AN INTRODUCTION To

INDUSTRIAL MICROBIOLOGY FOR THE GRADUATE AND POST GRADUATE STUDENTS OF DIFFERENT UNIVERSITIES IN MICROBIOLOGY AND BIOTECHNOLOGY

Dr. P.K. SIVAKUMAAR

Ph.D. Department of Microbiology Annamalai University Annamalainagar-608002

Dr. M.M. Joe

M.Phil., Ph.D. DST(ST-TN) Project Department of Microbiology Annamalai University Annamalainagar-608002

Dr. K. Sukesh

M.Phil., Ph.D. Department of Microbiology Malankara Catholic College Kaliakkavilai-629153

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PREF ACE TO THE FIRST EDITION PREFACE th

The 20 Century continued to spur on developments in Industrial Microbiology, since material shortages forced countries to look for biological sources of chemical feedstock. Currently numerous fermentation based products have hit the market. Recently Industrial Microbiology has been introduced in syllabus of most of the universities, at the graduate and postgraduate levels for microbiology and biotechnology students. The subject of industrial microbiology is quite vast with rapid scientific advances in a dazzling phase. This textbook “An Introduction to Industrial Microbiology” has various well-compiled chapters dealing with all aspects of industrial microbiology. Attempt has been made to provide the basic relevant information’s to serve the need of beginner’s. The subject matter has been prepared in simple language for the better understanding of the students. The fundamental concepts in all the chapters serve as a good foundation to understand and interpret the latest research and development going in the field of industrial microbiology. Chapter one deals with the history, concept and scope of industrial microbiology. In chapter two we have dealt with some basic aspects of fermentation technology such as fermentation media, aseptic techniques and fermentation process. Chapter three deals with some of the basic aspects of fermenter design, while the chapter four addresses the chemical engineering aspects of fermentation technology. Chapter’s five to nine are entirely devoted to different fermentation products ranging from organic acids, solvents, vitamins, enzymes, alcoholic beverages, antibiotics, etc and discusses various methods for the production. In chapter nine we have dealt with biofuels, petroleum, textile and certain aspects of environmental microbiology. In chapter ten an attempt has been made to address some industrial aspects in the production of microbial inoculants in the field of agriculture. The final chapter covers some aspects of post production process, fermentation economics, IPR, etc. In addition some basic concepts of metagenomics and nanobiotechnology are also discussed. This introductory book would be of immense help to undergraduate and postgraduate students of microbiology, biotechnology and allied sciences. We would like to thank the researchers, authors, editors and publishers whose publications have helped us in making out the text. We would to thank the Lord Almighty for his blessings showered on us and for leading us into the path of enlightment. The authors express their gratefulness to the vice-chancellor, Registrar and syndicate members of Annamalai University for permitting them to write this work. They express their sincere thanks to Dr. P. Narayanasamy, Dean Faculty of Agriculture, Annamalai University and Dr. SM. Muthukarrupan, Professor and Head, Department of Microbiology for their valuable suggestions and encouragement.

We would like to thank Mr. J. Danie Kingsley, Assistant Professor, School of Biosciences and Technology, VIT, Vellore for his contribution and constructive suggestions on chapter four. We thank our colleagues for their timely help and critical suggestion. Assistance rendered by our research scholars is also greatly acknowledged. Thanks are also due to Mr. K. Selvakumar, Laksha Computers, Annamalainagar for typing the manuscript and Mr. Santanu and Mr. Suryakanthan for drawing the diagrams. We would like to thank the publishers especially for providing us an opportunity to publish a book on a discipline like Industrial Microbiology that faces a dearth of quality publications. —Authors

ABBREVIA TION ABBREVIATION % ° C acetyl COA ADC ADH ADP AFM ARA ATCC ATP BCA BCG BOD Bp BPN Bt C cc CFSTR Cfu cm CMCase CMI COD CPBR cps Cry CSTF CSTR Cyt d DAC DSP dw EMP

