSO)


7. n-Hexane

8. Tryptone

9. Lactose

10. Maltose

11. Fructose

12. Yeast extract powder

13. Nutrient agar

14. Agar powder

15. ISP 2 to 7 media

16. Actinomycetes agar

17. Bennett’s agar

18. Nutrient broth

19. Sucrose

20. Silica gel 60-120 mesh

21. Silicagel powder

22. Chloroform

23. Ethyl acetate

24. Ferrous sulphate

25. Starch (NICE)

26. Magnesium sulphate

27. Manganese sulphate

28. Calcium carbonate


29. Potassium dihydrogen phosphate

30. Methanol

31. Silica gel TLC grade

32. Peptone

33. Water

34. Sodium hydroxide

35. Skimmed milk

36. Glucose


4.2. METHODS

4.2.1. ISOLATION AND CHARACTERIZATION OF SOIL

ISOLATES:

4.2.1.1. Collection of soil from different places:9,25

The soil samples were collected from places, in and around

Belgaum, Karnataka.

The soil samples were dried separately at 37?C for 1 hr in hot

air oven. Then they were cooled to room temperature. 1 gm of each

soil sample was added to a conical flask containing 100 ml of sterile

water and few drops of Tween-80 solution. All flasks were shaken for

30 minutes in orbital shaker incubator at 27?C. These flasks were

considered as stock cultures.

4.2.1.2. Screening of soil samples for actinomycetes, capable of

producing antibiotic by crowded plate technique: 9,35,36,38

A series of culture tubes containing 9 ml of sterile water were

taken. From the stock culture, 1 ml suspension was transferred

aseptically to the 1st tube (10-1), mixed well. From the 1st tube, 1 ml of

suspension was transferred into 2nd tube (10-2), mixed well. Similarly,

dilutions up to 10-5 were made (serial dilution technique). 0.1 ml of

suspension from each culture tube was spread on sterile soyabean-


casein digest medium (SBCD) plates, actinomycetes isolation agar

(AIA) medium plates and starch-casein agar medium plates

aseptically in Laminar-Air flow bench. The plates were incubated at

27?C (±2?C) for 84 hours. The plates were observed intermittently

during incubation.

After 72 hours, whitish pin-point colonies which is the

characteristic of actinomycetes with clear zone of inhibition around it,

were seen. The pinpoint colonies with inhibitory or clear zone of

inhibition were selected and purified into Actinomycetes agar slants.

The selected strains were further purified by multiple streaking

method. The stock cultures of each selected strain was prepared and

maintained in actinomycetes agar slants at +4?C.

The actinomycete colonies isolated from the crowded plate

were selected for the further study which were named as A1, A2,

A3…..A9 (Table 7).

4.2.1.3. Preliminary screening of crude antibiotic produced: 26

Agar Streak Method:-

The microbial sensitivity of the soil isolates were analyzed by

‘Agar streak method’. Each of the isolate was streaked as a straight

line on SBCD medium and incubated at 27?C for about 6 days (144


hours). After 6 days, different strains of microorganisms were

streaked at right angle, but not touching to the streak and incubated

at 37?C for 24 hours in case of bacteria and 27?C for 48 hours in

case of fungi. If, the organism is sensitive against the antibiotic

produced by actinomycetes, then it will not grow near the

actinomycetes.

The length of inhibition given against each test organism was

observed (table 8). The isolated actinomycetes were screened

against following microorganisms.

I) Gram positive Bacteria:

a. Bacillus subtilis

b. Staphylococcus aureus

II) Gram negative Bacteria:

a. Escherichia coli

b. Pseudomonas aeruginosa

III) Fungi:

a. Aspergillus niger

b. Aspergillus terreus

IV) Yeast:

a. Candida albicans

b. Saccharomyces cerevisae


Based on their antimicrobial properties, isolates were chosen

for the further biochemical characterization.

4.2.1.4. Taxonomical Characterization:- 7,28

i) Test for Melanoid pigment formation:-

This test is done to observe the production of pigments in the

mycelia of organisms and also the excretion of pigments into the

media. Pigment production is one of the most significant properties of

actinomycetes. Most of the soil actinomycetes produce melanin

pigments in Waksman medium. Many pigments are produced on

synthetic media which resulted in designation of many forms on the

basis of pigment character such as presence of pigment in the

vegetative or in aerial mycelium or distributed in and around the

colony or dissolved in the medium. These pigments vary greatly in

nature. It depends on the composition of different media, condition of

growth and age of culture. Thus, pigment production is one of the

easily recognizable characteristics of actinomycetes, when media of

known composition and definite conditions of culture are used.

The sterile slants of Waksman media were prepared in culture

tubes. Then, the isolates were streaked by simple streak method on


the slants aseptically and incubated for 96 hours at 27?C. During the

period of incubation, the melanin pigment formation was observed at

every 12 hour of interval for 96 hours (Table 9).

ii) Test for Nitrate Reduction:

Actinomycetes are having ability to reduce nitrate to nitrite. On

the basis of nitrate reduction property, they are divided into three

groups:

a) The actinomycetes which gives little or no reduction

b) The actinomycetes which gives moderate reduction

c) The actinomycetes which gives strong reduction

By considering the above property of actinomycetes, the soil

isolates were evaluated by using ‘organic nitrate broth’.

10 ml of sterile organic nitrate broth for each soil isolate was

prepared. Then loopful of an inoculum of soil isolates was added to

the broth aseptically and incubated at 27?C for 5 days. Its nitrate

reduction property was observed by using,

A) Naphthalene solution

B) Sulfonilic acid solution


To the broth under examination, 2 drops of reagent (A) and 2

drops of reagent (B) were added. A positive reaction shows pink-red

color (Table 9).

iii) Test for Proteolytic Activity:-

a) Milk coagulation and peptonization:-

The proteolytic activity of the soil isolates were evaluated using

pasteurized skimmed milk. The principle involved in this method is

the digestion of milk proteins by actinomycetes. The proteins which

are present in skimmed milk, if get digested, gives positive reaction.

All the soil isolates were inoculated aseptically into the different

sterile culture tubes containing sterile pasteurized skimmed milk and

incubated at 37?C for 48 hours. The tubes were observed daily for 48

hours. The tubes were observed for following reaction.

a) Reduction of litmus paper

b) Change in medium color


These changes take place due to the digestion of milk proteins

and change in the pH of the medium (Table 9).

b) Gelatin Liquefaction:

Soil actinomycetes can hydrolyze gelatin by its exoenzymes.

The protein gelatin is hydrolyzed by exoenzymes secreted by most of

the soil isolates. The nutrient gelatin medium employed in this

experiment will support the growth of most microorganisms. The solid

character of the medium depends upon the gelatin remaining in the

gel state. Many microorganisms produce exoenzymes that are

capable of hydrolyzing gelatin and liquefying the nutrient gelatin

medium.

The sterile slants of nutrient gelatin agar were prepared. The

soil isolates were inoculated into individual tubes of sterile nutrient

gelatin slants by stab culture method. The inoculated tubes were kept

at room temperature for 10 days (Table 9).

iv) Test for Amylolytic activity by starch hydrolysis:-

Amylolytic activity of soil isolates were studied by using starch

agar medium. Most of the soil actinomycetes have the ability to

hydrolyze starch rapidly by the action of amylolytic enzymes.


The sterile slants of starch agar media were prepared. The soil

isolates were streaked on the slants by simple streak method

aseptically, and incubated for a period of 5 to 7 days at 28?C.

Amylolytic activity was observed by using iodine solution which

indicates the hydrolysis of starch (Table 9).

v) Carbohydrate Assimilation Test:-

Type of carbohydrate source utilized by actinomycete is an

important biochemical property for the identification of actinomycete.

Assimilation is the utilization of carbon source by microorganisms in

the presence of oxygen.

Type of carbon source utilized by microorganism was identified

by change in pH of the carbon utilization agar medium. Positive

assimilation of growth indicated by color change from purple to yellow

induced by bromocresol purple dye present in the medium.

Sterile carbohydrate utilization agar (ISP No.9) with

bromocresol purple dye was prepared. It is, then, inoculated with 1 ml

of soil isolates and poured into sterile petridishes. After solidification,

sterile discs containing 3% of different carbon sources such as

dextrose, sucrose, starch, lactose, maltose were placed aseptically

on the surface of the medium and incubated at 27?C for 8-10 days.


Presence of growth around or/and under the discs along with

change in color of the medium from purple to yellow indicates the

type of carbon source utilized by isolated actinomycetes (Table 9).

vi) Acid production:-

The sterile glucose nutrient broth was prepared. It is then,

inoculated by the soil isolates and incubated at 28?C for 15 days. At

every 12 hours interval for change in color was observed. Blue to

yellow color change indicates the acid production (Table 9).

vii) Hydrogen sulfide (H2S) production:-

Numerous actinomycetes are able to ferment the proteins and

produce hydrogen sulfide gas. Cysteine is one of the components of

peptone contained in the H2S production medium. In the presence of

cysteine desulfurase enzyme, cysteine loses the sulfur atom and it is,

then, reduced by addition of H2 atom from water to form H2S

H2N - CH - COOH + H2 H2N - CH - COOH + H2S

CH2 - SH CH3

The sterile slants of hydrogen sulfide production media was

prepared and streaked with soil isolates and incubated at 37?C for 4

days. After incubation period, H2S production was observed by rotten


egg smell and change in color of the medium to greenish brown,

bluish black or black color (Table 9).

4.2.1.5. Morphological Characterization: 25,27

a) Cultural characterization:

Morphological and cultural characters of the selected

actinomycetes strains were studied by inoculating the selected strain

into sterile International Streptomycetes Project (ISP) media like,

? Tryptone – Yeast extract broth (ISP-1)

? Oatmeal agar (ISP – 3)

? Inorganic salts – Starch Agar (ISP – 4)

? Glycerol – Aspargine Agar (ISP – 5)

? Peptone – Yeast extract agar (ISP – 6)

? Tyrosine Agar (ISP – 7)

? Carbon utilization agar (ISP – 9)

The media were sterilized and poured into sterile petridishes.

