Microbial Biotechnology

Principles and Applications

Yuan Kun Lee

• Publisher: WSPC
• Year: 2006
• ISBN:
  9789814365543
• Topic: Biological Sciences
• Subtopic: Microbiology
• Index: Biological Sciences

In the second edition of this bestselling textbook, new materials have been added, including a new chapter on real time polymerase chain reaction (RTPCR) and a chapter on fungal solid state cultivation. There already exist a number of excellent general textbooks on microbiology and biotechnology that deal with the basic principles of microbial biotechnology. To complement them, this book focuses on the various applications of microbial-biotechnological principles. A teaching-based format is adopted, whereby working problems, as well as answers to frequently asked questions, supplement the main text. The book also includes real life examples of how the application of microbial-biotechnological principles has achieved breakthroughs in both research and industrial production.

Although written for polytechnic students and undergraduates, the book contains sufficient information to be used as a reference for postgraduate students and lecturers. It may also serve as a resource book for corporate planners, managers and applied research personnel.

Contents:

• Principles of Microbial Biotechnology:
  • Screening for Microbial Products (Alex Y L Teo and Hai Meng Tan)
  • Bioprocess Technology (Yuan Kun Lee)
  • Enzymology (Theresa May Chin Tan)
  • Manipulation of Genes (Azlinda Anwar, Heng-Phon Too and Kim Lee Chua)
  • Applications of Bioinformatics and Biocomputing to Microbiological Research (Paxton Loke and Tiow Suan Sim)
  • Real Time Polymerase Chain Reaction (Heng-Phon Too and Azlinda Anwar)
• Food Production Involving Microorganisms and Their Products:
  • Regulation of Foods Involving Genetically Engineered Microorganisms (Desmond K O'Toole and Yuan Kun Lee)
  • Fermented Foods (Desmond K O'Toole and Yuan Kun Lee)
  • Food Involving Yeast and Ethanol Fermentation (Desmond K O'Toole and Yuan Kun Lee)
  • Fungal Solid State Cultivation (Desmond K O'Toole)
  • Food Ingredients (Desmond K O'Toole and Yuan Kun Lee)
  • Enzyme Modified Food Products (Desmond K O'Toole and Yuan Kun Lee)
• Microbes in Agrobiotechnology:
  • Microbes and Livestock (Jimmy Kwang)
  • Transgenic Plants (Jian-Wei Liu)
• Microbes in Medical Biotechnology:
  • Diagnostic Clinical Microbiology (Vincent T K Chow, Tim J J Inglis and Keang Peng Song)
  • Microbes: Friends or Foes? (Vincent T K Chow, Tim J J Inglis and Keang Peng Song)
• Microbes in Environmental Biotechnology:
  • Municipal Wastewater Treatment (Lee Man Chu and P K Wong)
  • Industrial Wastewater Treatment (P K Wong and Lee Man Chu)
  • Municipal and Industrial Solid Waste Treatment (P K Wong and Lee Man Chu)
  • Regulatory Issues on Application of Natural and Genetic Engineered Microbes in Environmental Biotechnology (P K Wong and Lee Man Chu)
• Microbes in Alternative Energy:
  • Microorganisms and Production of Alternative Energy (Yuan Kun Lee)
• Patenting Microbial Biotechnology:
  • Patenting Inventions in Microbiology (John Candlish)

Readership: Primary Market: Undergraduates and teachers of tertiary institutions. Can also be used as a reference book for industry (corporate planners, managers and applied research personnel).

Part I

Principles of Microbial Biotechnology

The use of microorganisms for large-scale industrial purposes has a long history, which is long before the realization of the activities of the microorganisms. For centuries, beer, wine, vinegar, soy sauce and other fermented foods were produced through spontaneous fermentation of natural occurring microorganisms or the use of carry-over microbial seeds from the previous batch of production. The quality and productivity of these early products were very often inconsistent. The development of scientific screening and isolation methods allows the selection of desirable natural occurring or mutated microorganisms for specific purposes. These methods, coupled with the advancement of the technical know-how in large-scale sterilization of culture media, in provision of adequate oxygen supply and in mixing homogeneity of the culture systems, enable the exploitation of both anaerobic (yeast and some bacteria) and aerobic microorganisms (fungi and some bacteria). Common examples are: the development of large-scale processes for the production of citric acid, amino acids and antibiotics; in improved biotransformation of steroid hormones, as well as the mass production of many enzymes. The diverse catalytic activities of microorganisms are being used more and more widely to perform specific chemical reactions in the industrial production processes.