: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

Per cent Degree Centigrade Acetyl coenzyme alpha Analog to Digital Convertor Alcohol Dehydrogenase Adenosine diphosphate Atomic Force Microscopy Acetylene reduction assay American type culture collection Adenosine triphosphate Biocontrol Agents Bacillus of Calmette and Guren Biological oxygen demand Base pair Bacillus protease Novotype Bacillus thuringiensis Centigrade cubic centimeter Continuous flow stirred tank reactor Colony forming units centimeter Carboxymethyl cellulase Common wealth mycological institute Chemical oxygen demand Centrifugal packed bed reactor Cellular polysaccharides Crystal Continuous stirred tank fermenter Continuous Stirred tank Bioreactor Cytolytic Diameter Digital to Analog Convertor Down stream processing Dry weight Embden meyerhof parnas (vii)

Endo-PG Endo-PMG Exo-PG Exo-PMG F FAD FID FMN G g/L h ha HBV HMP HPLC IAA ICI IPR ISI K kDa Kg kPa -1 L lb LD50 m min ml MLF Mol/L mPa MPN MRI MSG VA n mole N NAD NDOM NFB medium

: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

Endopolygalacturanase EndoPolymethygalacturanase Exopolygalacturanase ExoPolymethygalacturanase Fahrenheit Flavin adinine dinucleotide Flame Ionization Detector Flavin Mononucleotide Gram Gram per liter Hour Hectare Hepatitis B virus Hexose monophosphate High Performance Liquid Chromatography Indole acetic acid Imperial chemical Industries Intellectual Property rights Indian Standard Institution Kelvin Kilo Dalton Kilogram Kilo pascal Per Litre Libra (Latin word, meaning scales and balances) Lethal Dose, 50% meter minute milligram Malolactic fermentation Moles per Litre milli pascal Most probable number Magnetic Resonance Imaging Monosodium Glutamate Volatile Acidity nanomole Normality Nicotinamide adenine dinucleate Near field scanning optical microscopy Nitrogen free bromothymol blue medium (viii)

NIR nm NMR NPV OPA PCT PDA per sq in PET PFR pH POB ppm PRTB psi PSM RBC RIA RNA SH Sq m SSF SBRAS STED TCA THF TLC USPTO UV V/V VAM W/V Wt YEMA μg μm

: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

Near Infrared Spectroscopy nanometer Nuclear Magnetic Resonance Nuclear Polyhedrosis Virus Ortho-phthalaldehyde Paris conventional treaty/ Patent cooperation treaty Potato dextrose agar Per Square Inch positron emission tomography Plug flow reactor Hydrogen ion concentration Poly occulative bodies parts per million Platinum Resistance Thermometer probe Pressure per square inch Phosphate solubilizing Microorganisms Rotating Biological contractors Regional Institute for Antibiotics Ribosomal nucleic acid Sulphydryll Square meter Single stage fermentation Sequencing batch reactor activated sludge Standard Emission Depletion Tricarboxylic acid cycle Tetrahydrofolate Thin layer chromatography United states patent and trade mark office Ultraviolet Volume per Volume Vesicular Arbuscular Mycorrhizae Weight per volume Weight Yeast extract manitol agar microgram micromole

(ix)

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CONTENTS 1.

2.

3.

4.

5.

6.

Industrial Microbiology : An Introduction Industrial microbiology—Definition and Scope History of Industrial Microbiology General Strategy of Metabolism General Concepts of Respiration and Fermentation Enzymes Key points Review questions Industrial Fermentation: Basics Isolation and Screening of Industrially Important Microorganisms Fermentation Medium Components of Media Aseptic Techniques Fermentation Types Types of Fermentation Process Fermentation Kinetics Key Points Review Questions Bioreactors—Design and Operation Principles Bioreactor Design of Bioreactor Bioreactor Types Key Points Review Questions Chemical Engineering Aspects of Fermentation Technology Fermentation Process Control Transport Phenomenon in Bioprocess Key Points Review Questions Organic Acids and Solvents Organic Acids Organic Solvents Key Points Review Questions Microbial Polysaccharides, Amino Acids and Enzymes Microbial Polysaccharides Amino-Acids Enzymes Key points Review Questions (xi)

...

1–20 1 4 5 10 14 19 20 21–46 21 27 28 31 34 35 41 45 46 47 – 62 47 49 54 62 62 63 – 85 63 76 85 85 86 –110 86 100 108 110 111 – 124 111 113 116 124 124

7.