After solidification of the media, culture of the selected strain was

streaked on the media surface by simple method aseptically and

incubated at 27?C for 7 days. Morphological characters such as

colony characteristics, type of aerial hyphae, growth of vegetative


hyphae, fragmentation pattern and spore formation were observed

(Table 10).

b) Microscopical characterization:- 25

Gram staining method:-

? Smear of the selected strain (A-4) was prepared on a clean

glass slide.

? Smear was allowed to air dry and heat fixed.

? Heat fixed smear was flooded with crystal violet.

? After one minute it was washed with water and flooded with

mordant Gram’s iodine.

? Smear was decolorized with 95% ethyl alcohol and then

washed with water.

? The smear was counter stained with safranin for 45 seconds.

? After washing with water, smear was dried with tissue paper

and examined under oil immersion (100x).

Among the soil isolated strains selected for study, strain A-4

showed characteristics of actinomycetes and also showed maximum

antibiotic activity. The over producing strains due to UV mutation

show a striking increase in the activity of enzymes involved in

antibiotic synthesis.

4.2.2.1. Strain Improvement:29,30,31


Strain improvement of the selected soil isolated strain i.e. A-4

was performed by UV-mutagenesis method to obtain hyper-antibiotic

producing stable UV mutants.

The selected strain of soil isolated actinomycete i.e. A-4 was

serially diluted with sterile water up to 10-3. Sterile Actinomycete agar

medium was prepared and poured into sterile petridishes. 0.1 ml

suspension from each culture tube (10-1 to 10-3) was spread on the

surface of actinomycete agar plate. Then, the plates were exposed to

UV irradiation of 324 nm at a distance of 25 cm for 5, 10, 15, 30, 60,

90, 120, 150 and 180 seconds in dark and then incubated at 27?C for

24 hours. (Tables 11 & 12)

4.2.2.2. Isolation of stable mutants:12

After 24 hours incubation, the plates were observed for the

mutant survivors. The mutant colonies were picked up based on the

diameter of the colony and screened for the antibiotic production.

The strains were stored in refrigerator and rechecked for the

antibiotic production. Many strains showed no antibiotic production

due to the dark repair mechanism i.e. unstable mutants. The mutation

procedure was repeated, as described above, to get the stable

antibiotic producing mutant strains.


The stable mutant strains were cultured in broth medium and

compared for the maximum production of antibiotic by cup-plate

method. The mutant strain showing large zone of inhibition was

selected for the further study.

4.2.2.3. Drug resistance study: 18,19,20

In drug resistance study, Streptomycin and rifampin were used

to mutate the UV strains. The range of concentration of streptomycin

and rifampin used in study was 5-7 mg/ml. The drug resistance study

is shown in table no.13.

4.2.2.4. Morphological and Microscopical Characterization of

Mutant A-4 Strain:25,27

Cultural Characterization:-

Morphological and cultural characters of the mutant A-4 strain

was studied by inoculating it into sterile international streptomycetes

project (ISP) media like,

? Tryptone: Yeast extract broth (ISP-1)

? Inorganic salts – starch agar (ISP-4)

? Glycerol – Asparagine agar (ISP-5)

? Peptone – Yeast extract agar (ISP-6)

? Tyrosine Agar (ISP-7)


All ISP media were sterilized and poured into sterile petridishes.

After solidification of the media, culture of the mutant A-4 was

streaked on surface of it and incubated at 27?C for 7 days.

Morphological characters of mutant A-4 strain were observed. (Table

14)

4.2.3.1. Optimization of fermentation process for antibiotic

production: 15,32,33,34

The optimization of fermentation medium is as important as

selection of an organism to obtain antibiotic production. The

components of the medium such as carbon, nitrogen, micronutrients

etc. should supply adequate amount of nutrients, energy for the

building of cellular constituents and biosynthesis of fermented

products. The source of carbon and nitrogen in the fermentation

media plays an important role, since microbial and fermented

products are largely composed of these elements.

It is usual that the production of antibiotic is promoted after

readily utilizable sugars as a carbon source. It is well known that

changes in the kind and concentration of nitrogen source influence

greatly antibiotic production and antibiotic production is inhibited by a

rapidly utilized nitrogen source (NH4+-, NO3

-, certain amino acids,


etc.). Therefore, considering all above points, the medium

optimization was performed for improved antibiotic yield.

4.2.3.2. Medium Formulation:-

4.2.3.3. Carbon Source: 15,39

According to the carbon assimilation test, the selected soil

isolated strain A-4 utilizes lactose as carbon source. Different

concentrations of lactose was used in the basal medium containing

0.3% yeast extract, 0.35% CaCO3 and 0.5% NaCl. The growth of

microorganisms and quantity of antibiotic production was measured

after 4 days at 28?C incubation period. The concentration of lactose

at which maximum growth of microorganisms and production of

antibiotics is high, is used for further studies. (Table 15)

4.2.3.4. Nitrogen Source:15,39

Different sources of nitrogen such as peptone, yeast extract,

soyabean meal, ammonium chloride, ammonium sulphate and

tryptone were tested for maximum productivity of antibiotic and

growth of microorganisms.

1% of each nitrogen source was added to basal medium

containing 4% lactose, 0.3% yeast extract, 0.35% CaCO3 and 0.5%

NaCl at neutral pH in six 250 ml flasks and sterilized. Then all the


flasks were inoculated with a culture of A-4 (selected strain) and

incubated for 4 days at 28?C in orbital shaker incubator at 150 rpm.

(Table 16)

4.2.4.1. Optimization of fermentation parameters:

4.2.4.2. Temperature:22.33

Optimal temperature for the productivity and growth of the

strain A-4 was determined by keeping the inoculated fermentation

media containing 4% lactose, 1% peptone, 0.3% yeast extract, 0.35%

CaCO3 and 0.5% NaCl at 27?C, 28?C, 29?C, 30?C, 31?C, 37?C and

38?C for 4 days in orbital shaker incubator.

After incubation period, growth of microorganisms and

productivity of an antibiotic were determined by packed cell volume

(PCV) and antimicrobial activity was determined using cup-plate

method. The optimum temperature giving maximum productivity and

growth was found to be 28?C for A-4 and 29?C for A-4 mutant strains

and the same was used for the future fermentation process. (Table

17)

4.2.4.3. pH: 22,23


Similarly, optimum pH required for maximum productivity of an

antibiotic and growth of microorganism was determined by adjusting

the pH of the fermentation medium at different pH ranges between 5

to 7.5. The pH values of the medium containing 4% lactose, 1%

peptone, 0.3% yeast extract, 0.35% CaCO3 and 0.5% NaCl were

adjusted by 0.1N NaOH and 0.1N HCl before sterilization. After the

sterilization, the culture of A-4 was inoculated and incubated at 28?C

for 4 days at 150 rpm in orbital shaker incubator.

After the incubation period, cultures were studied for its growth

and productivity of antibiotic by cup-plate method. The optimum pH

was determined to be 7.0 and the same was maintained for further

bioprocessing. (Table 18)

4.2.4.4. Dissolved oxygen concentration:33

For the optimization of dissolved oxygen concentration, the

laboratory fermenter model of Sartorius B-lite was used. It contains

detachable bioreactor made of Pyrex glass with autoclavable probes

and high torque motor driven by variable speed DV drive to maintain

agitator speed. It also contains a molecular solid state electronic

structure of PIP measurement control and display of temperature,

agitation and pH.


Following procedure was used for optimization of dissolved

oxygen concentration (DO2): 33

1. DO2 probe of polarographic type was calibrated between 0%

and 100%. 0% was adjusted by using saturated sodium

sulphite solution and 100% with air saturated water (Distilled

water aerated for half an hr).

2. pH probe of Ingold-USA was calibrated with standard buffers.

3. Then, the fermentation medium was filled in a fermentation

vessel and other anxillary such as temperature, DO2, antifoam,

pH probes and tubings were assembled and the whole

assembly was sterilized by autoclaving at 121?C for 20 minutes.

4. After sterilization, the assembly was carefully allowed to attain

the room temperature and pressure, and then the probes were

connected.

5. Parameters like pH (7), agitation (200 rpm), temperature (29?C)

were set and inoculated with 10% of 24 hours inoculum of

strain A-4.

6. The pH was maintained at 7 by using 0.1N NaOH and 0.1N HCl

with the help of peristaltic pumps.

7. To optimize the dissolved oxygen concentration, different

percentages (20%, 30%, 40%, 50%, and 60%) of DO2 were

varied and studied for its effect on growth and productivity of


antibiotic. During optimization of DO2, all other parameters such

as pH, agitation and temperature were maintained at the

constant levels.

8. It is found that, at 60% DO2 concentration maximum quantity of

antibiotic was produced. To determine the total duration of

process to be carried out in fermentor, a test batch was run by

keeping the DO2 concentration at 60% and the process was

continued for 4 days, in which the growth rate was monitored.

(Table 19)

4.2.4.5. Optimization of fermentation duration:22

The duration of fermentation process was optimized to obtain

the harvesting time of maximal antibiotic productivity.

By using above mentioned media formulation and fermentation

parameters conditions, the media was prepared and sterilized. It is

then inoculated with 10% of 24 hours incubated inoculum of A-4 and

incubated for 0 to 144 hours. The harvesting time of maximal

antibiotic productivity was determined by packed cell volume (PCV)

and antimicrobial activity using cup-plate method. (Table 20)

Following fermentation parameters were found to be ideal for

maximum antibiotic productivity using A-4 strain of actinomycete.


1) Temperature: 28?C

2) pH: 7.0

3) DO2 concentration: 60%

4) Harvesting time (Fermentation duration): 96 hrs

5) Inoculum concentration: 10%v/v

4.2.5.1. Down stream process:

The fermentation was carried out for 4 days, keeping all the

optimized fermentation parameters constant. After fermentation, the

medium was centrifuged at 10,000 rpm for half an hour at 4?C to

remove the biomass and cell debris. The supernatant was separated

(No.1). The cell pellet of biomass was triturated with sterile sand to

disrupt the cell wall. It was then washed with tris buffer (pH 7.4)

filtered and centrifuged. The supernatant which contained all

intracellular components was separated out (No.2). Both

supernatants (i.e. No.1 and No.2) were checked for its antimicrobial

activity. The supernatant (No.1) showed antimicrobial activity. This

indicates that antibiotics which can produce by strain A-4 is present

extracellularly. Purification of supernatant was carried out to get a

bioactive component in the following manner. (Figure 5)

4.2.5.2. Solvent Extraction:


1) 10 ml each of supernatant was taken in four separating funnel

and extracted each with 10 ml of petroleum ether, ethyl acetate,

n-butanol and methanol.