Microbial biotechnology was pushed to a new height in the eighties when continuous fermentation and airlift fermentation processes were developed for the production of food and feed grade microbial protein from industrial by-products, such as methanol and alkanes. These processes lead to considerable savings in capital, energy and labor costs.

Modern techniques of gene manipulation, and advanced bioinformatics and biocomputing are powerful tools for genomic and proteomic research. The scientific breakthroughs that ensued have made feasible the industrial manufacturing of non-microbial products, such as human growth hormone, interferon and viral vaccines.

It is the aim of Part I of this book to provide the readers with in-depth and comprehensive scientific knowledge in the areas as listed below, so as to facilitate the understanding of the various applications of microorganisms and the production of their bioactive molecules in the biotechnological systems:

• Screening for microbial products
• Bioprocess technology
• Enzymology
• Manipulation of genes
• Application of bioinformatics and biocomputing.

Chapter 1

Screening for Microbial Products

Alex Y. L. Teo and Hai Meng Tan

Kemin Industries (Asia) Pte Ltd 12 Senoko Drive, Singapore 758200

1.1. Introduction

If the discovery of penicillin marked the beginning of the antibiotics era, then the development of its fermentation has ushered in what might be called the golden age of industrial microbiology. The onset of this has resulted in the production of a large number of plant or microbial primary and secondary metabolites that are of commercial importance. Whereas primary metabolism is universal among living systems, secondary metabolism is mainly carried out by plants and microbes and is usually strain specific. The primary metabolites that are important in the bio-industry are the organic acids, vitamins, amino acids and purine nucleotides. However, of all the traditional products made by bioprocess, it is the secondary metabolites that are of great importance and value to the human health. Secondary metabolites, particularly from microbial sources, are selective in their actions on pathogenic bacteria and fungi. The success rate was so impressive that the pharmaceutical industry screened secondary metabolites almost exclusively for antibacterial, antifungal and antitumor chemotherapy as well as against diseases not caused by bacteria, fungi or tumors.

1.2. Screening for New Antibiotics

The main approach in which new antibiotics have been discovered has been by screening of groups of microorganisms such as Streptomyces, Penicillium, and Bacillus. In the screening approach, a large number of possible antibiotic-producing microorganisms are obtained from nature and pure isolates tested for antibiotic production by observing for diffusible materials that are inhibitory to the test or indicator bacteria. The classical method for testing potential antibiotic-producing microbial isolates is the cross-streak method, used by Fleming in his studies on penicillin. Isolates that demonstrate evidence of antibiotic production are then subjected to further studies to determine if the antibiotics they produce are new. When an organism producing a new antibiotic is discovered, it is produced in large quantity, purified and tested for cytotoxicity and therapeutic activity in infected animals. Most new antibiotics will fail the in vivo testings but a few of these new antibiotics that prove useful medically are then produced commercially. Since antibiotic-producing strains isolated from nature rarely produce the desired antibiotic at sufficiently high concentration, it is necessary to isolate new high yielding strains. In the commercial production of penicillin, the yield of this antibiotic was increased by 50,000 times using strain selection and appropriate medium development. Strain selection involves mutagenesis of the wild type culture, screening for mutants and testing of these mutants for enhanced antibiotic production.

1.3. Screening for Beneficial Cultures

Crude lactic cultures are known to inhibit some psychrotrophs in milk and ground beef. A large number of lactic acid bacteria, singly or in combination, have been shown to display varying degrees of antimicrobial activity against pathogenic microorganisms. In addition, viable cultures or components of lactic acid bacteria are useful in the treatment of displaced endogenous intestinal microflora, which are characteristic of many intestinal disorders and enhanced gut permeability of the host. Such bacteria are able to survive gastric conditions to colonize the intestine, at least temporarily, by adhering to the epithelium. They have been reported to improve the growth rate and feed utilization in pigs, chicken and calves and to improve their feed conversion ratio. There is significant decrease in the occurrence of diarrhea observed in pigs and calves fed with these beneficial bacteria. Lactic cultures are also believed to neutralize the effect of enterotoxin from E. coli, which are pathogenic for pigs. The beneficial effects of these bacteria ranged from displacement of harmful bacteria such as Clostridium perfringens to reduction of bacterial urease activity to synthesis of vitamins, stimulatory effects on the immune system and contribution to digestion.