8.

9.

10.

11.

12.

13.

Vitamins, Growth Factors and Hormones Vitamins Growth Factors Hormones Key Points Review Questions Alcoholic Beverages and Other Microbial Food Products Alcoholic Beverages Microbial Food Products Key Points Review Questions Antibiotics and Vaccines Antibiotics Vaccines Key Points Review Questions Agricultural Microbial Inoculants: Industrial Production Aspects Biofertilizer Bio-control Agents Biopesticides Bio-Herbicides Inoculant Formulations Key Points Review Questions Biofuels, Textile, Petroleum and Environmental Microbiology Biogas Production Textile Microbiology Petroleum Microbiology Biosorption Bioremediation Bioleaching Composting Technology Key Points Review questions Dairy and Food Microbiology Dairy Microbiology Food Microbiology Food Preservation Detection of Food-Borne Pathogens Key Points Review Puestions Fermentation—Post Production Techniques and the Future Post Production Process The Future Key Points Review Questions References (xii)

125 – 136 125 130 132 135 136 137 – 163 137 146 163 163 164 – 183 164 173 182 183 184 – 207 184 195 197 201 202 206 207 208 – 228 208 212 213 214 217 220 222 227 228 229 – 268 229 250 261 264 267 268 269 – 302 269 295 301 302 303 – 307

Chapter

1

INDUSTRIAL MICROBIOLOGY : AN INTRODUCTION A. INDUSTRIAL MICROBIOLOGY—DEFINITION AND SCOPE

Use of microorganisms, usually grown on a large scale, to obtain valuable commercial products by certain chemical transformations is called industrial microbiology. The terms industrial microbiology and fermentation technology are virtually synonymous in their scope, objectives and activities. Fermentation is the oldest of all biotechnological processes. The term is derived from the Latin verb fevere, to boil-the appearance of fruit extracts or malted grain acted upon by yeast, during the production of alcohol (Stanbury et al., 1997). Fermentation is a process of chemical change caused by organisms or their products, usually producing effervescence and heat. Microbiologists consider fermentation as a process for the production of a product by means of mass culture of microorganisms. Biochemists consider fermentation as an energy generating process in which organic compounds act both as electron donors and acceptors; hence fermentation is an anaerobic process where energy is produced without the participation of oxygen or other inorganic electron acceptors. This discipline dates back and originated with bread and wine making fermentation processes (alcoholic fermentation) and subsequently expanded in the area of production of pharmaceuticals (e.g., antibiotics), food additives (e.g., amino acids), organic acids (e.g., butyric acid and citric acid), enzymes (e.g., amylases, proteases), and vitamins. All these products are obtained by enhancing the metabolic reactions that microorganism were already capable of carrying out in natural conditions. The services generated by microorganisms also include the degradation of organic wastes, detoxification of industrial wastes and toxic compounds, to the degradation of petroleum and management of oil spills, etc. Industrial microbiology also encompasses activities like production of biocontrol agents, inoculants used as biofertilizers, etc. In broadest sense, industrial microbiology, is concerned with all aspects of business that relate to microbiology. In a more restricted sense, industrial microbiology is concerned with (i) employing microorganisms to produce a desired product, and with (ii) preventing microbes from diminishing the economic value of various products. The activities in industrial microbiology begin with the isolation of microorganisms from nature, their screening for product formation, improvement of product yields, maintenance of cultures, mass culture using bioreactors, and usually end with the recovery of products and their purification. The major microorganisms used to carry out important chemical transformation (biocatalytic processes) are the microfungi (yeasts and moulds) and certain prokaryotes (e.g., Streptomyces). The ultimate source of all strains of microorganisms used in biocatalytic 1