2) The organic solvent layer was concentrated and was subjected

to antimicrobial studies.

3) Among four solvent layers, only ethyl acetate layer was found

to possess antibiotic property. Then the remaining fermented

broth was also extracted with ethyl acetate.

4) Then the ethyl acetate layer was concentrated at vacuum at

37?C to get dried product. It was further purified by extracting

with various solvents in increasing polarity i.e., petroleum ether,

n-butanol and methanol.

5) Antimicrobial activity of each solvent concentrate was checked.

Methanol soluble part showed maximum antimicrobial acivity.

Methanolic extract was subjected to further purification.

6) The dried active product obtained from the concentration of

methanolic fraction was dissolved in cold methanol which gave

two portions, an amorphous powder precipitate and a soluble

part. Both portions were again tested for its antimicrobial

activity.

7) Only the amorphous portion showed antimicrobial property but

soluble part did not show any activity.


8) Further, the purified amorphous product was analyzed for its

purity by thin layer chromatography using different solvent

systems.


4.2.5.3. Thin Layer Chromatography: 40

The chromatographic parameter used in Thin Layer

Chromatography is as shown below.

Stationary Phase:

- Silica gel GF

Mobile Phase:

- Methanol : Acetic acid (9.2 : 0.8)

- Methanol : Chloroform (9.4 : 0.6)

- n-butanol : Acetic acid : Water (4 : 1 : 0.5)

The TLC plates were prepared and spotted with the sample.

Then, plates were developed by running the mobile phase and

observed under UV light at 254 nm and also observed by developing

spot with iodine vapour. (Table 22)

4.2.6.1. Structural Identification of the Antimicrobial Compound:

The structure of the isolated compound was characterized as

follows:-

1) UV Spectroscopy:4,40,41

The purified antibiotic from A-4 was subjected to UV analysis by

using Methanol as a solvent and ? max using UV spectrometer.


2) I.R. Spectroscopy: 4,41

Purified antibiotic was subjected to IR analysis by using KBr

pellets and the peaks obtained were observed and interpreted. The

IR spectrum of the compound is shown in figure 6 and their data are

shown in table 26.

3) NMR Spectroscopy:4,41,42

The purified compound was subjected to NMR analysis by

proton resonance spectroscopy using NMR spectroscopy. Its

spectrum was observed, interpreted and shown in figure 7, and their

data are shown in table 27.

4.2.7.1. Physical properties of purified product:43,44,45,46

The purified product was studied to determine its physical

properties like,

a) Color

b) Consistency

c) Melting point

d) Solubility

The results are tabulated in table 25.


4.2.8.1 Determination of Antimicrobial Activity:26.47

Determination of the minimum inhibitory concentration (MIC) in liquid

medium:-

Requirements:-

1) Nutrient broth / Sabouraud dextrose broth

2) Assay tubes

3) Standardized culture of test organism

4) Sterile pipettes and petridishes

5) Cyclone mixer

6) Test compound

Test organisms used for the study are as follows:

I) Gram positive:

1. Bacillus subtilis

2. Staphylococcus aureus

II) Gram negative:

1. Pseudomonas aeruginosa

2. Klebsiella pneumonia

III) Fungi:

1. Candida albicans

2. Aspergillus flavus


Procedure:26,47

4.2.8.2. Standardization of Inoculum:

1) 24 hr old cultures of all the test organisms were diluted by 10

fold serial dilution technique using sterile water from 10-1 to 10-8.

2) All the dilutions were plated in sterile nutrient agar (in case of

bacteria) and Sabouraud dextrose agar (in case of fungi).

3) Calories are counted from a petridish where countable numbers

of colonies are present. Colony forming units (CFUs) present in

1ml of the initial broth was calculated.

4.2.8.3. Preparation of Stock Solution:

100 mg of the test substance was dissolved in 10 ml of DMSO

to prepare a stock solution of concentration 10 mg/ml.

4.2.8.4. Preparation of seeded broth:

The stock culture was diluted approximately with nutrient broth

for bacteria and Sabouraud dextrose broth for fungi to get a working

concentration of 106 CFU/ml.

4.2.8.5. Two-fold serial dilution method: (Macrodilution method):

? 0.2 ml of 10 mg/ml stock solution (using DMSO) of the test

substance was added to 1.8ml of seeded broth to produce a

concentration of 100 ? g/ml (1st tube) of the test substance.


? 1 ml from the first tube was transferred to second tube

containing 1ml of seeded broth to get 500 ? g/ml of test solution.

Likewise the test compounds and the standard drugs were

serially diluted in two-fold manner to obtain different

concentrations ranging from 1000 to 7.56 ? g/ml.

? Then it is incubated at 37?C for 24 hours and 27?C for 48 hours

for bacteria and fungi respectively.

? The minimum inhibitory concentration (MIC) was found out by

visual comparison method (presence of turbidity due to growth).

? In experimental terms the MIC is the concentration of the drug

present in the clear tube i.e. in the tube having the lowest

antibiotic concentration in which growth is not observed.

? Apart from this, one positive control (without test substance)

and one negative control (sterile broth) were kept.

? The results of MIC are recorded in tables 23 and 24.


RESULT AND DISCUSSION

5.1. CHARACTERIZATION OF SOIL SAMPLES:

5.1.1. Screening of Soil Samples:

In the course of screening for novel antimicrobial substances

(antibiotics) from soil samples, antibiotic-producing actinomycete

cultures were recorded from soil samples taken in Belgaum,

Karnataka. Actinomycetes have provided many important bioactive

compounds of high commercial value and continue to be routinely

screened for new bioactive substances.

In the present study, about eighteen actinomycetes were

isolated from soil samples, with isolation media as soyabean-casein

digest medium (SBCD), actinomycetes isolation agar (AIA) and

starch-casein agar, having pinpoint colonies with zone of inhibition,

cultured by crowded plate technique. Isolation of actinomycetes from

the isolation media was carried out by multiple streak method in

actinomycetes agar media to obtain the pure cultures of nine

actinomycetes strains. Each purified strain was preserved in

actinomycetes agar slants at 4?c.

The soil samples were collected aseptically and processed

within two hours after the heat treatment which inhibits growth of


unwanted bacteria and fungi. Addition of CaCO3 and heat treatment

helped in the making up of actinomycetes populations in the soil.

The presence of relatively large populations of actinomycetes in

soil samples of Belgaum indicates that it is an eminently suitable

ecosystem which helps to isolate actinomycetes from screening

programs. The details of collection of soil samples are shown in table

7. (Plate 1)


Table No.7: Collection of Soil Samples and Number of Actinomycetes in Isolation Plates

No. of actinomycetes colonies on isolation

media

Sr. No.

Dilution of soil

samples (10-5)

Heat treatment (45?C, 1

hr) SBCD AIA SCA

Nature of soil sample

pH of Soil

1

2

3

4

5

6

7

8

9

10-4

10-4

10-3

10-4

10-3

10-3

10-4

10-5

10-5

NA

NA

AP

AP

AP

AP

AP

AP

AP

--

--

4

1

--

--

17

33

48

--

--

6

3

--

--

38

48

50

--

--

--

--

--

--

15

36

32

Water logged mud

Water logged mud

Loamy

Sandy

Mud

Mud

Red-dry soil

red-dry soil

Black-dry soil

7.5

7.5

6.3

7.0

8.0

7.8

5.3

5.7

5.4

NA: Not applied, AP: Applied SBCD – Soyabean casein digest medium AIA – Actinomycetes isolation agar SCA – Starch casein agar


It is obvious to conclude that actinomycetes numbers generally

decreased with heavy rainfall with great moisture content of soil.

Actinomycetes preferred neutral or slightly alkaline soil to grow. It is

found that actinomycetes number is less in the surface soil than 11-

15 cm deep because of the most favorable combination of pH value

and water content. The number of actinomycetes in great amounts

was found in black-alkaline sandy soil.

5.1.2. Test for Microbial Sensitivity:

The isolated strains of actinomycetes were tested for microbial

sensitivity against five bacterial strains and three fungal strains by

agar streak method (Table 8).

Out of nine actinomycetes screened, six strains namely A1, A2,

A3, A4, A5 and A6 showed significant antimicrobial activity against

both gram-positive and gram-negative organisms. However, A4

showed a very broad spectrum with higher scores than all other

strains.

These six actinomycetes strains, selected through the microbial

sensitivity test, were further taken for the taxonomical

characterization. (Plate 2)


5.1.3. Taxonomical Characterization:

A classification of any microbial order is a temporary and man-

made arrangement in which similar individuals, sharing certain

common features, are grouped together as taxonomic units at

different levels in a taxonomic hierarchy. The most stable

classifications are likely to be those in which the relationships

between taxa are based upon all kinds of data e.g. genetic, phenotic,

serologic etc.

Taxonomical characterization of six selected soil isolates were

done by testing for melanoid formation, nitrate reduction, milk

coagulation, and peptonization, gelatin liquefaction, starch hydrolysis

H2S production, and carbon assimilation.


Table No.8: Sensitivity of different microorganisms towards the soil isolates by agar streak method

Soil isolates B. subtilis S. aureus E. coli K.

pneumoniae P.

aeruginosa A. niger A. terreus C. albicans

A1

A2

A3

A4

A5

A6

A7

A8

A9

++

+

-

++

+

+

++

-

-

+

++

-

++

-

-

-

+

+

+

+

++

+++

+

+

+

+

-

-

-

-

++

+

-

+

+

+

++

++

++

+

+

-

+

-

-

-

-

-

+

-

-

+

+

-

-

-

+

-

-

-

-

+

-

+

+

++

++

-

-

+

-

-

? +++ = Better inhibition, ++ = Good inhibition, + = Moderate inhibition, - = No inhibition


Table No.9: Taxonomical Characterization of Soil Isolates

Soil isolates

Melanoid formation

Nitrate reduction

Proteolytic activity

Gelatin liquefication

Starch hydrolysis

Carbon assimilation

Acid production

H2S production

A-1 Light brown pigmentation

+ Clear with acidic reaction

+ + Glucose + -

A-2 Only growth + Clear with acidic reaction

+ + Glucose + -

A-3 Only growth + Not clear with slightly alkaline reaction

+ + Fructose + +


+ Not clear with acidic reaction

+ + Lactose + +

A-5 Only growth + - + + Lactose + -


+ - + + Maltose + -

‘+’ Positive reaction ‘-’ Negative reaction


All the six strains namely A1, A2, A3, A4, A5 and A6 showed

positive results in starch hydrolysis and nitrate reduction tests. Strain

A1, A4 and A6 showed positive results in pigment production test in

Waksman medium (light brown colored pigment) and A2, A3, A5

showed negative results in melanoid formation test but they produced

good growth in above said test medium.

