BSc Industrial Microbiology Notes Study Material

BSc 2nd Year Industrial Microbiology Notes Study Material: BSc is a three-year program in most of the universities. Some of the universities also offer BSc Honours. Out of those, there are BSc 2nd Year Study Material, BSc Sample Model Practice Mock Question Answer Papers & BSc Previous Year Papers. At gurujistudy.com you can easily get all these study material and notes for free. Here in this post, we are happy to provide you Industrial Microbiology Notes Study Material.

BSc Industrial Microbiology Notes Study Material

Various commercial products of economic value made by microbes are (i) medicines i.e. pharmaceuticals, including antibiotics, steroids, human protein, vaccines, and vitamins, (ii) organic acids, (iii) amino acids, (vi) enzymes. (v) alcohols, (vi) organic solvents and (vii) synthetic fuels. In addition to these, quite recently potential of microbes could also be realised in (viii) recovery of metals from ores through bioleaching, (ix) recovery of petrol, and (x) single all protein production.

The term fermentation in industrial microbiology is used in a wider sense to include any chemical transformation of organic compounds carried out by using microbes and their enzymes. Production methods in industrial microbiology bring together the raw materials (substrates), microorganisms (specific strains or microbial enzymes) and a controlled favourable environment (created in a fermentor) to produce the desired substance. The essence of an industrial process is to combine the right organism, an inexpensive substrate, and the proper environment to produce high yields of a desired product.

Microbes possess a wealth of metabolic equipment that brings about diverse chemical transformations. This characteristic of microbes could be exploited in obtaining some valuable products of daily use. The cheap raw materials available in nature as a waste, may be converted into useful commercial products by the activity of microbes. Microbes thus serve a dual purpose. Firstly, they are good agents of disposal of these wastes, and secondly the resultant end products of their breakdown are useful commercial products.(BSc Industrial Microbiology Notes & Study Material)

Industrial microbiology is an important area of applied microbiology. It refers to the use of microorganisms in commercial enterprises. Cheap raw materials are converted to valuable products through the metabolism of microbes. Microbes for this purpose could be exploited in different ways. For instance this includes (i) synthesis of fermentation products as acids, alcohols or other organic compounds, (ii) transformation of one compound into another desired type, (iii) the production of enzymes, antibiotics, or insecticides, or (iv) the use of microbes themselves as food.

Development Phases

It is possible to distinguish different phases in the development of industrial microbiology. The first phase, relatively ancient, involves fermentations for production of alcoholic beverages and traditional dairy products and bread. These processes are non-septic and rely either on a large inoculum or on the good housekeeping to prevent microbial spoilage of product. They all also reflect the synthesis of a primary product of metabolism by the organism e.g. the enzyme rennin in cheese-making, lactic acid in curd, CO₂ in bread, and alcohol in beers and wines.

The second phase was the development of large scale relatively sterile culture facilities in which an inoculum is added. This included processes as large scale cultivation of yeast cells for brewing and baking industries. Efforts were made to fermentation vessels and pipelines. Steam was often used for this purpose. The growth media were often by-products of other industries i.e. complex mixture of molasses products come from butanol, and hence the industry that grew up was known as the fermentation industry. The third phase involved the batch culture of microbes under aerobic conditions and aseptic state. Strains of the microbes that are more efficient in antibiotic production were selected. Strains of Penicillium in current use produce as much as ten thousand times as much penicillium per unit volume of fermentation liquor than does Fleming’s original isolate.

During the 1960s, the fourth phase, interest in the use of microorganisms as bulk sources of protein for animal and human food, the so-called single-cell protein, led to the exploitation of continuous culture methods on industrial scale. This required the development of sophisticated fermentation equipment and the complete aseptic conditions.

Finally, the most recent fifth phase has been the biotechnology-a term often being used indiscriminately and fashionably. It can be defined as “the application of biological organisms, systems or processes to the manufacturing and service industries”. There has been some tendency to equate gene-cloning and recombinant DNA technology with biotechnology, through many other areas are included in it. Other important areas are the use of enzymes and immobilized cells as catalysts and sensors, the manipulation of cell physiology for the maximum production of metabolites and the use of cell fusion to generate either monoclonal antibodies or new cell lines.(BSc Industrial Microbiology Notes Study Material)

Microbes As Ideal Organisms

Microorganisms possess several characteristics, which make them most ideal organisms for industrial processes. They possess a broad variety of enzymes making an array of chemical conversions possible. They have a relatively high metabolic activity which allows conversions to take place rapidly. They possess a large surface area for quick absorption of nutrients and release of end products. Moreover, they have a high rate of multiplication, as a 20-minute generation time for E. coli under ideal conditions.(BSc Industrial Microbiology Notes Study Material)

In an industrial process, a microorganism in fact represents a mini-chemical factory. The efficiency of this factory depends upon the liberation of large amount of a single product that may be efficiently isolated and purified. The microbe should be easily cultivated and maintained and should have genetic stability with infrequent mutations. Its value enhances if it can grow on inexpensive, readily available substrate that may be the by-product of other industrial processes. For example, a large amount of whey is produced in cheese production. If there are other microbes which could convert the whey components to lactic acid, this will add to an overall profit of cheese industry.(BSc Industrial Microbiology Notes Study Material)

Of the many species of microbes, relatively few possess the genetic information needed to produce economically useful products. Some microbial species used for production of commercial products.

Production Process

After selecting the suitable microbe for the purpose, it is necessary to develop a fermentation process that optimises conditions for desired microbial activity and that should yield maximum amount of the product with highest economic profit. The commercial process occurs in a stepwise manner, initially using small flasks, then small fermentors (under 10 gallons), intermediate-size fermentors (upto several hundred gallons), and finally, large-scale fermentors (thousands of gallons). The organic and inorganic composition of the medium, pH, temperature and O₂ concentration are the main factors that are varied to maximise the efficiency of production process.