1.4. Screening for Antimicrobial Peptides

Microbial peptides with pronounced antimicrobial activity are commonly isolated from animals, plants, microbes and in fermented food. They are small and cationic with molecular masses of between 3,000 and 6,000 daltons. Post-translational modification of precursor peptides has been shown to introduce intramolecular thioether bridges to cationic peptides such as Pep 5, nisin, and subtilin. Although these peptides offer an important potential safety advantage over chemically synthesized preservatives when incorporated into food, many peptides are not suitable owing to the pathogenic nature of the producer-strains. Peptides such as colicins, epidermin and Pep 5 may be useful in topical application in creams and salves, but are unlikely to be approved for use in foods because of the nature of the producer-strains. Bovine lactoferrin, an antimicrobial component of colostrum and milk, helps in the protection of infants from gastrointestinal infections. Porcine pepsin cleavage of native lactoferrin produces low molecular weight peptides inhibitory to some Gram-negative and Gram-positive bacteria. In addition, hydrolysis of native lactoferrin at pH 2 and 120°C produces active peptides, which are bactericidal.

Methanol-acetone extracts of lyophilized fermented milk by lactic cultures have been reported to contain ninhydrin-positive materials. These proteinaceous materials do not lose antimicrobial activity when exposed to 100°C for 10 minutes, are active at pH 5.4 and not inactivated by pepsin treatment. Purified cationic, low molecular weight material isolated from Streptococcus diacetylactis, has been found to be heat stable and active towards several Pseudomonas species up to pH 6.0. Acidophilin, a low molecular weight (approximately 700 daltons) nitrogenous compound from Streptococcus diacetylactis and Leuconostoc citrovorum cultures, has potent antimicrobial activity. Acidophilin is also produced by Lactobacillus acidophilus. Antimicrobial compound(s) from Streptococcus thermophilus are likely to be amines of low molecular weights, which are heat stable. An antimicrobial substance from Lactobacillus sp. strain GG is inhibitory to Gram-positive as well as Gram-negative bacteria but not lactobacilli. It is active between pH 3 and 5, heat stable and resistant to proteinase K, a-chymotrypsin, trypsin, and carboxypeptidase A. It resembles a microcin from some of the characteristics studied. The antimicrobial substance from Lb. sp. strain GG has a molecular weight of less than 1,000 daltons and is soluble in acetone-water.

1.5. Screening for Low Molecular Weight Peptides

Microcins are low molecular weight amino-acid-oligopeptide antibiotics, which are resistant to some proteases, extreme pH values and heat, and soluble in methanol. In contrast, bacteriocins are larger proteins, which can be inactivated by proteases. Microcin A15 (< 500 daltons) is a bacteriostatic compound with a methionine moiety. Microcin B17, with a molecular weight of 4000 daltons, is sensitive to pronase, subtilisin, and thermolisin. Microcin C7, which is about 900 daltons, is sensitive to trypsin and subtilisin but loses little activity when exposed to 100°C for 30 minutes. Microcin C7 is a linear structure consisting of acetyl-methionine, arginine, threonine, glycine, asparagine, alanine and a supposed ethanolamine entity at the carboxyl-terminal end. Microcins D15, D93, and D140 are highly hydrophilic, basic and small peptides.

1.6. Screening for Potential Bacteriocins

Bacteriocins are an extremely heterogeneous group of substances. The original definition of bacteriocins referred to proteins of the colicin type produced by Escherichia coli. Bacteriocins are proteinaceous compounds usually with bactericidal activity against species closely related to the producer bacterium. These secondary metabolites also possess fungicidal, metal-chelating and immuno-modulating properties. Most bacteriocins are produced by Gram-positive bacteria such as Bacillus, lactic acid bacteria and Streptomyces against pathogenic and food spoilage microorganisms such as Staphylococcus aureus and Listeria monocytogenes. Produced by strains of Gram-positive and Gram-negative bacteria, they are characterized by lethal biosynthesis, intraspecific activity, and adsorption to specific receptors. Bacteriocins are active macromolecules possessing a narrow inhibitory spectrum of activity, protein in nature, plasmid encoded and without effect on producer cells. Reports have shown that most bacteriocins produced by Gram-negative bacteria act on closely related species. On the other hand, inhibition of Gram-negative bacteria has not been clearly demonstrated by purified bacteriocins from Gram-positive organisms. In this regard, the presence of a lipoteichoic acid receptor for pediocin in the host cell wall suggested ...