The six strains showed positive result in gelatin liquefication

and H2S production test. The six strains were able to produce the

acid in the acid production test.

All the strains showed positive results for proteolytic activity

except A5 and A6. These physicochemical properties help to

differentiate the genus of actinomycetes. Therefore based on

taxonomical characterization, A1, A2, A3, A4 were classified under

the genus actinomycetes and strains A5 and A6 under the genus

nocardia.

Among all the isolated strains of actinomycetes, strain A4

showed promising results for an effective antibiotic production which

is selected for the further detailed investigation regarding the

optimization of fermentation media and large-scale bioprocessing of

its antibiotic production.


Different carbon sources such as glucose, lactose, maltose,

sucrose and dextrose were tested for the source of carbohydrate

(Table 9, Plates 3 to 8).

5.1.4. Morphological and cultural characterization of A-4 strains:

The morphological and cultural characteristics of the strain A-4

were studied on International Streptomyces Project (ISP) media. The

different ISP media used for the morphological study were ISP-1,

ISP-3, ISP-4, ISP-5 and ISP-7. The growth characteristics, presence

of aerial mycelium and soluble pigments were observed (Table

10, Plates 9 to 14).

Table No.10: Morphological and cultural characterization of the strain

A-4

Sr.

No.

Medium Used A4

1. Tryptone-yeast

extract broth (TSP-1)

Growth occurs by the pellicle formation.

2. ISP-2 Creamish white colored colonies with clear

zone around it were observed.

3. (Oatmeal agar) ISP-3 Slight black – creamish color thick colonies;


no aerial mycelium formation was observed.

4. Inorganic salt-starch

agar (ISP-4)

Blackish-brown colored thick colonies with

waxy margin and convex surface was

observed.

5. Glycerol asparagines

agar base (ISP-5)

Whitish colored thin colonies striated surface;

with less aerial mycelium and filamentous

growth was observed.

6. Peptone yeast extract

iron agar (ISP-6)

Thin transparent colonies with black colored

soluble pigments were seen. No filamentous

growth was seen.

7. Tyrosine agar base

(ISP-7)

Cream colored, lobe shape, convex surface,

little mycelium growth was observed.

8. Carbon utilization

agar (ISP-9)

Thin yellowish golden colored colonies with

little mycelium growth were observed.

Morphological characteristics of A-4 strains in different ISP

media, showed the filamentous growth in ISP-5 and ISP-7 media and

the pigmentation was seen in ISP-6 medium.

The morphological characters of strain A-4 were also studied by

microscopical observation after Gram-staining method.


The observations revealed that A-4 strain is gram positive and

rod shaped microorganism. The microscopical characteristics were

observed under 10x and oil-immersion (100x).

By studying the morphological, cultural and taxonomical

characteristics, it is observed that strain A-4 is belong to the genus

actinomycetes.

5.2. IMPROVEMENT OF A-4 STRAIN BY MUTAGENESIS:

From the literature survey, it is seen that natural isolates usually

produce commercially important products in trace amounts and

therefore every attempt is made to increase the productivity of

selected microorganisms. With advances in biochemistry,

engineering and genetic manipulative techniques, scientists took a

more targeted approach towards developing microbial cultures for the

production of bioactive compounds with therapeutic value, thereby

evolving towards the establishment of “new biotechnology”.

Classical strain development has typically relied on mutation

and random screening of improved strain. This empirical approach

has a long history of success, best exemplified by the improvements

achieved by fungal or actinomycete cultures capable of

overproducing metabolites in quantities as high as 80g/l. Thus,


application of strain improvement to new fermentation processes

continues to be documented in the literature, despite the age of the

technology.

Improvement of the microbial strain offers the greatest

opportunity for cost reduction without significant capital outlay. The

desired result is the ability of a manufacturing process to meet

additional demands without adding more production scale fermenters

or bioreactors. Several procedures are employed to improve

microbial strains, all of which bring about changes in the DNA

sequence, which may ultimately evolved in the formation of

auxotrophic strain. Auxotrophic mutants do not have feedback

mechanism to control the biosynthesis of secondary metabolite,

which results in the overproduction of bioactive compounds.

Increase yield of bioactive compounds (antibiotic) may be

achieved by optimizing the culture medium and fermentation

parameters and also by strain improvement. The potential

productivity of the organism is controlled by its genome. The genome

contains antibiotic genes and regulatory mechanisms which directs

the biosynthesis of antibiotic. Therefore, the genome must be

modified to amplify the antibiotic genes to increase the potential yield

by applying various techniques.


In the strain improvement study, the genetic modification of the

strain A-4 was carried out by UV irradiation method and drug

resistant method.

5.2.1. UV Mutation:

A 5 ml sample of spore suspension from solid culture of A-4

strain was transferred to aseptic plates of actinomycetes agar. It was

done in duplicates.

The experiments of UV irradiation were conducted by exposing

the A-4 strain to UV rays of 324 nm at 35 cm distance apart in

different time intervals. Then the plates were incubated at 27?C in

BOD incubator for 4 days. The survival strains after mutagenesis

were picked and tested for the antibiotic production. It is found that,

the microorganisms which were exposed to UV irradiation for 150

seconds showed strain improvement. The improvement in the genetic

modification was tested by the ability of the mutant strains to produce

the antibiotic in large quantities.

The auxotrophic mutants were selected by comparing the

amount of antibiotic produced (mg/L) by each mutant strain and were

named as P1, P2, P3…… P10.


Each muant strain isolated from the plates were exposed to the

UV rays and assayed for the antibiotic production by inoculating the

mutant strain in medium containing 5% lactose, 0.3% yeast extract,

0.35% CaCO3 and 0.5% NaCl in 250 ml flasks for 96 hours on a

orbital rotatory shaker at 150 rpm, a 2 ml portion of the seed culture

was used to inoculate 150 ml of production media for 48 hours. (Table 11

and 12)


Table No.11: Antibiotic Productivity by UV Mutant Strains

Sr.

No.

Source of

microorganisms

Colonies

before UV

mutation

UV

exposure

time (sec)

Colonies

after UV

mutation

Antibiotic

producing

mutants

1

2

3

4

5

6

7

8

9

5 ml spore

suspension plated

on each

actinomycetes

agar plates

75

80

85

84

100

107

79

77

86

0

5

10

15

30

60

90

120

150

--

69

65

62

35

38

49

30

26

--

5

3

6

5

6

8

2

9


Table No.12: Comparison of UV mutant strains for the antibiotic

production

Sr. No.

Auxotrophic mutants UV exposure time (sec)

Potency (mg/L)

Selected auxotrophic

mutant

1. 2 M1 M2

5 sec 1.9 2.3

M2

2. 2 M1.1 M2.2

10 sec 2.1 2.3

M2.2

3. 4 M1.1.1 M1.1.2 M1.1.3 M1.1.4

15 sec 1.6 1.5 1.3 2.0

M1.1.4

4. 1 M2.1 30 sec 2.0 M2.1

5. 3 M3 M4 M5

60 sec 1.9 2.1 1.7

M4

6. 2 M3.1 M3.2

90 sec 2.3 2.4

M3.2

7. 1 M4.1 120 sec 2.5 M4.1

8. 2 M5.1 M5.2

150 sec 2.5 2.4

M5.1

9. 3 M6 M7 M8

180 sec 2.9 3.2 3.5

M8


5.2.2. Drug Resistance: (Streptomycin and Rifampin)

The auxotrophic strain screened from UV mutant strains

namely M2, M2.2, M1.1.4, M2.1, M4, M3.2, M4.1 and M5.1 were

selected for the drug resistant study.

Physiological differentiation (in antibiotic biosynthesis) in

microorganisms usually starts when cells encounter adverse

environmental conditions. Thus for the drug resistance study, it is

observed that 2-8 mg/ml concentration of antibiotics (i.e.

Streptomycin and Refampin) were sufficient to produce spontaneous

Streptomycin-Rifampin-resistant (str - rif) mutants. The frequency of

mutants producing increased antibiotic is higher as compared to the

UV mutation.

As in the mutation study, the UV mutant strains (auxotrophs)

were again treated to obtain the drug resistant mutants which showed

the superior ability of antibiotic production. The drug resistant

(Streptomycin and Rifampin) auxotrophic resulted in further increase

in (2.5 to 7 fold higher) antibiotic productivity.

The amount of antibiotic produced by each UV-drug-resistance

mutagenesis auxotroph was determined by inoculating 2ml of seed

culture of each strain into 100ml of production medium containing 5%


lactose, 0.3% yeast extract, 0.35% CaCO3, and 0.5% NaCl, in 250 ml

flasks for 96 hours in orbital shaker incubator and then by solvent

extraction method using ethyl acetate.

In drug resistant study of various UV mutants as listed in table

no.13, it is observed that the UV mutant strain M5.1 after the effects

of drugs namely streptomycin and rifampin in concentration of 7mg/ml

each, showed the maximum antibiotic production as 4.9 mg/ml and it

is then designated as A-4 mutant strain. A-4 mutant strain is used for

the further fermentation studies.


Table No.13: Effect of Streptomycin and Rifampin on UV mutants and

antibiotic productivity of UV-drug resistant mutants

Sr.

No.