Essential nutrients in the medium for microbial growth include C, N, P sources shown below. The choice can be made on economic and biological grounds.(BSc Industrial Microbiology Notes Study Material)

Aeration

Of all conditions for growth, aeration is most important and difficult to maintain. Many industrial fermentations are aerobic and therefore it is important to achieve the optimal oxygen concentration to permit microbial growth with maximum product yield. The development of fermentors for the growth of obligately aerobic microbes in a broth (submerged aerobic culture) requires careful design in order to achieve optimal O₂ concentration. Many fermentor designs have mechanical stirrers to mix the solution, baffles to increase turbulence and ensure adequate mixing and forced aeration to provide required oxygen.

Batch versus Continuous flow process

A fermentation process may be designated as a batch process which is analogous to inoculating a flask containing a broth with a microbial culture, or as a continuous flow process, analogous to that of a chemostat. In a batch process, a large tank with a volume of upto 100,000 gallons of medium is used. The tank is known as a fermentor, since industrial microbiologists use the term fermentation for any aerobic or anaerobic process catalysed by microorganisms. The medium is sterilised with steam or with a gas as SO₂. Microorganisms, about 2 to 4% of the total volume are added to the fermentor. Oxygen is bubbled if the process is aerobic but omitted if anaerobic conditions are required. The microbial growth is allowed to take place for days, weeks or months. Microbes are removed after fermentation is complete. The product is isolated from the materials in the tank.(BSc Industrial Microbiology Notes Study Material)

As compared to batch process, flow-through fermentors are more prone to contamination. However, flow-through design has the advantage of producing a continuous supply of the product for commercial distribution. In this method, the medium is added continuously to the tank to replace that which has been fermented. The isolation and purification of product from the medium is thus an on-going process. An instrument called a chemostat is used to provide a constant flow, keeping microorganisms in the logarithmic phase of growth. In some cases a turbidostat is used. This instrument measures the turbidity of the microbial population to indicate the level of growth. When a certain level is reached, it adds new medium to the tank while withdrawing the spent material. The fermentation is thus maintained at a constant level.

Immobilised enzymes

The use of use of immobilised enzymes is an attractive alternative for production of a desired product. Microbial enzymes and/or microbial cells are adsorbed or bonded to a solid surface support, such as cellulose. The bonded and thus immobilised enzymes act as a solid-surface catalyst. The solution containing the biochemicals to be transformed by enzyme is then passed across the solid surface. Temperature, pH and O₂ levels are set optimal to achieve maximum rates of conversion. The use of immobilised enzymes makes an industrial process far more economical, as it avoids expense of continuous flow growth process and discards unwanted biomass. When whole cells rather than cell-free enzymes are employed care should be taken in maintaining the viability of the microbes during the process. This generally requires addition of necessary growth substrates.

Recovery of product

Several methods are used for recovery of the products. These include distillation, centrifugation, filtration and chromatography separation. The recovered product is packaged and marketed.

1. Medicines (Pharmaceuticals)

Pharmaceutical manufacturing industry is a major source of employment for industrial microbiologists. Microbes have maximum application in medicines which include antibiotics, steroids, vitamins and vaccines.

[1] Antibiotics

Here emphasis will be on their source and production industry. There are thousands of antibiotics produced by microbes in nature. Of these, relatively few are produced commercially. The major antibiotics used in medicines and their microbial sources are given below:

Penicillin. An inoculum of Penicillium chrysogenum is produced by inoculating dense spore suspension onto a wheat bran-nutrient solution, incubated at 24°C for one week or so and then transferred to inoculum tank. Tanks are agitated with forced aeration for 1-2 days to have heavy mycelia growth for subsequent inoculation into a production tank. The typical medium used for producing penicillin today has total glucose or molasses (continuous feed) -10%; cornsteep liquor solids – 4-5%; total phenylacetic acid (cont. feed) – 0.5-0.8%; total vegetable oil (cont. feed) – 0.5%. Phenylacetic acid is the precursor used to form the benzene ring side chain of penicillin G molecule. The pH of the medium after sterilisation is approx. 6.0. It takes about 7 days in fermentation.

After fermentation is complete, the concentration of penicillin reached at maximal achievable levels, the liquid medium containing penicillin is separated from the fungal cells, using a rotating vacuum filter. Fungal biomass is scraped from the surface of the filter drum, dried and marketed as an animal feed supplement. Penicillin is recovered from the filtrate using various extraction methods.

The penicillin G thus obtained can be further modified to form various penicillin derivatives. The modification can be done chemically or by using microbial enzymes. For example 6- aminopenicillanic acid (6-APA) can be formed by fermentation, using bacterial acylase enzymes in an aqucous solution at 37°C. The same transformation of penicillin G to 6-APA by using chemical solvents, anhydrous conditions and low temperature, can also be done in three steps.

Cephalosporins. Similar semisynthetic approaches can be used for manufacture of other antibiotics. Cephalosporin C is made as the fermentation product of Cephalosporium acremonium. However, this form not potent for clinical use. Its molecule can be transformed by removal of an alpha-aminoadipic acid side chain to form 7-alpha aminocephalosporanica (7-ACA), which can be further modified by adding side chains to 10 clinically useful broad-spectrum antimicrobials. Various side chains car added to as well as removed from both 6-APA and 7-ACA to produce biotics with varying spectra of activities and varying degrees of resist inactivation by enzymes produced by pathogenic microbes. Thus  we have entered into the so-called third-generation cephalosporins, such as moxalactam, developed for control of bacteria that produce enzymes capable of degrading penicillins and cephalosporins.

Streptomycin. Streptomycin and various other antibiotics are produced using strains of Streptomyces griseus. Spores of this actinomycete are inoculated into a medium to establish a culture with a high mycelial biomass for introduction into an inoculum tank, with subsequent use of the mycelial inoculum to initiate the fermentation process in the production tank. The medium contains soybean meal (N- source), glucose (C- source) and NaCl. Process is carried out at 28°C and the maximum production achieved at pH range of 7.6-8.0. High agitation and aeration are needed. Process lasts for about 10 days. The classic fermentation process involves three phases. During the first phase there is rapid growth of the microbe with production of mycelial biomass. Proteolytic activity of the microbe releases NH₃ to the medium from the soybean meal, causing a rise in pH. During this initial fermentation phase there is little production of streptomycin. During second phase there is little additional production of mycelium, but the secondary metabolite, streptomycin accumulates in the medium. The glucose and NH₃ released are consumed during this phase. The pH remains fairly constant-between 7.6 and 8.0. In the third and final phase, when carbohydrates become depleted, streptomycin production ceases and the microbial cells begin to lyse. pH increases and process normally ends by this time.

