Biotechnology In Microbial Processes

BIOTECHNOLOGY IN MICROBIAL PROCESSES

OLANIYI RASHEED ABIODUN

UNVERSITY OF LAGOS, LAGOS. NIGERIA MATRIC NO: 089071021

MICROBIAL TECHNOLOGY (MIC 801)

BIOTECHNOLOGY IN MICROBIAL PROCESSES The word biotechnology was coined in 1919 by Kark Ereky to apply to the interaction of biology with human technology (Narins, 2003). Brian (2001) defined biotechnology as Industrial processes requiring the use of biological systems, including genetic engineering, fermentation technology, hybridoma technology and agricultural technology. There are many microbial processes in which biotechnology has been engaged. This range from genetic engineering, enzyme production, production of metabolites of industrial significance, production of different fermented foods, bioremediation, microbial digestion of cellulose employed in biopulping, microbial degradation of biomass to produce biofuels, production and use of encapsulated and immobilized cells, e.t.c.

GENETIC ENGINEERING Recombinant DNA technology, genetic modification/manipulation (GM) and gene splicing are all terms used together with or to refer to genetic engineering. It is a branch of biotechnology where organisms are genetically manipulated to produce new products, alter virulence, increase plant production, e.t.c. (Gloria and Lewis 2002). These are done by various techniques of molecular biology, using methods such as mutation, cloning, gene amplification, protoplast fusion, e.t.c. Genetic engineering techniques have found some successes in numerous applications. Some examples include the manufacture of synthetic human insulin through the use of modified bacteria, the manufacture of biodegradable plastics by the use of Polyhydroxybutyric acid (PHB) produced by Alcaligenes eutrophus, and the production of new types of experimental mice such as the oncomouse (cancer mouse) for research. ENZYME PRODUCTION

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Production of enzymes by microorganisms has been put to use more on the industrial scale; this is because as catalysts they enhance the rate of reactions and more so, as biocatalysts they do not have side effects. This is the reason why many chemical processes that involve the use of chemical compounds as catalysts are now being replaced by microbial enzymes. Examples of enzymes that are useful for industrial processes and which have enjoyed the use of biotechnology are: Amylase, pectinase, protease, cellulase lipase,esteraces, e.t.c.

Enzymes

Source or Type

Carbohydrases

Applications Laundry and dishwashing detergents, industrial pipe/tank cleaners, textiles, pulp and paper, fermentation ethanol

Alpha-amylase

Bacterial a-amylase

Textiles, starch syrups, laundry and

(e.g., Bacillus

dishwashing detergents, paper desizing,

subtilis), Fungal a-

fermentation ethanol, animal feed

amylase (e.g., Aspergillus niger), Alkaline a-amylase ß-amylase

From a strain of

Brewing, maltose syrup

Bacillus Cellulase

Dishwashing detergents, animal feed, textiles, bioenergy production

Proteases

Brewing, baking goods, protein processing, distilled spirits, laundry and dishwashing detergents, lens cleaners, leather and fur, chemicals

Acid proteinase

Endothia parasitica,

Baking, improves dough handling

Rhizopus, Aspergillus niger, A. oryzae Alkaline

Bacillus subtilis,

protease

Bacillus

Detergents, leather and fur

licheniformis

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Bromelain

Pineapple stem

Food industry

Pepsin

Porcine or bovine

Cheese production

stomach Peptidases Aminopeptidase Lactococcus lactis

Food and animal feed

Endo-peptidase Lipases and

Phospholidases,

Esterases

pregastric esterases,

Cleaners, leather and fur, dairy, chemicals

phosphatases Aminoacylase

Porcine kidney,

Optical resolution of amino acids

Aspergillus melleus Glutaminase

Bacillus, Aspergillus Conversion of glutamine to glutamate

Lysozyme

Chicken egg white,

Antibacterial (germicidal in dairy industry)

Saccharomyces cerevisiae, Pichia pastoris Penicillin

Bacillus megaterium, Chemical synthesis

acylase

Escherichia coli

Source: Diversa & Novo Nordisk BIOFUEL Biofuels are fuels made from biomass or waste products of animals like cow dung. One of the most important ways of making biofuels is the use of the degrading ability of microorganisms. The microorganisms degrade the raw materials to produce fuels of different types such as biodesiel, biogas, bioalcohols, e.t.c. Biogas- Biogas is produced by the process of anaerobic digestion of organic material by anaerobes. It can be produced either from biodegradable waste materials or by the use of energy crops fed into anaerobic digesters to supplement gas yields.