Strain Concentration

of streptomycin

used for

selecting

Streptomycin

mutants (? g/ml)

Concentration

of Rifampin

used for

selecting

Rifampin

mutants (? g/ml)

Frequency (% of

Streptomycin &

Rifampin

mutants

producing

increased

antibiotic)

Highest

productivity

detected

(mg/ml)

1

2

3

4

5

6

7

8

9

M2

M2

M2.2

M2.2

M1.1.1

M1.1.1

M2.1

M2.1

M4

5

7

5

7

5

7

5

7

5

5

7

5

7

5

7

5

7

5

48 (48/100)

34 (34/100)

3 (3/100)

41 (41/100)

11 (11/100)

15 (9/60)

8 (4/48)

9 (9/100)

50 (45/90)

3.2

4.0

2.8

1.7

2.9

3.2

3.0

3.3

3.9


5.2.3. Morphological and cultural characteristics of A-4 mutant

strain:

The morphological and cultural characteristics of the strain A4

mutant were studied on International Streptomyces Project (ISP)

media. The different ISP media used for the morphological study

were ISP-1, ISP-3, ISP-4, ISP-5 and ISP-7. The growth

characteristics, presence of aerial mycelium and soluble pigments

were observed (Table 14, Plates 15 to 21)


Table No.14: Morphological and cultural characterization of the strain

A-4 mutant

Sr.

No.

Medium Used A-4 mutant

1. (Ootmeal agar) ISP-3 Growth occurs by pellicle formation. Aerial

formation was observed.

2. Inorganic salt-starch

agar (ISP-4)

Thick creamish colored colonies. No soluble

pigments observed. Aerial growth was

observed.

3. Glycerol asparagines

agar base (ISP-5)

Thin striated white colored colonies with

dense aerial formation was observed.

4. Peptone yeast extract

iron agar (ISP-6)

Golden colored colonies with less aerial

formation and brownish black colored soluble

pigments was observed.

5. Tyrosine agar base

(ISP-7)

Thin striated white colored colonies with no

pigmentation observed. Aerial formation was

observed.


5.3. OPTIMIZATION OF FERMENTATION PROCESS FOR

ANTIBIOTIC PRODUCTION:

5.3.1. Medium formulation:

Medium formulation is necessary to each fermentation process.

It is necessary to optimize each and every component of fermentation

media by varying the concentration of media constituents in order to

achieve the maximum antibiotic production. The purpose of media

optimization is to support the efficient growth of microorganisms.

Different combinations of medium constituents and sequences of

optimized fermentation conditions need to be investigated to

determine the growth conditions, which produce the biomass with

best suited physiological state constituted for antibiotic production.

For the optimization of fermentation process, the basal medium

containing 0.3% yeast extract, 0.35% CaCO3 and 0.5% NaCl for the

antibiotic production as suggested by the literature survey. In order to

achieve maximum antibiotic production, experiments were conducted

to optimize the various parameters such as carbon source, nitrogen

source, temperature, pH, DO2, and micronutrients etc.


5.3.2. Optimization of Carbon Source:

It is observed that rapid utilizable carbon source do not support

the antibiotic production as the antibiotic synthesis do not totally

depend on the growth of microorganism, in fact, it depends upon the

adverse conditions imposed by the medium or surroundings.

Similarly the carbon assimilation test, as described in

taxonomical characterization, was performed for A-4 mutant strain. It

is observed that lactose is the good source of carbon for growth as

well as antibiotic production for A4 mutant strain.

To optimize the concentration of lactose for growth and

antibiotic productivity, different concentrations were studied (Table

15, figures 6&7).


Table No.15: Optimization of Carbon Source

A-4 A-4 Mutant Sr.

No.

Lactose

concentration

(%) Packed

cell

volume

(PCV) %

Zone of

inhibition

A4 [d in

cm]

Packed cell

volume

(PCV) %

Zone of

inhibition

A4 mutants

(cm)

1

2

3

4

5

6

1%

2%

3%

4%

5%

6%

0.07

0.1

0.33

0.4

0.5

0.4

1.9

2.1

2

2.4

2.4

2.2

0.07

0.02

0.36

0.46

0.53

0.44

1.8

2.1

2.3

2.4

2.5

2.2

d = diameter


It was observed that 5% concentration of lactose as a carbon source is

optimal for the production of antibiotic for A-4 strain and 4%

concentration for lactose for A-4 mutant strain.

The packed cell volume (PCV) and zone of inhibition were also

studied for the same and found better sources.

5.3.3. Optimization of Nitrogen Source:

The effect of nitrogen source on induction and secretion of

antibiotic was studied by using different nitrogen sources in the basal

medium. The nitrogen sources used for the experiments were

ammonium chloride, ammonium sulfate, peptone, soybean meal and

yeast extract.

The effect of nitrogen source on antibiotic productivity of A-4

and A-4 mutant strains were observed (Table 16, figures 8&9).


Table No.16: Optimization of Nitrogen Source

A-4 A-4 mutant Sr.

No.

Nitrogen source

(1% each) Packed

cell

volume

(%)

Zone of

inhibition

(diameter in

cm)

Packed

cell

volume

(%)

Zone of

inhibition

(diameter

in cm)

1

2

3

4

5

Ammonium

chloride

Ammonium

phosphate

Peptone

Soybean meal

Yeast extract

0.1

0.1

0.4

0.3

0.3.

1.5

1.3

3.6

2.9

2.1

0.1

0.1

0.6

0.4

0.3

1.7

1.5

4.0

3.2

2.4

Well diameter = 0.9 cm

Organism used = K.pneumoniae

By determining its packed cell volume (PCV) and zone of

inhibition of all nitrogen sources, peptone at concentration of 1% was

found to be good source of nitrogen for the selected A-4 and A-4

mutant strains.


5.4. OPTIMIZATION OF FERMENTATION PARAMETERS:

5.4.1. Temperature:

Optimum temperature for maximum growth and productivity of

actinomycetes A-4 and A-4 mutant was determined by studying their

packed cell volume (PCV) and zone of inhibition (Table 17, figures

10&11).


Table No.17: Optimization of Temperature

A-4 A-4 Mutants Sr.

No.

Temperature

(in ?C) Packed

cell

volume

(%)

Zone of

inhibition

(diameter

in cm)

Packed cell

volume (%)

Zone of

inhibition

(diameter in

cm)

1

2

3

4

5

6

28?C

29?C

30?C

31?C

37?C

38?C

0.6

0.5

0.5

0.4

0.2

0.2

3.0

2.6

2.6

2.5

2.4

--

0.6

0.7

0.5

0.5

0.3

0.3

2.6

3.2

3.0

2.8

2.2

--


Organism used = K.pneumoniae


From the temperature optimization experiments it is observed

that the temperature adequate for growth is not always the same as

that for antibiotic production.

From the temperature optimization study, it is observed that A-4

strain and A-4 mutant strain showed good growth and maximal

antibiotic production at 28?C and 29?c respectively.

The each incubated medium in temperature optimization study

was tested against different bacteria and fungi as S.aureus, E.coli

and C.albicans.

For the further studies of fermentation processes of both A-4

and A-4 mutant strains, optimum temperatures were adjusted at 28?C

and 29?C respectively and the same is used for fermentation

process.

5.4.2. pH:

The pH of the fermentation affects not only the growth but the

production of antibiotic as well as to the medium constituents and the

temperature.

Optimum pH for maximum growth and productivity of

actinomycetes A-4 and A-4 mutant was determined by studying the

packed cell volume (PCV) and zone of inhibition (Table 18, figures 12&13).


Table No.18: Optimization of pH for optimum growth and antibiotic

productivity

A-4 A-4 mutants Sr.

No.

pH

PCV

(%)

Zone of

inhibition

(diameter in

cm)

PCV

(%)

Zone of

inhibition

(diameter in

cm)

1

2

3

4

5

6

7

8

5

5.5

6

6.5

7

7.5

8

8.5

0.2

0.2

0.5

0.5

0.6

0.4

0.4

0.1

1.5

1.8

2.1

2.3

2.9

2.4

2.3

--

0.3

0.2

0.4

0.6

0.8

0.4

0.3

0.3

1.7

2

1.9

2.6

3.5

2.4

2.1

1.5


Organism used = Klebsiella pneumoniae


The incubated medium which was kept at different pH levels in pH

optimization experiments was tested against K.pneumoniae and its

activity was determined.

From the obtained results pH 7 was found to be suitable for

both the selected actinomycetes A-4 and A-4 mutants. The selected

optimum pH was kept constant and the same is used for further

process of fermentation.

5.4.3. Dissolved oxygen concentration (DO2):

Optimum DO2 for maximum growth and productivity of

actinomycetes A-4 and A-4 mutants was determined by studying the

packed cell volume (PCV) and zone of inhibition. The DO2

optimization study was carried out in Sartorius B-Lite fermenter

(laboratory) of 3 lit capacity. (Plate 22)

The volume of media used in DO2 study was of 1 liter and the

same was kept constant during the study at different DO2

concentrations. The agitation speed of the fermenter was 200 rpm for

each concentration and which is kept constant for each DO2

concentration used in DO2 optimization experiments (Table 19,

figures 14&15).


Table No.19: Optimization of Dissolved Oxygen Concentration (DO2)

A-4 A-4 mutants Sr.

No.

DO2

PCV

(%)

Zone of

inhibition

(diameter in

cm)

PCV

(%)

Zone of

inhibition

(diameter in

cm)

1

2

3

4

5

6

30%

40%

50%

60%

70%

80%

0.3

0.3

0.5

0.8

0.7

0.3

1.8

1.9

2.2

2.6

2.4

1.4

0.4

0.5

0.7

0.9

0.8

0.5

1.7

1.9

2.5

3.2

2.9

1.8




The incubated medium which was kept at different DO2 levels in

fermenter was tested against Klebsiella pneumoniae and its activity

was determined.

From the obtained results DO2 level at 60% was found to be

optimum for maximum growth and antibiotic production for both A-4

and A-4 mutant strains and the same was used for the further

process of fermentation.

5.4.4. Duration of fermentation:

The fermentation batch processed containing optimized

medium formulation and fermentation conditions, was studied to

determine the maximum duration of fermentation and results are

shown in the table 20. (figures 16 & 17)


Table No.20: Optimization of duration of fermentation for the

maximum growth and antibiotic production

A-4 A-4 mutants Sr.

No.