After the process is complete, mycelium is separated from the broth by filtration and the antibiotic recovered. In one method of recovery and purification, streptomycin is adsorbed onto activated charcol and eluted with acid alcohol. It is then precipitated with acetone and further purified by use of column chromatography.

[II] Steroids

Microbial biotransformation of steroids is very important in the pharmaceutial industry. Steroids are used in treatment of various disorders and also involved in regulation of sexuality. Chemical synthesis of steroids in very complex because of the requirement to achieve the necessary precision of substituent location. For example cortisone can be synthesised chemically from deoxycholic acid but the process requires 37 steps, many of which must be carried out under extreme conditions of temperature and pressure with the resulting product costing over $200 per gram. The most difficult is introduction of oxygen atom at number 11 position of the steroid ring, but this can be accomplished by some microorganisms. The fungus, Rhizopus nigricans for example hydroxylates progesterone, forming another steroid with the introduction of oxygen at the number 11 position. The fungus Cnninghamella blakesleeana similarily can hydroxylate the steroid cortexolone to form hydrocortisone with the introduction of oxygen at number 11 position. Other transformations of the steroid nucleus carried out by microbes include hydrogenations, dehydrogenations, epoxidations and removal and addition of the side chains. The use of such microbial biotransformations in the formation of cortisone has lowered the original cost over 400-fold, so that in 1980 the price of cortisone in the U.S. was less than 50 cents per gram, compared to the original $200.

In a typical steroid transformation process, the microbe, such as Rhizopus nigricans is grown in a fermentation lank using an appropriate growth medium and incubation conditions to achieve a high biomass. In most cases agitation and aeration are done to have rapid growth. After the growth of the microbe, the steroid to be transformed is added (as progesterone here) to the fermentor containing R. nigricans that has been growing for one day or so and the steroid is hydroxylated at number 11 to form 11-alpha-hydroxyprogesterone. Product is recovered by extraction with methylene chloride or other solvents, purified chromatographically and recovered by crystallization. A large number of similar transformations are carried out to produce a great variety of steroid derivatives for different medicinal uses.(BSc Industrial Microbiology Notes Study Material)

[III] Human proteins

Genetic engineering has expanded the industrial applications of microorganisms including production of human proteins. By using recombinant DNA technology, human DNA sequences that code for various proteins have been incorporated into the genomes of bacteria. By growing these recombinant bacteria in fermentors, human proteins could be produced commercially. Human insulin, for instance, is produced by a recombinant E. coli strain and marketed as humulin. Other strains are used to produce human growth hormone, tumor necrosis factor (TNF), interferon (human recombinant beta interferon-trade name, Betaseron), and interleukin-2 (human recombinant interleukin-2, trade name- Proleukin). Humulin is used in treatment of diabetes in individuals allergic to insulin harvested from cattle. Human growth factor is used in treatment of dwarfism, and interleukin-2, interferon and TNF are important components of human immune system.

[IV] Vaccines

Production of vaccines involves growing the microbes possessing the antigenic properties needed to elicit a primary immune response. Mutant strains and attenuated or inactivated virulent pathogens (without removing antigens) are used for producing vaccincs. Prophylactic treatment of serious pathogenic viruses and bacteria could become possible only by vaccines. Viruses are grown in embryonated eggs or tissue cultures. The rabies vaccine, produced earlier in embryonated duck eggs with painful side effects has now been replaced by a vaccine produced in human fibroblast tissue cultures.

[V] Vitamins

Microbial fermentations are used for commercial production of several essential animal vitamins. Some important vitamins and their microbial sources are shown below.

Vitamin B₁₂ can be produced as a by-product of Streptomyces antibiotic fermentation. A cobalt salt is added to the fermentation reaction as a precursor to vitamin B₁₂. The vitamin accumulates in high concentration, but not toxic to streptomyces. It can also be produced by direct fermentation using Propioni bacterium shermanii or Pseudomonas denitrificans.(BSc Industrial Microbiology Notes Study Material)

2. Organic Acids

Several organic acids including acetic, gluconic, citric, itaconic, gibberellic and lactic acids are produced commercial through microbial transformations.(BSc Industrial Microbiology Notes & Study Material)

Gluconic acid is produced by various bacteria, including Acetobacter spp and several fungi including Aspergillus and Penicillium spp. A niger converts glucose to gluconic acid in a single enzymatic reaction. Submerged culture is needed. A niger is initially grown to form enough mycelial biomass. Later part is enzymatic. A typical growth medium consists of 25% glucose, various salts, CaCO₃ and a boron source. Fermentation is carried out at 30°C with aeration and agitation. Gluconic acid is recovered by adding calcium hydroxide to form crystalline calcium gluconate. Free gluconic acid can be recovered by adding the acid.

Citric acid is also produced by Aspergillus niger. Citric acid is used as food additive, especially in soft drinks, as a metal chelating and sequestering agent, and as a plasticizer. It is essential to limit the growth of the fungus so that high levels of citric acid can accumulate. This is possible by having a deficiency of a trace element or phosphate in the growth medium. The medium contains molasses, ammonium nitrate, MgSO₄ and potassium sulphate. Acid is added to lower the pH. Metals added to the medium are removed from solution by cation exchange resins.

Itaconic acid is used as a resin in detergents. The transformation of citric acid by Aspergillus terreus can be used for commercial production of itaconic acid. Fermentation process involves a well-aerated molasses-mineral salts medium at a pH, below 2.2. At higher pH this microbe degrades itaconic acid. Like citric acid, low levels of trace metals must be used to achieve acceptable product yields.