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Bioalcohols- are produced by the action of microorganisms and enzymes through the fermentation of sugars or starches (easiest), or cellulose. FERMENTATION Fermentation is the metabolic process in which small amounts of Adenosine Triphosphate (ATP) are produced from glucose in an anaerobic condition by microorganisms and sometimes create by-products of ethyl alcohol or lactic acid. Fermentation is considered one of the most important forms of biotechnology and can be traced back thousands of years. The benefits of fermentation are numerous, this is why it is used in many industrial processes like pharmaceutical, brewing, food, e.t.c. This include; the conversion of food to different varieties with unique tastes and flavours; making inedible foods edible, increase in shelf life, e.t.c. BIOPOLYMERS Biopolymers are microbially produced polymers used to modify the flow characteristics of liquids and to serve as gelling agents (Prescott et al., 2002). These are employed in many areas of the pharmaceutical and food industries. The advantage of using microbial biopolymers is that production is independent of climate, political events that can limit raw material supplies, and the depletion of natural resources. Production facilities also can be located near sources of inexpensive substrates (e.g., near agricultural areas). At least 75% of all polysaccharides are used as stabilizers, for the dispersion of particulates, as film-forming agents, or to promote water retention in various products. Biopolymers include (1) dextrans, which are used as blood expanders and absorbents; (2) Erwinia polysaccharides that are in paints; and (3) polyesters, derived from Pseudomonas oleovorans, which are a feedstock for specialty plastics. Cellulose microfibrils, produced by an Acetobacter strain, are used as a food thickener. Polysaccharides such as scleroglucan are used by the oil industry as drilling mud additives. Xanthan polymers enhance oil recovery by improving water flooding and the displacement of oil. This use of xanthan gum, produced by Xanthomonas campestris, represents a large potential market for this microbial product. Cyclodextrins can be used for a wide variety of purposes because these cyclical molecules bind with substances and modify their physical properties. For example, cyclodextrins will increase the solubility of pharmaceuticals, reduce their bitterness, and mask chemical odors.

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Cyclodextrins also can be used as selective adsorbents to remove cholesterol from eggs and butter or protect spices from oxidation. BIOSURFACTANTS Many surfactants that have been used for commercial purposes are products of synthetic chemistry. At the present time there is an increasing interest in the use of biosurfactants. These are especially important for environmental applications where biodegradability is a major requirement. Biosurfactants are used for emulsification, increasing detergency, wetting and phase dispersion, as well as for solubilization. These properties are especially important in bioremediation, oil spill dispersion, and enhanced oil recovery (EOR). The most widely used microbially produced biosurfactants are glycolipids. These compounds have distinct hydrophilic and hydrophobic regions, and the final compound structure and characteristics depend on the particular growth conditions and the carbon source used. Good yields often are obtained with insoluble substrates. These biosurfactants are excellent dispersing agents and have been used with the Exxon Valdez oil spill. (Prescott et al., 2002) BIOCONVERSION PROCESSES Bioconversions, also known as microbial transformations or biotransformations, are minor changes in molecules, such as the insertion of a hydroxyl or keto function or the saturation/desaturation of a complex cyclic structure, that are carried out by nongrowing microorganisms. The microorganisms thus act as biocatalysts. Bioconversions have many advantages over chemical procedures. A major advantage is stereochemical; the biologically active form of a product is made. In contrast, most chemical syntheses produce racemic mixtures in which only one of the two isomers will be able to be used efficiently by the organism. Enzymes also carry out very specific reactions under mild conditions, and larger water-insoluble molecules can be transformed. Unicellular bacteria, actinomycetes, yeasts, and molds have been used in various bioconversions. The enzymes responsible for these conversions can be intracellular or extracellular. Cells can be produced in batch or continuous culture and then dried for direct use, or they can be prepared in more specific ways to carry out desired bioconversions. A typical bioconversion is the hydroxylation of a steroid. In this example, the water-insoluble steroid is dissolved in acetone and then added to the reaction system that contains the pregrown microbial cells. The course of the modification is monitored, and the final product is extracted from the medium and purified.