Time in

hrs PCV

(%)

Zone of

inhibition

(diameter in

cm)

PCV

(%)

Zone of

inhibition

(diameter in

cm)

1

2

3

4

5

6

7

8

9

0

12

24

36

48

60

72

84

96

--

0.1

0.2

0.25

0.4

0.4

0.7

0.9

1.5

--

--

2.0

2.3

2.2

2.4

2.5

2.7

2.7

--

0.02

0.1

0.3

0.4

0.6

0.9

1.5

3.3

--

--

--

2.1

2.4

2.7

2.8

3.2

3.6




From the data obtained, the time vs. packed cell volume (PCV)

was plotted which revealed that the origin of stationary phase was

from the fourth day.

Based upon the above consolidated results, fermentation

medium and fermentation conditions were optimized for the maximum

production of antibiotic and they are given as follows. (Plates 23 to

26)

Table No.21: Composition of Fermentation Medium

Ingredients Quantity

Lactose

Peptone

Calcium carbonate

Magnesium sulphate (MgSO4 . 7H2O)

Potassium dihydrogen phosphate (KH2PO4)

Sodium chloride

Ferrous sulphate

4%

1%

0.5%

0.025%

0.1%

0.05%

0.001%


Fermentation conditions:-

Temperature : 28?C

pH : 7

DO2 : 60%

Duration of : 96 hours

fermentation

5.5. THIN LAYER CHROMATOGRAPHY:

Thin layer chromatography of the purified compound was

performed. The spots on the plates were developed and observed

under UV light at 254 nm and also observed by developing spot with

iodine vapors and Rf values are reported. Only one spot was seen

after developing TLC plates (Table 22).


Table No.22: Thin layer chromatography of purified antimicrobial

compound

Sr. No. Solvent Systems Rf values

1.

2.

3.

Methanol : Acetone acid (9.2 : 0.8)

Methanol : Chloroform (9.4 : 0.6)

n-butanol : Acetic acid : Water (4 : 1 : 5)

0.65

0.77

0.87

5.6. DETERMINATION OF ANTIMICROBIAL ACTIVITY:

The antimicrobial activity in terms of minimum inhibitory

concentration (MIC) of an isolated compound from A-4 and A-4

mutant fermentation broth was studied.

The antibiotic showed broad spectrum of activity against gram

positive and gram negative organisms and the MIC was found to be

in the range of 100-125 ? g/ml. (Table 23)

The compound also possess significant antifungal activity

against fungal strains tested, and the MIC was found to be 125 ? g/ml.

(Table 24)


Table No.23: Determination of MIC for an isolated antimicrobial

compound against bacteria

MIC of isolated compound in

? g/ml Test microorganisms (Bacteria)

A-4 A-4 mutant

A. Gram positive organisms

1. Staphylococcus aureus

2. Bacillus subtilis

125

125

100

100

B. Gram negative organisms

1. Escherichia coli

2. Pseudomonas aeruginosa

3. Klebsiella pneumoniae

125

100

100

100

62.5

62.5


Table No.24: Determination of MIC for an isolated antimicrobial

compound against fungi

MIC of isolated compound in

? g/ml Test microorganisms (Fungi)

A4 A4 mutant

1. Candida albicans

2. Saccharomyces cerviciae

125

125

125

62.5

Table No.25: Physical properties of purified antimicrobial compound

Sr. No. Physical

Properties

Antimicrobial compound

1.

2.

3.

4.

Color

Consistency

? max

Solubility

Yellowish cream color

White amorphous powder

216

Soluble in water, methanol

Sparingly soluble in DMSO

Insoluble in petroleum ether, ethyl

acetate


5.7. STRUCTURAL IDENT IFICATION OF ANTIBIOTIC:

In an attempt to establish the chemical structure of an antibiotic

produced by strain A-4, spectral studies such as UV, IR and NMR

were performed and are shown in figures 18 and 19.

The ? max of the isolated compound by UV analysis was 216

nm. The basic peak obtained by IR spectroscopical study and their

corresponding groups are given in table 26.

The basic data obtained by NMR spectroscopical study and

their corresponding groups are given in table 27.


Figure 18: IR Spectrum for an Isolated Antibiotic

A-4

615.

91

871.

23

1099

.8314

14.1

6

1677

.79

3424

.50

-10

-5

0

5

10

15

20

25

30

35

40

45

50

55

60

%T

500 1000 1500 2000 2500 3000 3500

Wavenumbers (cm-1)


Figure 19: NMR Spectrum for an Isolated Antibiotic


Table No.26: IR spectroscopical data and their functional groups

Sr. No. Wave number Functional groups

1

2

3

4

5

3424

1414

1677

1099

3147

Secondary amines

Alkyl groups

Aldehydic groups

Alcoholic groups

Aromatic groups

Table No.27: NMR Spectroscopical data and their functional groups

Sl. No. ? values Functional group

1. 1.24 Methyl H-shift

2. 2.86 Methyl H-shift

3. 3.29 Aldehyde

4. 3.48 Alcohols

5. 3.7 Esters

6. 5.2 Alkene


7. 6.9 Aromatic-H or Heterocyclic-H

From the data of solubility study and spectroscopical studies may be

classified under the group of aminoglycoside.


Plate No.1: Photograph Showing Crowded Plate Method

Plate No.2: Test for Microbial Sensitivity


Plate No.3: Test for Melanoid Pigment Formation

Plate No.4: Test for Nitrate Reduction

Plate No.5: Test for Milk Coagulation and Peptonization


Plate No.6: Test for Gelatin Liquefication

Plate No.7: Test for Amylolytic Activity by Starch Hydrolysis


Plate No.8: Carbohydrate Assimilation Test

Plate No.9: Morphology of A-4 Strain on ISP-1 and ISP-7

Plate No.10: Morphology of A-4 Strain on ISP-3 & ISP-5


Plate No.11: Morphology of A-4 Strain on ISP-4

Plate No.12: Morphology of A-4 Strain on ISP-6


Plate No.13: Microscopy of A-4 Strain Plate No.14: Microscopy of A-4 Strain under 10X under 100X

Plate No.15: Microscopy of A-4 Mutant Plate No.16: Microscopy of A-4 Mutant Strain under 10X Strain under 100X

Plate No.17: Morphology of A-4 Mutant Strain on ISP-3


Plate No.22: Photograph showing Laboratory Fermenter of 3L

Capacity of Sartorius B-Lite Company

Plate No.23: Photograph showing Antimicrobial Activity of Broth

Collected at an interval of 24 hr during bioprocess


Plate No.24: Photograph showing Antimicrobial Activity of Broth Collected at an interval of 48 hr during bioprocess



SUMMARY AND CONCLUSION

Screening of antibiotics has been widely performed for about

last 50 years and new antibiotics are still being found. In screening of

new antibiotics, new approaches are required and following three

factors must be considered i.e. detection of antibiotic producing

microorganisms, selection of producing microorganisms and

cultivation methods. These are closely related to each other, and their

efficient combination is essential for successful screening of an

antibiotic.

Though the production of antibiotic is sometimes evident during

growth of the microorganisms, usually the production is actively

carried out after growth reaches stationary phase. The morphological

and physiological properties of the strain are greatly changed before

and after antibiotic accumulation begins. Particularly trophophase, in

which respiration is high and vegetative growth is accelerating by

utilizing constituents of the medium. In idiophase, growth stops and

antibiotic production reaches at maximum. Thus 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.


As the antibiotics are secondary metabolites, they are

synthesized in trace amounts in an ordinary fermentation. Moreover

the synthesis of antibiotic is regulated by tight metabolic and genetic

regulation. Therefore it is the task to the biotechnologists to modify

the wild type strain and to provide cultural conditions to improve the

productivity of antibiotics. Improvement of the microbial strain offers

the greatest opportunity for cost reduction without significant capital

investment. The desired result of strain improvement is the ability of a

manufacturing process to meet additional demands without adding

more production scale fermenters. With the consideration of above

basic criteria, the production of antibiotic using ‘Actinomycetes’

species was studied by initial isolation of actinomycetes strains from

soil by ‘Crowded Plate Technique’.

From crowded plate, 09 actinomycetes strains were isolated

and tested for antibiotic production. According to the antimicrobial

spectra against chosen 5 bacteria and 3 fungi, one strain is selected,

showing broad spectrum of antimicrobial activity for the further

bioprocess studies.

The selected strain is designated as A-4 and its taxonomical

characterization is performed. From taxonomical characterization

results, A-4 is classified as actinomycete. Then, its morphological,


cultural and microscopical characters were studied on various ISP

media, which ultimately concluded that A-4 is an actinomycete.

For the strain improvement, the A-4 strain was mutated by UV

irradiation and drug resistant treatment. In the drug resistant

treatment, streptomycin and rifampin (1-10 ? g/ml) were used.

As in UV mutation study, it is found that, the rate of evolution of

mutant strains is less as compared to the drug resistant study. After

the mutational studies, the selected mutants by random screening,

were tested for the ability of synthesizing antibiotics. The one mutant

strain showed highest production of antibiotic is selected for further

bioprocess studies and is designated as A-4 mutant.

The production of antibiotic from both A-4 and A-4 mutant was

compared using batch fermenter under optimized media composition

and process conditions. When the growth of the microorganisms

reached to idiophase, the fermented broth was collected, filtered and

centrifuged for separation of cell debris, and supernatant was

collected. The same is performed for A-4 mutant fermentation.

From the supernatant which is separated from the fermentation

broth was subjected to the solvent extraction using various organic

solvents like petroleum ether, chloroform, ethyl acetate etc. The cell


biomass collected during fermentation broth filtration, is subjected to

cell disintegration using sterile sand to check the intra-cellular

antibiotics.

In solvent extraction study, same proportion of organic solvent

and fermentation broth were extracted. Each solvent was studied,

initially, for its antimicrobial properties using cup-plate method.

It is found that ethyl acetate is the good solvent for the isolation

of antibiotic from fermentation broth as it did not modify the antibiotic

and maximum concentration of antibiotic was extracted using ethyl

acetate from fermentation of broth as compared to the other organic

solvents.

The crude antibiotic from the ethyl acetate was collected by

evaporating the solvent at 30?C. The obtained crude antibiotic was

purified by using cold methanol. The purified antibiotic in powder form

from methanol was collected and its physicochemical properties were

determined.

The purified antibiotic showed significant andpotent antibiotic

activity. The product resembles macrolide group of antibiotics. Efforts

to establish the complete structure of the antibiotic is in progress.