Gibberellic acid and related gibberellins are important growth regulators of plants. Commercial production of these acids helps in boosting agriculture. This acid is formed by the fungus. Gibberella fujikuroi (imperfect state, Fusarium moniliforme) and can be produced commercially using aerated submerged cultures. A glucose-mineral salt medium, incubation at 25°C and slightly acidic pH are used for fermentation. It takes normally 2-3 days.

Lactic acid is used as preservative in foods, in leather production and textile industry. Various other forms of lactic acid are also used for other purposes – in resins as polylactic acid, in plastics, electroplating as copper lactate and baking powder and animal feed. Fermentation is carried out by Lactobacillus, Streptococcus and Leuconostoc spp. The typical medium normally contains 10-15% glucose or other sugar, 10% CaCO₂ to neutralize the lactic acid formed, and ammonium phosphate and nitrogen sources in trace. Corn sugar, beet molasses, potato starch and whey are often used as carbohydrate sources for fermentation; temp. of 45- 50°C and pH 5.5-6.5. Agitation is needed but not aeration. Process is complete within 5-7 days with approximately 90% of the sugar conversion to lactic acid.(BSc Industrial Microbiology Notes Study Material)

3. Amino Acids

Microbial production of lysine and glutamic acid every year has increased for world-wide sales. Animal feeds are deficient in amino acids which are to be added to them. Lysine produced by microbial fermentation and methionine produced synthetically are used as animal feed supplements. In the microbial production of amino acids only the desired L-isomer is formed, whereas their chemical synthesis produces a racemic mixture that requires expensive separation methods to remove the bio-active D-isomer half of the mixture. Through use of genetically-enginereed strains of microbes some of the limitations of fermentation processes have been overcome. The direct production of L-lysine from carbohydrates uses a homoserine requiring auxotroph of Corynebacterium glutamicum.

The blocking of homoserine synthesis at the level of homoserine dehydrogenase results from feed-back inhibition of that enzyme and leads to the accumulation of lysine. Cane molasses is generally used as the substrate, pH near 7.0 by adding NH₃ or urea. About 50 g/l of lysine can be produced in 2-3 days by use of this auxotroph of C. glutamicum L-glutamic acid and monosodium glutamate (MSG) can be produced by direct fermentation using strains of Brevibacterium, Arthrobacter, and Corynebacterium Cultures of C. glutamicum and Brevibacterium flavum are widely used for large scale production of MSG. A glucose mineral salts medium, with periodic addition of urea, pH 6-8 and temperature of 30°C and well aeration, are used for fermentation. There are several methods for inducing leaky membranes that permit excretion of the product from the cell. One way is to grow C. glutamicum in a medium with suboptimal concentration of biotin or adding fatty acids or detergents. These allow secretion of glutamic acid by membranes.

4. Enzymes

Enzymes have important applications in industry and these are produced by different microbes, mostly by fungi.

A generalised scheme for microbial production of commercial enzyme. Enzymes produced for industry include proteases, amylases, glucose isomerases, glucose oxidases, rennin, pectinases and lipases. Of these, proteases, glucamylase, alpha-amylase and glucose isomerase are produced extensively.

Proteases attack peptide bonds of proteins. The largest application of microbial proteases is in the laundry, in modern detergent formulations. They are used for removal of spots of milk, eggs, blood. These are heal-stable. Proteases are largely produced by Bacillus licheniformis. Other alkaline proteases are being developed using recombinant DNA technology, to function over a wide range of pH and temperature. One such recombinant strain is Bacillus sp. Gx 6644, active for milk casein. Another strain Bacillus sp. GX 6638 produces several alkaline proteases. Through this technology a recombinant strain of a bacterium has also been developed. It produces the enzyme kerazyme, used for dissolving hair and opening hair-clogged drains. In baking industry also these enzymes have important application. Microbial proteases reduce mixing time and improve the quality of loaf. Fungal proteases are mainly obtained from Aspergillus spp. and bacterial from Bacillus spp. These enzymes are used as meat- tenderizer and in leather industry for bating of hides.

Amylases are used for preparation of sizing agents in textile industry, preparation of starch sizing pastes for use in paper industry, bread production, chocolate and corn syrups and removal of spots in laundry. There are various types of amylases -alpha-, -Beta– and glucamylases. Aspergillus oryzae, A. niger, Bacillus subtilis and B. diastaticus are principally used. The conversion of starch to high-fructose syrup utilises amylases, i.e. in producing sweeteners. Various other enzymes produced by different microbes also have industrial applications. These are rennin used in cheese production and Mucor pussilus is used for its commercial production. Fungal pectinases are used in clarification of fruit juices. Glucose oxidase is used to remove oxygen from soft drinks, salad dressings etc.(BSc Industrial Microbiology Notes Study Material)

Microbial enzymes are also used for production of synthetic polymers. Plastic industry mostly uses chemical methods for producing alkene oxides used in the production of plastics. It is now possible to synthesise alkene oxides by using microbial enzymes and genetically-engineered strains would make commercial production feasible. The synthesis of alkene oxides from alkenes requires sequential action of three enzymes: pyranose-2-oxidase from the fungus Oudmansiella mucida, a haloperoxidase from the Caldariomyces, and an epoxidase from a Flavobacterium sp.