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Biotransformations carried out by free enzymes or intact nongrowing cells do have limitations. Reactions that occur in the absence of active metabolism—without reducing power or ATP being available continually —are primarily exergonic reactions. If ATP or reductants are required, an energy source such as glucose must be supplied under carefully controlled nongrowth conditions. When freely suspended vegetative cells or spores are employed, the microbial biomass usually is used only once. At the end of the process, the cells are discarded. Cells often can be used repeatedly after attaching them to ion exchange resins by ionic interactions or immobilizing them in a polymeric matrix. Ionic, covalent, and physical entrapment approaches can be used to immobilize microbial cells, spores, and enzymes. Microorganisms also can be immobilized on the inner walls of fine tubes. The solution to be modified is then simply passed through the microorganism-lined tubing; this approach is being applied in many industrial and environmental processes. These include bioconversions of steroids, degradation of phenol, and the production of a wide range of antibiotics, enzymes, organic acids, and metabolic intermediates. One application of cells as biocatalysts is the recovery of precious metals from dilute-process streams. (Prescott et.al, 2005) PRODUCTION OF ANTIBIOTICS Microorganisms produce a huge number of small molecular weight compounds that are broadly described as secondary metabolites. A traditional approach to the discovery of new, naturally occurring bioactive molecules utilizes “screens.” A screen is an assay procedure that allows testing of numerous compounds for a particular activity. Tens of thousands of secondary metabolites and other compounds have been examined for biological activity in various organisms and many have proved invaluable as antibacterial or antifungal agents, anticancer drugs, immunosuppressants, herbicides, tools for research, and the like. Genetically modified microorganisms have been engineered to produce such compounds in large amounts. Among these, antibiotics are the secondary metabolites considered among the most important to human therapeutics, and the most extensive use of screens is in the search for compounds with selective toxicity for bacteria, fungi, or protozoa. It is estimated that natural microbial antibiotics provide the starting point for over 75% of marketed antimicrobial agents (Glazer et al., 2007). Examples include Penicillin, Streptomycin, Ampicillin, Tetracycline, e.t.c. One of them is discussed below.

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Streptomycin Streptomycin is a secondary metabolite produced by Streptomyces griseus, for which changes in environmental conditions and substrate availability also influence final product accumulation. In this fermentation a soybean-based medium is used with glucose as a carbon source. The nitrogen source is thus in a combined form (soybean meal), which limits growth. After growth the antibiotic levels in the culture begin to increase under conditions of controlled nitrogen limitation. The field of antibiotic development continues to expand. At present, 6,000 antibiotics have been described, with 4,000 of these derived from actinomycetes. About 300 new antibiotics are being discovered per year (Prescott et al., 2005) BIOREMEDIATION Bioremediation is the field of biotechnology that is responsible for the process of cleaning up environmental sites contaminated with chemical pollutants by using living organisms; such as, bacteria, fungi, and plants, to degrade hazardous materials into less toxic substances for the environment. There are various techniques or methods used in bioremediation, the most commonly used are Bioenhancement (also known as biostimulation), Bioaugmentation, and Bioventing (Gareth and Judith, 2003). Biostimulation/Bioenhancement - concentrates solely on the existing microfauna, stimulating their activity by the manipulation of local environmental conditions. Bioaugmentation, by contrast, requires the deliberate introduction of selected microbes to bring about the required clean-up. These additions may be unmodified ‘wildtype’ organisms, a culture selectively acclimatised to the particular conditions to be encountered, or genetically engineered to suit the requirements. Enzyme or other living system extracts may also be used to further facilitate their activity. Some land remediation methods simultaneously bioenhance resident bacteria and bioaugment the process with the addition of fungi to the soil under treatment.

Bioventing

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Bioventing is a technique used to remediate contamination above the water table boundary, by aeration. The intention is to stimulate accelerated breakdown of the pollutants present. The aeration increases the dissolved oxygen levels of the soil water and thus facilitates uptake by the native microorganisms. (Gareth and Judith, 2003).

Fig. 1. Bioventing HYBRIDOMA TECHNOLOGY Hybridoma cells are cells that have been engineered to produce a desired antibody in large amounts. Hybridoma is a hybrid cell produced by the fusion of a tumor cell with an ordinary antibody-producing cell, which then proliferates and yields large amounts of a monoclonal antibody (Gosh et al., 1983) To produce monoclonal antibodies, B-cells are removed from the spleen of an animal that has been challenged with the relevant antigen. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer). This fusion is performed by making the cell membranes more permeable. The fused hybrid cells (called hybridomas), being cancer cells, will multiply rapidly and indefinitely and will produce large amounts of the desired antibodies. They have to be selected and subsequently cloned by limiting dilution. Supplemental media containing Interleukin-6 (such as briclone) are essential for this step. The production of monoclonal anti-bodies was first invented by Cesar Milstein, Georges J. F. Köhler and Niels Kaj Jerne in 1975.