Optimization of the parameters of the bioreactors and medium

formulation helped in increasing productivity of the product in

laboratory scale and may help in production scale.

Based on the above experimental study, we have arrived the

following conclusions:-

? The strain A-4 and A-4 mutant was found to be having better

antimicrobial activity in comparison with other soil isolates of

actinomycetes, which have been investigated.

? The strain A-4 was mutated by UV radiation technique and drug

resistance technique and the more stable strain, A-4 mutant

showing maximum antibiotic production, was isolated by

random screening method; and used for further experiment.

? The morphological, biochemical studies of strain A-4 showed

the characteristic features of the family Actinomycetaceae.

? The fermentation medium and parameters were optimized for

maximum production of antibiotic from both A-4 and A-4 mutant

strain using a lab scale fermenter.

? The potency of the antibiotic from both A-4 and the A-4 mutant

strains were compared using zone of inhibition. It is found that,

A-4 mutant showed maximum potency of the antibiotic.


? After the downstream processing, the antibiotic from the

fermentation broth was extracted by using ethyl acetate as a

organic solvent and purified by cold methanol.

? The UV, IR and NMR data showed the antibiotic belongs to the

macrolide group.


BIBLIOGRAPHY

1) Selman A. Waksman. The Actinomycetes. 1st edition, Watham,

MASS, USA. 1954.

2) John E. Smith. Perspective in biotechnology and applied

microbiology. 1989; 105-134.

3) John Bu’Lock. Basic Biotechnology. Academic Press 1987; 425-

448.

4) Erick J. Vandemme. Biotechnology of Industrial Antibiotics. Dekker

Series, Vol. 22, Marcel Dekker Inc., New York 1948; 3-42.

5) Egorov NS. Antibiotics – A Scientific Approach. MIR Publishers,

Moscow, 1992; 62-75 & 132-176.

6) Actinomycetals: Characteristics and practical importance. Edited

by G. Syker and FA Skinner. Academic Press, London & New

York 1973; 1-91 and 231-247.

7) Edward G. Jaff. Bergey’s Manual of Derminative Bacteriology. 9th

edition. WMC Brown Publishers, USA, 712-829.

8) William R. Strohl. Biotechnology of Antibiotic. 2nd edition, Vol.82.

Marcel Dekker Inc., New York, 1-63.

9) Casida LE. Industrial Microbiology, 3rd edition. Wiley Easter Ltd.,

1984; 3-437.


10) Brun YV, Skimkets LJ. Prokaryotic development. ASM Press,

2000; p.11-31.

11) ST Williams, R. Locci A. Beswick, DI Kurtboke, VD Kuznetsov, FJ

Lemonnior, PF Long. Detection and identification of novel

actinomycetes. Research in Microbiology 1993; 144(8):653-656.

12) S. Parekh, VA Vinci, RJ Strobel. Improvement of microbial strains

and fermentation processes. Applied Microbiology and

Biotechnology 2000; 54:287-301.

13) Stanbury PF, Whitaker A, Hall SJ. Principles of Fermentation

Technology. 2nd edition, Pergamon, 1995; 93-116, 43-65.

14) Yuzura Iwai and Satoshi Omura. Culture conditions for screening

of new antibiotics. Journal of antibiotics 1981; 25(2): 123-141.

15) S. Chatterjee and LC Vinning. Nutrient utilization in Actinomycetes.

Induction of ? -glucosidases in Streptomyces venezuelae.

Canadian Journal of Microbiology 1981; 27:639-645.

16) JC Ensign, P. Normand, JP Burden and CA Yallop. Physiology of

some actinomycete genera. Research in Microbiology 1993;

144(8):657-660.

17) Penicillins and other antibiotics by Gcos Andrews and J. Miller.

Tood Publishing Group 1949; 42-51.


18) Yoshiko Okamoto-Hosoya, Susumu Okamoto and Kozo Ochi.

Development and antibiotic-overproducing strains by site-directed

mutagenesis of the rpsl gene in Streptomyces lividans. Applied

and Environmental Microbiology 2003; 4256-4259.

19) Haifeng Hu and Kozo Ochi. Novel approach for improving the

productivity of antibiotic-producing strains by inducing combined

resistant mutations. Applied and Environmental Microbiology

2001; 1885-1892.

20) Yoshiko Hosoya, Susummu Okamoto, Hideyaki Muramatsu and

Kozo Ochi. Acquisition of certain Streptomycin-resistant (str.)

mutations enhances antibiotic production in bacteria. Antimicrobial

Agents and Chemotherapy 1998; 2041-2047.

21) Jun Xu, Haifeng Hu, Noboru Otake and Kozo Ochi. Innovative

approach for improvement of an antibiotic – overproducing

industrial strain of Streptomyces albus. Applied and Environmental

Microbiology 2003; 6412-6417.

22) PDA James, C. Edwards and M. Dawson. The effects of

temperature, pH and growth rate on secondary metabolism in

Streptomyces thermoviolaceus grown in a chemostat. Journal of

General Microbiology 1991; 137:1715-1720.


23) V. Gesheva, V. Ivanova, R. Gesheva. Effects of nutrients on the

production of AK-111-81 macrolide antibiotic by Streptomyces

hygroscopicus. Microbiological Research 2005; 160:243-248.

24) Juan Soliveri, Alfonso Mendoza and Maria-Enriqueta Arias. Effect

of different nutrients on the production of polyene antibiotics PA-5

and PA-7 by Streptoverticillium sp 43/16 in chemically defined

media. Applied Microbiology and Biotechnology 1988; 28:254-257.

25) Haque SFK, Sen SK, Pal SC. Screening and identification of

antibiotic producing strains of Streptomyces. Hindustan Antibiotics

Bulletin 1992; (3-4):76-83.

26) Haque SKF, Sen SK, Pal SC. Antimicrobial spectra and toxicity of

antibiotics from Streptomyces antibioticus sr 15-4. Indian Journal

of Microbiology 1996; 36:113-114.

27) Rong-Yang Wu and Ming-Ho Chen. Identification of the

Streptomyces strain KS3-5. Bot Bull Acad Sin 1995; 36:201-205.

28) James A. Matson, Charles Claridge, James A. Bush, Jaffery

Titers, William T. Bradner and Terrence W. Doyle. AT2433-Al,

AT2433-A2, AT2433-B1 and AT2433-B2. Novel antitumor

antibiotic compounds produced by Actinomadura melliaura.


Taxonomy, fulmentation, isolation and biological properties. The

Journal of Antibiotics 1989; XLII(11): 1547-1555.

29) JIN Zhi-Hua, CEN Pei-Lin. Improved production of spiramycin by

mutant Streptomyces ambofaciens. Journal of Zhejiang University

Science 2004; 5(6):689-695.

30) Sima Ray and AK Banik. Development of a mutant-strain of

Aspergillus niger and optimization of some physical factors for

improved calcium gluconate production. Indian Journal of

Experimental Biology 1994; 32:865-868.

31) MJ Butler, E. Takano, P. Bruheim, S. Jovetie, F. Marinelli, MJ

Bibb. Deletion of scbA enhancer antibiotic production in

Streptomyces lividans. Applied Microbiology and Biotechnology

2003; 61:512-516.

32) Ahmed Lebrihi, Driss Lamsaif, Garard Lefebure and Pierre

Germain. Effect of ammonium ions on spiramycin biosynthesis in

Streptomyces ambofaciens. Appl Microbiol Biotechnol 1992;

37:382-387.

33) Shigetoshi Okada and Sinroku Iwamatu. Scale-up production of

Milbemycin by Streptomyces hygroscopicus subspecies

aureolacrimosus with control of internal pressure, temperature.


Aeration and Agitation. J Chem Technol Biotechnol 1997; 70:179-

187.

34) Praveen Kumar Jain and PC Jain. Production of heptaene

antifungal antibiotic by Streptomyces purpeofuscus CM-1261.

Indian Journal of Experimental Biology 2005; 43:342-345.

35) Masayuki Hayakawa, Yuka Momose, Takayuki Kajiura, Toyohiko

Younazaki, Tomohiko Tamura, Kazunori Hatano and Hideo

Nonomura. A selective isolation method for Actinomadura viridis in

soils. Journal of Fermentation and Bioengineering 1995;

79(3):287-289.

36) Masayuki Hayakawa, Tomohiko Tamura, Hiromitsu Lino and

Hideo Nonomura. Pollen-baiting and drying method for the highly

selective isolation of Actinoplanes spp. from soil. Journal of

Fermentation and Bioengineering 1991; 72(6):433-438.

37) Masayuki Hayakawa, Takayuki Kajiura and Hideo Nomomura.

New methods for the highly selective isolation of

Streptosporangium and Dactylosporangium from soil. Journal of

Fermentation and Bioengineering 1991; 72(5):327-333.

38) SK Augustine, SP Bhavsar, M. Baserisaleni and BP Kapadnis.

Isolation, characterization and optimization of antifungal activity of


an actinomycete of soil origin. Indian Journal of Experimental

Biology 2004; 42:928-932.

39) Anurag Pandey, Anupam Shilka and SK Mujumdar. Utilization of

carbon and nitrogen sources by Streptomyces Kanamyceticus

M27 for the production of an antibacterial antibiotic. African

Journal of Biotechnology 2005; 4(9):909-910.

40) Slavica B. Llic, Sandra S. Konstantinovic, Zoran B. Todorovic.

UV/VIS Analysis and antimicrobial activity of Streptmyces isolates.

Medicine and Biology 2005; 12(1):44-46.

41) Beckett AH, Stenlake JB. Practical Pharmaceutical Chemistry. 3rd

edition, Vol. II. CBS Publishers 1986; 76-149.

42) Harald Gunther. NMR spectroscopy. 2nd edition, John Wiley and

Sons : New York, p.15-52.

43) Naoki Matsumoto, Toshio Tsuchida, Masato Maruyama, Naoka

Kinoshita, Yoshiko Homma and Tomio Takeuchi. Lactonamycin, a

new antimicrobial antibiotic produced by Streptomyces rishiriensis

MJ 773-88K3. Taxonomy, fermentation, isolation, physicochemical

properties and biological activities. The Journal of Antibiotics 1999;

52(3):269-275.