5. Solvents

Most organic solvents are synthesised chemically. But a few solvents can also be produced commercially by microbial fermentation. Ethanol, although produced by fermentation for beverages and gasohol, industrial alcohol for use as solvent is mostly produced chemically. The production of acetone and butanol by fermentation was discovered by Chaim Weizmann, a Polish-born chemist working in England. The microbial production of acetone and butanol uses anaerobic Clostridium spp. The fermentation process uses conversion of starch to acetone by C. acetobutylicum. Another species, C. saccharoacetobutylicum is able to convert the carbohydrates in molasses to acetone and butanol. These bacteria first synthesize acids (acetic and butyric) which are then converted to acetone and butanol. The solvents produced by fermentation are recovered by distillation. Glycerol is an important solvent in flavouring and food colouring, and also used in production of explosives and propellants. Through fermentation, glycerol production in Germany was an important factor during World War I. Microbial production uses addition of sodium sulphite to a yeast-ethanol fermentation process. Sodium sulphite reacts with CO₂ to produce sodium bisulphite, which prevents the reduction of acetaldehyde to ethanol. Saccharomyces cerevisiae, and bacteria as Bacillus subtilis are used.(BSc Industrial Microbiology Notes Study Material)

6. Fuels

Synthetic fuels produced by microbes should help meeting energy- crisis world over. Useful fuels produced by microbes include ethanol, methane, hydrogen and hydrocarbons. Right strains are able to do the job. For microbial production of fuels, waste materials such as sewage and municipal garbage are used as fermentation substrates.

Ethanol production by microbes has become very popular in those areas where plant residues (agricultural and other wastes) are available in abundance. Brazil produces and uses large amounts of ethanol as automotive fuel. Gasohol, a 9 : 1 blend of gasoline and ethanol, has become popular fuel in USA. Despite some problems with the ethanol-fuel, several processes are employed for its commercial production. The most efficient microbes are Zymomonas mobilis (fermenting carbohydrate and producing alcohol twice as rapidly as yeasts) and Thermoanaerobacter ethanolicus, a thermophilic bacterium. Corn sugar and plant starch are used as substrates. A two-step fermentation is used for conversion of cellulose to ethanol, (i) conversion of cellulose to sugars, generally by Clostridium spp, followed by (ii) conversion of these sugars to ethanol by yeasts, Zymomonas or Thermo- anaacrobacter spp.

Methane produced by methanogenic bacteria is also another potential energy source. Methane is used for generation of mechanical, heat and electrical energy Anwerobic decomposition of waste materials produces large amounts of methane.

Biogas, a mixture of different gases is produced by anaerobic microbes using domestic and agricultural wastes. Bulk (about 50-70%) of biogas is CH₄ and other gases are in low proportions. These include CO₂ (25-35%), H₂ (1-5%), N₂ (2-7%) and O₂ (0-0.1%). In India a large number of gobar gas plants are already in operation in rural areas. Left overs of these plants are good food fertilisers also. Animal waste is first hydrolysed by hydrolytic bacteria. It is followed by acid formation by a group of acetogenic bacteria, which convert monomers into simple compounds like NH₃, CO₂ and H₂. Finally methanogens reduce acetate and/or CO₂, to CH₄. In India, cattle dung is the chief source of biogas.

Other fuels include hydrogen that could be developed as a major fuel produced by microbes in future. Photosynthetic microbes produce H. They are able to convert solar energy into a fuel that can be stored. The photoproduction of hydrogen is very attractive. Some higher molecular weight hydrocarbons are produced by some algae. However, a thorough understanding of basic mechanisms of microbial hydrocarbons formation and the formation of petroleum deposits should permit the development of genetically engineered microbes and fermentation processes to produce synthetic sources of petroleum hydrocarbons.

7. Alcoholic Beverages

Alcoholic fermentations have been well known since ancient time, though little was known of the nature of the process. Louis Pasteur could show that yeasts were the organisms responsible for such a process. Later details became available about the chemistry of fermentations, which were exploited for industrial manufacture of alcoholic beverages. Microorganisms, principally, yeasts in the genus Saccharomyces are used to produce various types of alcoholic beverages. The process relies on alcoholic fermentation conversion of sugar to alcohol by microbial enzymes.(BSc Industrial Microbiology Notes & Study Material)

1. Beer. The fermentation of beer is usually a batch process. In some countries the production of beer is carried out in a continuous flow- through process. Beer is a product of the fermentation of barley grains by yeasts. Barley seeds are allowed to germinate. During germination the naturally occurring amylases convert the grain starch to sugars, most of which being maltose. The process is called malting and the digested grain as malt. The next step is to mash the grain with water and remove the fluid portion called the wort. Dried petals of a vine, Humulus lupulus, called hops are then added to the wort to give it flavour, colour and stability. Hops also prevent contamination of the wort, due to presence of two antimicrobial substances in petals. At this stage the fluid is filtered and yeast is added in large quantities.(BSc Industrial Microbiology Notes & Study Material)

The yeast, commonly used in fermentation of wort is one of the many strains of Saccharomyces cerevisiae, developed by brewers. Generally, the yeast is taken from a previous batch of beer. Some yeasts give a uniform cloudiness to the beer and are carried to the top of the fermentation vat by foaming carbon-dioxide. Such yeasts are called top yeasts and the product as ale. Other yeasts ferment the beer more slowly and produce with less alcohol than ale. These yeasts are known as botton yeasts and their product as lager beer. Saccharomyces carlsbergensis, used in U.K. is a bottom fermenter.(BSc Industrial Microbiology Notes & Study Material)

Generally, one week’s time is needed for normal fermentation to take place. After a week, the young beer is transferred to vats for primary and secondary ageing. It may take about six months more. Some yeast is left in the beer that is to become keg beer and the product is refrigerated to preserve it. The thick wall of the keg traps CO₂ produced during continual fermentation. For canning or bottling the beer is to be pasteurised at 140°F for 13 min. to kill the yeast or may be filtered to remove the yeasts. Some yeast is used to seed new wort, the rest for animal feed or pressed to tablets for human consumption (single cell protein or SCP). Alcoholic content of beer is roughly 4 per cent.

Sake, though commonly known as rice wine, is somewhat like a rice beer. It is obtained from steamed rice. The rice starch is converted first to sugar by Aspergillus oryzae or Rhizopus sp. Later Saccharomyces spp. ferment the sugar until the alcohol level is approximately 14 per cent, when sake is ready for consumption.

2. Wine. Wine fermentations are carried by controlled cultures of Saccharomyces ellipsoideus, a variety of Saccharomyces cerevisiae or with the strains of yeast naturally occurring on grapes. The former method is common in U.S.A., the latter in Europe.