2

AN INTRODUCTION TO INDUSTRIAL MICROBIOLOGY

processes is nature. Actually, all such microbial strains are normally obtained from the “wild-type” condition that existed when the strain was first isolated. These isolates may further improved by genetic manipulation via mutagenesis and selection or recombinant DNA technology or protoplast fusion (fungi). To be useful in industrial microbiology, an organism must: (a) Produce usable substance(s) or effect(s) (b) Be available in pure culture (c) Be genetically stable, but amenable to genetic manipulation (d) Produce spores or other reproductive structures to allow easy inoculation (e) Grow rapidly and produce the desired products quickly in large-scale cultures. (f ) Grow in such a way that the cells are easily separated from the product (g) Not be harmful to humans or plants and animals, etc. Once the valuable industrial microorganism comes to hand, it is maintained either in microbiology laboratory in industry or in a microbial culture collection centers at international or national level. At present, in addition to this traditional industrial microbiology, a new era of microbial biotechnology is fast expanding in which the genes of the microorganisms responsible for such and other metabolic reactions are being manipulated to give to many new products at commercial level. Over all the fermentation technology aims at combining the right organism, an in expensive substrate, and the proper environment to produce high yields of a desired product. Some important products of fermentation process are discussed below:

1. Microbial Biomass Microbial biomass is produced commercially as single cell protein (SCP) using unicellular algae as species of Chlorella or Spirulina for human or animal consumption, or viable yeast cells needed for the baking industry. Bacterial biomass is also used as an animal feed.

2. Micr obial Met abolit es Microbial Metabolit abolites (a) Primary metabolites: During the log or exponential phase microorganisms produce a variety of substances essential for their growth, such as nucleotides, nucleic acids, aminoacids, proteins, carbohydrates, lipids, etc, or by-products of energy yielding metabolism such as ethanol, acetone, butanol, etc. These products are usually called as primary metabolites and the phase at which it is produced is termed as trophase. Examples of some primary metabolites are given in Table 1.1. Table 1.1 Primary metabolite

Organism

Industrial importance

Citric acid

Aspergillus niger

Solvent

Acetone & butanol

Clostridium acetobutylicum

Food industry

Lysine Glutamic acid

Corynebacterium glutamicum

Riboflavin

Ashbya gossipii

Vitamin B12

Pseudomonas denitrificans

Nutritional additive Flavour enhancer Nutritional

3

INDUSTRIAL MICROBIOLOGY: AN INTRODUCTION

(b) Secondary metabolites: Organisms produce a number of products, other than the primary metabolites. The phase during which products are produced have no obvious role in the metabolism of the organism and this phase is called as idiophase. The products produced in this phase are called as secondary metabolites. Many secondary metabolites are produced as an end product of primary metabolism (Table 1.2). Table 1.2 Metabolite

Organism

Penicillin

Penicillium chrysogenum

Streptomycin

Streptomyces greseus

Erythromycin

Streptomyces erythreus

Gresofulvin

Penicillium griseofulvin

Gibberelin

Gibberella fujikuroi

Significance Antibiotics

Growth hormone

Enzymes are either produced in the trophase or in the idiophase and hence they do not belong to either of the group. Examples of some commercially produced enzymes are given in Table 1.3. Table 1.3 Enzyme

Organism

Amylase

Aspergillus oryzae

Cellulase

Trichoderma reesii

Protease

Bacillus sp

Pectinase

Aspergillus sp

Significance Brewing Food industry

3. Food Industr oducts Industryy Pr Products A wide variety of food products, such as sour cream, yogurt, cheese, fermented meat, bread, alcoholic beverages, vinegar etc are produced through microbial fermentation products.

4. Recombinant Products Recombinant DNA technology has made it possible to introduce genes from any organism into microorganism, resulting in the production of transgenic organisms capable of producing the product of the introduced gene. Genetically modified Escherichia coli, Saccharomyces cerevisiae, yeast and filamentous fungi are now being used to produce interferon, insulin, human serum albumin and several other products.

5. Bioconver sion Bioconversion Production of a structurally similar compound from a particular one, during the fermentation process is called as biotransformation or bioconversion. The oldest instance of this process is the production of acetic acid from ethanol. One example is the conversion of digoxin from digitoxin with the aid of Digitalis lanata.

6. Elicitor Elicitorss It is possible to induce production or to enhance the production of a compound in culture by elicitors, which may be by microorganisms. For example the production of morphine and codeine by Papaver somniferum has increased 18 times by Verticillium dahliae.