44) Masao Tsukamoto, Shigeru Nakajama, Hiroharu Arakawa,

Yasuyuki Sugiura, Hajime Suzuki, Mioko Hirayama. A new

antitumor antibiotic, BE-19412A, produced by a Streptomycete .

The Journal of Antibiotics 1998; 51(10): 908-914.

45) EO Stapley, M. Jackson, S. Hernadez, JM Mata. Cephamycins, a

new family of ? -Lactam antibiotics. Antimicrobial agents and

chemotherapy. 1972; 2(3): 123-131.

46) MS Puar, TM Chan, D. Delgarno, E. Barrabee, B. Pramanik, M.

Patel. Sch 486058: A novel cyclic peptide of Actinomycete origin.

The Journal of Antibiotics 2005; 58(2):151-154.

47) Noemi Antal, Hans-Peter Fiedler, Erko Stackebrandt, Winfried

Beil, Karsten Stroch, Axel Zeeck. Retymicin, Cyaltamycin B,

Saguayamycin Z and Ribofuranosyllumichrome. Novel secondary

metabolites from Micromonospora sp. Tii 6368. Taxonomy,

fermentation, isolation and biological activities. The Journal of

Antibiotics 2005; 58(2):95-102.

48) Richard A, Nelson, Joseph A. Pope, Jr., George M. Luedemann,

Lloyd E. McDaniel and Carl P. Schaffner. Crisamicin A, A new

antibiotic from Micromonospora 1. Taxonomy of the producing

strain, fermentation, isolation, physicochemical characterizatiion


and antimicrobial properties. The Journal of Antibiotics 1986;

39(3): 335-344.

49) Teruaki Ozasa, Kenichi Suzuki, Miho Sasamata, Konichi Tanaka,

Masato Kobori, Shigenobu Kadota, Koji Nagai, Takeshi Saito,

Shunichi Watanabe and Masaru Isanami. Novel antitumor

antibiotic phospholine 1. Production, isolation and

characterization. 2. Structure determination. The Journal of

Antibiotics 1989; XLII(9): 1331-1343.

50) WM Maiese, MP Lechevalier, HA Lechevalier, J. Korshalla, N.

Kuck, A. Fantini, MJ Wildey, J. Thomas and M. Cyreenstein.

Calicheamicins, a novel family of antitumor antibiotics: Taxonomy,

fermentation and biological properties. The Journal of Antibiotics

1989; XLII(4):558-563.

51) Li-H, Lu W, Zhang Y, Jin W, Pao P, Liu X, He Y, Zeng Y. Isolation

and characterization of Mutactimycin-producing mutant. Weisheng

Wu Xue Bao 1992; 32(5):353-358.

52) Masayuki Takizawa, Rita R. Colwell and Russel T. Hill. Isolation

and diversity of actinomycetes in the Chesapeake Bay. Applied

and Environmental Microbiology 1993; 997-1002.


53) Ogata K, osawa H and Tani Y. Production of an antibiotic by

Streptomyces sp. A-60. J Ferment Technol 1977; 55(3):285-289.

54) Judith Schimana, Hans-Peter Fiedler, Ingrid Growth, Roderich

Siibmuth, Winfried Beil, Martina Walker and Axel Zeeck.

Simocyclinones, Novel cytostatic angucyclinone antibiotics

produced by Streptomyces antibioticus Tii 6040, 1. Taxonomy,

fermentation, isolation and biological activities. The Journal of

Antibiotics 2000; 53(8): 779-787.

55) Moncheva P, Tishkova S, Dimitrova N, Chipeva V, Nikolova SA

and Bogatzevska N. Characteristics of soil actinomycetes from

Antartica. Journal of Culture Collections 2000-2002; 3 : 3-14.

56) Klaus Gebhardt, Rudiger Pukall and Hans-Peter Fidler.

Streptocidins A~D, novel cyclic decapeptide antibiotics produced

by Streptomyces sp. Tii-6071, Taxonomy, fermentation, isolation

and biological activities. The Journal of Antibiotics 2001; 54(5):

428-433.

57) Marcelo Bertasso, Meike Holzenkampfer, Axel Zeeck, Erko

Stackebrandt, Winfried Beil and Hans-Peter Fiedler. Ripromycin

and other polycyclic macrolactums from Streptomyces sp. Tii

6239. Taxonomy, fermentation, isolation and biological properties.

The Journal of Antibiotics 2003; 56(4): 364-371.


58) Tomotsu Furumai, Keiichi Takagi, Yasuhiro Igarashi, Noriko Saito

and Toshikazu Oki. Arisostatins A and B, New members of

tetrocarcin class of antibiotics from Micromonospora sp. TP-

A0316: Taxonomy, fermentation, isolation and biological

properties. The Journal of Antibiotics 2000; 53(3227-232.

59) Lintskarova DG, Stoilova-Disheva MM, Peltekova VT. Increase in

tylosine production by a commercial strain of Streptomyces

fradiae. Prikl Biokhim Mikrobiol 2005; 41(2):189-193.

60) Hiromi B. Maruyama, Yasuji Suhara, Junko Suzuki-Watanabe,

Yoshifumi Maleshima, Nobuko Shimizu, Michiko Ogura-Hamada,

Hideo Fujimoto and Kouichi Takano. A new antibiotic,

Fumaramidmycin: production, biological properties and

characterization of producer strain. The Journal of Antibiotics

1975; XXVIII(9): 636-647.

61) Masayuki Hayakawa, Hiromitsu Lino, Satoshi Takeuchi and

Toyohiko Tamazaki. Application of a method incorporating

treatment with chloramine-T for the selective isolation of

Streptosporangiaceae from soil. Journal of Fermentation and

Bioengineering 1997; 84(6):599-602.

62) Hideo Nonomura and Yuwao Onara. Distribution of Actinomycetes

in soil. Journal of Fermentation Technology 1971; 49(1):1-7.


63) R. Thakur, MK Roy, NN Dutta and Rl Bezbaruah. Coordinate

production of Cephamycin C and clavulanic acid by Streptomyces

clavuligerus. Indian Journal of Experimental Biology 1999;

37:1031-1033


ANNEXURE

The following the compositions of the media employed for strain

preservation and morphological studies.

1. Actinomycetes Agar Gms/Lt

Heart infusion broth 25.0

Casein enzymic hydrolysate 4.0

Yeast extract 5.0

Dextrose 5.0

Cysteine HCl 1.0

Soluble starch 1.0

Potassium phosphate 15.0

Ammonium phosphate 1.0

Magnesium sulphate 0.2

Calcium chloride 0.02

Agar 20.0

2. Carbon utilization agar (ISP-9)

Ammonium phosphate 2.64

Monopotassium sulphate 2.38


Dipotassium sulphate 5.65

Magnesium sulphate 1.0

Copper sulphate 0.006

Ferrous sulphate 0.0011

Magnesium chloride 0.0079

Zinc sulphate 0.0015

Agar 15

pH – 7

3. Glucose nutrient broth

Meat extract 3.0

Peptone 5.0

Glucose 10.0

pH – 7

4. Glycerol-Asparagine Agar Medium (ISP-5)

L-asparagine (anhydrous) 1.0

Glycerol 10.0

K2HPO4 1.0

Trace salts solution 1.0 ml

Distilled water 1.0 ml]

pH – 7.2 to 7.4


5. Hydrogen Sulphide Production Medium

Ferric ammonium nitrate 0.5

Dibasic potassium phosphate 1.0

Na2S2O4 0.02

Yeast extract 1.0

Agar 20

pH – 7

6. Inorganic salt – starch agar medium (ISP-4)

Solution I

Soluble starch – 10 gm was made into paste with the

small amount of cold water and then the volume was made up

to 500ml.

Solution II

K2HPO4 1.09

NaCl 1.09

Ammonium sulphate 2.09

Calcium carbonate 2.09

Trace salt solution 1.0 ml


Distilled water 500 ml

pH – 7.0 to 7.4

Solution I & II were mixed and 20.09 of was added.

7. Nutrient Gelatin

Peptone 5.0

Beef extract 3.0

Gelatin 120.0

pH – 6.8

8. Organic Nitrate Broth

Peptone 0.5

Meat extract 0.3

KNO3 0.1

pH – 7.0

9. Peptone – Yeast Extract Iron Agar (ISP-1)

Peptone 20.0

Ferric ammonium citrate 0.5

K2HPO4 1.0

Na2S2O35H2O 0.08


Yeast extract 1.0

Solution II

K2HPO4 1.0 g

NaCl 1.0 g

Ammonium sulphate 2.0 g

Calcium carbonate 2.0 g

Trace salt solution 1.0 ml

Distilled water 500 ml

pH – 7.0 to 7.4

Solution I & II were mixed and 20.0 g of water added

10. Nutrient Gelatin

Peptone 5.0

Beef extract 3.0

Gelatin 120.0

pH – 6.8

11. Organic Nitrate Broth

Peptone 0.5

Meat extract 0.3

MHO3 0.1


pH – 7.0

12. Peptone – Yeast Extract Iron Agar (ISP-6)

Peptone 20.0

Ferric ammonium citrate 0.5

K2HPO4 1.0

Na2S2O35H2O 0.08

Yeast extract 1.0

Distilled water 1 litre

Agar 20.0

pH – 7.0

13. Soybean Casein Digest Agar

Casein enzymic hydrolysate 17.0

Peptic digest of soybean meal 3.0

Sodium chloride 5.0

Dipotassium phosphate 2.5

Dextrose 2.5

pH 7.1 ± 0.2

14. Tyrosine Agar (ISP-7)


L-Tyrosine 0.5

Glycerol 15.0

L-Asparagine 1.0

K2 HPO4 – 7H2O 0.5

MgSO4 7H2O 0.5

NaCl 0.5

FeSO4 . 7H2O 0.5

Trace salts solution 1.0 ml

Distilled water 1 lit

Agar 20.0

pH – 7.2

15. Waksman No.42 Medium

Yeast Extract 1.0

L.tyrosine 1.0

Sodium chloride 8.5

Agar 16.0

pH – 6.8

Dooccttoorr ooff PPhhiilloossoopphhyy -...

Documents

Transcript of Dooccttoorr ooff PPhhiilloossoopphhyy -...