Wine is made from ripe fruit, fruit juice, or plant extract such as dandelions. Fermentation usually begins with crushing of the fruit to produce must. Sulfurdioxide may be added to control the process. In natural fermentations, SO₂ is not used, and the yeasts begin to digest sugar. Oxygen may be supplied to promote aerobic growth of the yeast. However, anaerobic conditions are established later. Alcohol production occurs within few days, though ageing may take months or years. During this period, secondary fermentations develop the flavour, aroma and bouquet of the wine. Red wine becomes red as alcohol extracts the colour of grape skin. For red wine, fermentation is carried out at 24-27°C for 3-5 days and white wine takes 1-2 weeks at 10-21°C. Additional carbondioxide production yields Champagne and other sparkling wines which are naturally carbonated. Sherry wines result from inoculation with special yeasts to have unique flavours. In dry wines, most or all of the sugar is metabolised, whereas in sweet wines, the fermentation is stopped before entire sugar is consumed. The strongest natural wines have about 16 per cent alcohol (yeasts cannot tolerate higher levels than this). Most table wines average about 10-12 per cent alcohol, with fortified wines reaching 22 percent alcohol. In fortified wines, brandy or other spirits are added to produce port, sherry and cocktail wines. For mass production, the wine is pasteurised, filtered and bottled.

3. Distilled spirits. They contain considerably more alcohol than beer or wine. Alcoholic content is shown by a proof number which is the twice the actual percentage of alcohol. The process of distilled spirit begins with same type as for wine and beer, except that after the fermentation process the alcohol is collected by distillation, to allow a higher concentration of alcohol. The raw product is first fermented by Saccharomyces, then aged and finally matured in casks. At this point the process differs. The alcohol is concentrated by a distillation apparatus using heat and vacuum. During further maturing unique flavours from the chemicals as aldehydes, ethers and volatile acids are added. The alcohol content is then standardised by diluting it with water before bottling. There are four basic types of spirits: brandy is made from fruit juice, and rum from molasses, whisky from malted cereal grains as for instance, scotch from barely, rye from rye-grain and bourbon from corn. The neutral spirits, as vodka is made from potato starch and left unflavoured, and gin flavoured with juniper oil.

8. Vinegar

Vinegar production involves an initial anaerobic fermentation to convert carbohydrates by Saccharomyces cerevisiae to alcohol, followed by a secondary oxidative transformation of the alcohol to form acetic acid by Acetobacter and Gluconobacter. The starting materials for vinegar may be fruits (grapes, oranges, apples, pears), vegetables (potatoes), malted cereals Charley, rye, wheat, corn) and sugary syrups (molasses, honey, maple syrup). Wine vinegar comes from grapes and cider vinegar from other fruits. In slow methods of vinegar production, still used in some European countries, an initial natural alcohol fermentation achieves an alcohol concentration of 11-13%. After producing the alcoholic liquid, acetic bacteria are seeded into the solution and allowed to convert the alcohol slowly to acetic acid. In the Orleans process, a barrel is filled about 1/4th full with raw vinegar from a previous run to provide the active inoculum. A wine, hard cider, or malt liquor is then added as a substrate. Air is left in the barrel, acetic acid bacteria grow as a film on the top of liquid, and conversion to acetic acid occurs in several weeks to several months to complete at 21-29°C.

To increase the rate of acetic acid production, a vinegar generator can be used in which alcohol-containing liquid is trickled over a surface film of acetic acid bacteria. The film of bacteria is maintained on wood chips. Alcohol liquid is sprinkled over the wood chips and during this slow-trickling the liquid down through the generator, the alcohol is converted to sc acid. Air enters the generator from the bottom. Today, though in industrial production of vinegar submerged culture reactors are used, forced aeration is used to maximise the rate of acetic acid production. The bacteria grow in the fine suspension created by air bubbles and the fermenting liquid. Here 8-12% alcoholic liquid is inoculated with Acetobacter sp. at 24-29°C with controlled aeration. The vinegar thus formed is clarified by passing through a filter and allowed to age for taking final taste.

9. Enhanced Recovery of Metals (Bioleaching)

Since high-grade ore deposits are easily accessible, these become rapidly depleted. It thus becomes necessary to recover mineral resources from low-grade ore deposits. However, no appropriate technology is still available for recovery of metals from low- grade deposits. It is encouraging to find some microorganisms who could do it efficiently. This potential of microbes could only be realised recently and efforts are being made to use them for enhanced recovery of mineral resources from natural deposits. Microbes have been used for recovery of two important natural resources – metals and petroleum.

It was in 1957 than a relationship between the presence of Thiobacillus ferrooxidans and the dissolution of metals in copper-leaching operation was recognised by American microbiologists. T. ferrooxidans and T. thiooxidans are thermoacidophilic archaebacteria. They are autotrophs and grow in acidic and hot environments. It has been demonstrated that these Thiobacillus spp. can be used for extraction of copper and uranium from insoluble minerals. This implication of microbial activity in weathering, leaching and deposition of mineral ores could develop into a recent field of biotechnology – biohydrometallurgy. Biomineralisation is the deposition of metals as insoluble oxides and sulphides due to microbial activity.

Microbial mining is the process of bioleaching recovers metals from ores that are not suitable for direct smelting due to their low metal content. Bioleaching uses microbes to alter the physical or chemical properties of a metallic ore so that the metal can be extracted. Metals can be extracted economically from low-grade sulphide or sulphide-containing ore by exploiting metabolic activities of thiobacilli, particularly T. ferrooxidans. Under optimal conditions in the laboratory, as much as 97% of the copper in low-grade ores has been recovered by bioleaching, but such high yields are not achieved in actual mining operations. The process is at present commercially used for recovery of copper and uranium from low-grade ores. Laboratory experiments could show that recovery of other metals such as Ni, Zn, Co, Sn, Cd, Mb, Pb, Sb, As and Se from their low- grade sulphide-containing ores is also possible through bioleaching. The leaching process can also be used to separate the insoluble lead sulphate (PbSO₄) from other metals that occur in the same ore.