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MODERN BIOTECHNOLOGICAL APPROACHES TO TRADITIONAL PROCESSES Recombinant Chymosin It is a fact that good-quality cheese can only be made with good-quality rennet, derived from the stomach of unweaned calves. Chymosin is the proteolytic enzyme in rennet that catalyzes the milk clotting activity, but it is often contaminated with other enzyme activities and microorganisms that may cause quality problems. The availability of this enzyme is limited and can be costly. The calf chymosin gene has been isolated by several research groups and cloned in microorganisms such as Escherichia coli(Beppu, 1983), Kluyveromyces marxianus , and Aspergillus niger . The K. marxianus chymosin is excreted into the fermentation broth and is easily purified. The final product is free of other activities or microorganisms and has been on the market now for some time with excellent results (Jenson, 1993). Phage Resistance Bacteriophages are viruses that infect and kill bacteria, including cheese and fermented milk starter cultures, which leads to low production, low-quality products, and economic losses. The discovery that some starter strains have phage resistance mechanisms encoded on plasmids has resulted in the development of molecular cloning techniques to develop new resistant strains, especially in Lactococcus lactis. Currently, research in this subject is divided among three main areas: (1) the study of the mechanisms of infection (Valyasevi et al.,1991; Monteville et al., 1994) and resistance (McKay and Baldwin, 1984; Sing and Klaenhammer, 1990; Garvey et al., 1995, 1996); (2) the study of restriction and modification systems in lactic acid bacteria (Sing and Klaenhammer, 1991; Su et al., 1999); and (3) the study of bacteriophage genomes (Brown et al., 1994; Djordjevic and Klaenhammer, 1997). Something similar is also being developed in the case of yogurt for Streptococcus thermophilus (Brussow et al., 1994). Nisin Resistance Potential food-grade selectable markers from the lactococci include genes associated with carbohydrate metabolism, bacteriophage resistance, and nisin production or resistance (Froseth and McKay, 1991). Nisin is an important antimicrobial agent produced by some Lactococcus lactis subsp. lactis strains and is very effective against a variety of Gram positive bacteria (Froseth et al., 1988). In one of the studies related to the cloning of nisin

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resistance, the objective was to obtain a genetically modified strain of L. lactis subsp. lactis lac(–) for potential use in accelerating the ripening rate of cheese. A very efficient new foodgrade cloning system for industrial strains of Lactococcus lactis has just been developed that allows the overexpression of many technologically important cloned genes in industrial strains. This new vector (pFG200) has many advantages, including (1) easy cloning of genes into a versatile polylinker region, (2) a small and stable multicopy vector that is introduced by electroporation into Lactococcus with high efficiency, and (3) a selection system allowing selection and maintenance in milk, which allows genetic modification, cloning, and overexpression of Lactococcus DNA in a food-grade manner. The plasmids generated with this vector are stably maintained in the host cells for more than 35 generations in media, including milk (Sørensen et al., 2000). Other Applications Other applications of biotechnology in microbial processes that cannot be expantiated in this write up include: Biomolecular engineering, Molecular bioinformatics, Organic acid production, Vaccine production.

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REFERENCES Alexander N. Glazer and Hiroshi Nikaido (2007). Microbial Technology Second Edition. Cambrige University Press Beppu, T. (1983) The cloning and expression of chymosin (rennin) genes in microorganisms,Trends Biotechnol., 1(3), 85–89. Brigham Narins (2003). World of Immunology and Microbiology. Vol. 1 and 2 Brussow, H., Fremont, M., Bruttin, A., Sidoti, J., Constable, A., and Fryder, V. (1994) Detection and classification of Streptococcus thermophilus bacteriophages isolated from industrial milk fermentation, Appl. Environm. Microbiol. , 60, 4537–4543. Gareth M.E and Judith C.F. (2003) Environmental Biotechnology. Theory and Application Coffey, A.G., Daly, C., and Fitzgerald, G. (1994) The impact of biotechnology on the dairy industry, Biotechnol. Adv., 12, 625–633. Ghosh, AK, Mason, DY, Spriggs, Al (1983). Immunocytochemical staining with monoclonal antibodies in cytologically "negative" serous effusions from patients with malignant disease. J Clin Pathol, 36, 1150-1153. Gustavo F. and Gustavo V. (2003). Food Science and Food Biotechnology. CRS Press. Washington. Jenson, I. (1993) Biotechnology and the food supply: food ingredient products of biotechnology, Food Australia, 45(12), 568–571. Prescott , Harley, and Klein (2002). Microbiology 5th Edition. McGraw-Hill. Robert W. Hutkins (2006). Microbiology and Technology of Fermented Foods. Blackwell Publishing. Thieman, William (2004). Introduction to Biotechnology. San Francisco, CA: Pearson Education, Inc. Valyasevi, R., Sandine, W.E., and Geller, B.L. (1991) A membrane protein is required for bacteriophage c2 infection of Lactococcus lactis subsp. lactis C2, J. Bacteriol., 173, 6095–6100.

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Sørensen, K.I., Larsen, R., Kibenich, A., Junge, M.P., and Johansen, E. (2000) A foodgrade cloning system for industrial strains of Lactococcal lactis, Appl. Environm. Microbiol., 66, 1253–1258.

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