The general process carried out by T. ferrooxidans (T.f.) and related species can be shown by the following equation.

MS + 20₂ → MSO₄ where M is divalent metal.

Because metal sulphide is insoluble and metal sulphate usually water-soluble, this transformation produces a readily leachable form of the metal. T.I., a chemolithotroph derives energy through oxidation of either a reduced sulphur compound or ferrous iron. It exerts its bioleaching action by oxidising the metal sulphide being recovered either directly converting S^2- to SO₄^2- and/or indirectly by oxidising the ferrous iron content of the ore to ferric ion. The ferric ion, in turn, chemically oxidises the metal to be recovered to a solub form that can be leached from the ore.

It is possible to leach the ore in situ without first mining it, if there formation is porous and overlays a water-impermeable stratum. A pattern of boreholes is established with some of the holes used for injecting the leaching liquour and others for the recovery of leachate. More frequently, however, this bioleaching process is used after the ore is mined, broken up and piled in heaps on a water-impermeable formation or on a specially constructed apron. Water is then pumped to the top of ore heap and trickles down through the ore to the apron. A continuous reactor leaching operation for recovery of copper from its low-grade sulphide ore is shown in Fig.

The leaching water and ore usually supply enough dissolved mineral nutrients required by T.f., but in some cases NH₃ and PO₄ may be added. The leached metal is extracted with an organic solvent and then removed from solvent by stripping. Both the leaching liquor and the solvent are recycled.(BSc Industrial Microbiology Notes & Study Material)

Copper is generally in short supply. Low-grade copper ore contains 0.1-0.4% Cu. The pregnant leaching solution may contain 1 to 3 g of Cu/l. In copper leaching operations, Thiobacillus involves both, direct oxidation of CuS and indirect oxidation of CuS via generation of ferric ions from ferrous sulphide, present in most of the important copper ores such as chalcopyrite (CuFeS₂). In the latter case, copper replaces iron i.e. CuSO₄ + Fe^x → Cu^x + FeSO₄. In 1980s various firms began to utilise bioleaching for extracting copper.(BSc Industrial Microbiology Notes & Study Material)

The recovery of uranium, a nuclear fuel, can also be enhanced by microbial activities, which should help overcoming global energy crisis. Moreover, the current controversies about nuclear plants may also be diluted/solved, atleast from economics point of view, if not safety. Insoluble tetravalent uranium oxide (UO₂) occurs in low-grade ores. There is no evidence for direct oxidation. But UO₂ can be indirectly converted to leachable hexavalent form (UO₂SO₄) by T.f., which oxidises ferrous iron in pyrite (FeS), that often accompanies uranium ores. The oxidised iron as an oxidant converts UO₂ to UO₂.SO₄ chemically, which can be recovered by leaching. In Canada, bioleaching was first employed in 1970 for extraction of uranium.(BSc Industrial Microbiology Notes & Study Material)

Recovery of copper and uranium through bioleaching depends on several factors, such as type of the geological formations, ore characteristics and prevailing conditions under which the concerned microbe is to grow. Also, oxidative activity of Thiobacillus results into high temperature, and other bacteria like Sulpholobus (obligate thermophile and acid tolerant) can be useful that can oxidise ferrous iron and sulphur in a manner similar to thiobacilli. Sulpholobus has been used for bioleaching of molybdenite (molybdenum sulphide), whereas Thiobacillus is intolerant of high concentrations of molybdenum, mercury and silver.

Besides bioleaching, some microbes including fungi are able to accumulate metals in their cells at concentrations higher than in the surrounding media. Such bioconcentration has the potential for extracting rare metal ores from dilute solutions and for recovery of metals (gold, silver) from industrial effluents. Rhizopus binds uranium from low-grade ores and nuclear wastes. Theoretically, microbes could be used to recover gold from the sea. In South Africa efforts are being made for extraction of gold through bioleaching.(BSc Industrial Microbiology Notes & Study Material)

10. Enhanced Recovery of Petroleum

Besides metals, microbes can also be used to enhance recovery of petroleum hydrocarbons. The tertiary recovery of petroleum (the use of bio. logical and chemical means to enhance oil recovery), and the enhanced recovery of hydrocarbons from oil shales are important due to depletion of recoverable oil resources. Tertiory recovery of oil uses solvents, surfactants and polymers to dislodge oil from geological formations. Xanthan gums produced by some bacteria, such as Xanthomonas campestris, are useful compounds in oil recovery. These polymers have high viscosity and flow characteristics that allow them to pass through small pores in rock layers containing oil deposits. Xanthan gums are added during water flooding operations (water is pumped into oil reservoirs to force out oil). These help push the oil toward the production wells. The polymers are produced by conventional fermentation in which X. compestris is grown and the gums are recovered. Many oil shales contain large amounts of carbonates and pyrites and their removal increases the porosity of shale, enhancing recovery of oil. Acid dissolves the carbonates and these can be produced by Thiobacillus spp. growing on sulphur and iron in the pyrite. Thus bioleaching of oil shales by microbes has also the potential for enhancing the recovery of hydrocarbons.

Recombinant DNA Technology and Industrial Microbiology

It is genetically possible to “tailor” the microorganisms for the production of any microbial metabolite-vitamin, amino acid or enzyme. Gene cloning extends the genome of the microorganism by allowing the introduction of novel genes from comparatively unrelated species. The cloning of genes from higher eukaryotes, particularly from man and his domestic animals has been seen to offer even greater industrial potential. Which microbes should then be used as universal recipients for such genes and hence as production organisms? The two most ideal are the prokaryote, Escherichia coli and the eukaryote, Saccharomyces cerevisiae. Some of the important products which gene cloning may make available in near future are as follows:

The above proteins could be obtained on large scale through fermentation by methods, relatively more cheaper than the conventional ones. For example, human growth hormone was previously extracted from the pituitary gland of cadavers and was mostly in short supply. Now, increase in supply should help more patients. Equally important is the development of new vaccines through gene-cloning. Genes for single antigens can be cloned and expressed by bacteria and a purified antigen which has not been derived directly from the pathogenic organism or virus may be used as a vaccine. In this way, vaccines for viral hepatitis and foot-and-mouth disease have been developed.

Immobilisation of Enzymes and Cells – Relevance to Industrial Microbiology

Besides gene-cloning, in microbial biotechnology several commercial processes have used immobilised microbial cells and enzymes in the last few years. Enzymes have been used in industry for over 70 years, initially in detergents that is still perhaps one of the largest bulk users of proteases and lipases. In conventional biological industries also microbial enzymes have been used, e.g. proteases and amylases in malting and rennet in cheese manufacture. However, during last 15 years or so, immobilised cells and enzymes have been used as production systems. Why enzymes are preferred over chemical processes?

(1) Enzymes carry out stercospecific reactions with high accuracy, whereas chemical technology results into many side-products from which the desired product is to be purified.

(2) Enzymes are cheap and carry out reactions at low temperature and at atmospheric pressure, whereas chemical catalysts require special expensive conditions.

(3) Since there is much diversity in microbial world, an enzyme for a desired reaction can be easily found out by merely screening a range of microbes.

(4) Mutants with altered enzyme function can be isolated with appropriate genetic methods. Thus enzymes having different substrate specificities or with different physical properties (as temperature resistance) can be isolated. Also conventional genetics and gene-cloning can be used for making specific changes in genes to increase the expression of the desired enzyme.(BSc Industrial Microbiology Notes & Study Material)

More recently, whole microbial cells have been used for specific chemical transformations. This should not be confused with anaerobic fermentation or conventional secondary metabolite production. Here only part of the cell’s metabolism is now being utilised, usually a single pathway, and sometimes only a single enzyme. The advantage of using cells is that the expense of purifying the enzyme is avoided and, in some cases, the enzyme is more stable in its natural environment than after purification. Frequently, cells and enzymes are subjected to immobilisation on an inert support.(BSc Industrial Microbiology Notes & Study Material)

The stability of an enzyme is improved after its immobilisation. Immobilisation affords a simple way of separating the enzyme or cell from the products when the reaction is complete and is likely to prove invaluable in the development of biological sensors (biosensors) – special electrodes based upon the selectivity and high affinity of enzymes for their substrates.

In recent years the technology of enzyme and whole cell immobilisation could have much impact on industrial production of several products. Immobilisation means “imprisonment or confinement of a biocatalyst in a distinct phase to a suitable inert support, where it can act upon its natural substrate repeatedly and continuously, and can be removed conveniently”. For a biocatalyst (enzyme/cell) the substrate is disposed in a bulk phase. The physically entrapped or covalently bonded biocatalyst is chemically bonded to an inert, insoluble matrix (support), which is a high molecular weight polymer, such as glass beads, starch, cellulose, polyacrylamid. Since 1916, when the phenomenon was reported for the first time by J.M. Nelson and E.G. Griffin, this has been largely used in several manufacturing processes. They had initially reported the immobilisation (adsorption) of an invertase on charcol/alumina without loss of activity. It was during 1950s and 1960s that the technique became very popular.

Why immobilisation?

Immobilisation of enzymes has some advantages over free enzymes. These are (i) improved stability of the enzyme due to its binding to a support, (ii) recovery of enzyme at the end of reaction for repeated use, (iii) better efficiency of enzyme and manipulations of the catalysed reactions and (iv) higher purity and yield of product with conservation of resources and minimising pollution risks.

Why whole cells?

Sometimes use of immobilised enzymes becomes troublesome due to some reasons. For instance, extraction and purification of enzymes can be quite expensive and tedious. Some reactions are catalysed by more than one enzymes, thus with more enzymes, it becomes difficult to manage the reactor conditions. Moreover, immobilised enzymes can not be used where the enzyme needs continuous cycling of cofactors. Hence these problems can be overcome by using whole cells.

How to immobilise?

A number of methods have been developed for immobilisation of enzymes, which except a few can also be used for cells as well. Basically, it is (i) entrapment, or (ii) binding. Entrapment can be accomplished by (i) fibre, (ii) microencapsulation, or (iii) gel. In gel entrapment, the processes involved are, (i) polymerisation, (ii) ionic network formation, or (iii) precipitation. Binding may be (i) cross-linking, or (ii) carrier- binding. In carrier-binding, the processes involved are (i) adsorption, (ii) chelation, or (ii) covalent binding. These methods belong to the following five general categories.

1. Adsorption. The enzymes are adsorbed to several kinds of adsorbents with charged or neutral surfaces. Such materials are used for separation of proteins by adsorption chromatography. Calcium phosphate gels, carbon, carboxymethylcellulose (CMC), carboxymethyl sephadex, collagen, silica, gel, titania, alumina etc. are used.

2. Co-valent binding. Enzyme is bound covalently to a support material using any of the various methods. The enzyme forms a covalent link with active groups of support material.

3. Cross-binding. Enzymes may be cross-linked to a multifunctional without any solid support. Diazobenzidine, glutaraldehyde, toluene 2, 4-di-iso thiocyanate, hexamethylene di-isocyanate are some of the reagents used.

4. Entrapment. Enzyme is entrapped inside a cross-linked gel matrix. The gel is allowed to develop in an aqueous solution containing one or more enzymes.

5. Microencapsulation. This is modified entrapment where enzyme is immobilized within microcapsules prepared from organic polymers.

Industrial applications of immobilized systems

Immobilised systems (enzymes and cells) possess important practical applications in industry. Immobilised systems have been used in antibiotic productions. At least in case of patulin and penicillin G production, immobilisation system can be operated on a continuous basis. Immobilised mycelia of Penicillium chrysogenum are used.

Quite recently immobilised fungal systems have also been applied to environmental problems. Microbial biotechnology has important applications in biodegradation, disposal of wastes and renewable sources of energy.(BSc Industrial Microbiology Notes & Study Material)

BSc Industrial Microbiology Notes & Study Material

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