The Biology Topics of ecology involve studying the relationships between living organisms and their environment.
Food Industrial Production of Monosaccharides Using Microbial, Enzymatic, and Chemical Methods
Useful activities of various bacteria are employed in the production of a number of industrial products. Large-scale productions of useful substances are possible using suitable strains of microorganisms and liquid media in fermenters or bioreactors.
A fermenter is a large vessel made up of aluminium or steel. It contains an agitator for mixing medium and a tank for the addition of nutrients, buffer, etc. The automatic control system controls the entire process of fermentation. All the operations before starting the fermenter are called upstream processes. The operations after fermentation are called downstream processes. Downstream processes include a collection of the product, its concentration, purification, and further processing such as removal and proper treatment of waste matter, collection of by-products, etc. The most common products obtained through these processes are alcohols, antibiotics, organic acids, enzymes, etc. Production of such microbial substances on a large scale involves scaling up the technology, i.e., the technology has to pass through three stages of trial, viz. laboratory scale, pilot plant scale, and manufacturing unit.
A. Laboratory Scale
As soon as a new microorganism is discovered with the ability to produce a novel substance, the maximum number of strains of that microbe is tested. The most suitable strain is selected and cultured in a laboratory-scale glass fermenter. The suitable parameters for the fermentation process are worked out like optimum levels of nutrients, pH, aeration, disposal of evolved CO2, temperature, and effects of byproducts formed whether they inhibit the process or stimulate it. The downstream processes are also worked out like separation and purification of the products. Ultimately, the final process passes to the next level.
B. Pilot Plant Scale
Here, the production quality and cost-effectiveness are tested. The glass vessels are replaced with metallic containers. The optimum conditions if varying, are correctly adjusted.
C. Manufacturing Unit
The economic evaluations done at the previous stage are used to determine the appropriate size of the manufacturing unit. The reaction is carried out on a trial basis in three ways.
- Support growth system where the microbes grow on the surface of the nutrient medium.
- Suspended growth system where the microbes are suspended in the nutrient medium.
- Column or immobilized system where microbes are immobilized in calcium alginate beads.
1. Leather Industry
Many acids produced from microorganisms by fermentation are used in leather tanning. Some enzymes and microorganisms’ names are listed below:
|1. Protease||Bacillus subtilis, other microorganisms, fungi, etc.||Tanning|
|2. Keratinase||Streptomycetes||Degradation of hair of animal|
|3. Lipase||Pseudomonas sp. and fungi||Tanning|
|4. Pectinase||Aspergillus niger||Fruit juice and brewery|
|5. Streptokinase||Streptococcus sp.||Unclothing of blood in blood vessels|
2. Alcoholic Fermentation
Alcoholic fermentation is also referred to as ethanol fermentation. Ethanol fermentation is a biological process in which sugars such as glucose, fructose, and sucrose are converted into cellular energy and thereby produce ethanol and carbon dioxide as metabolic waste products. Because yeast performs this conversion in the absence of oxygen, alcoholic fermentation is considered an anaerobic process. Alcoholic fermentation occurs in the production of alcoholic beverages and ethanol fuel and in the rising of bread dough. The bacteria species used in alcoholic fermentation are Clostridium acetobutylicum, Klebsiella pneumonia, Leuconostoc mesenteroides, Sarcina ventriculi, Zymomonas mobilis, Lactobacillus, etc.
Some microorganisms involved, product names, uses and industries are mentioned below:
|1. Alcohol||Sarcina Ventriculi||Beer, Wine||Brewery|
|2. Alcohol||Zymomonas mobilis||Rum, Whiskey||Brewery|
|3. Alcohol Butanol||Clostridium acetobutylicum||Alkyl Butanol Production||Brewery and Chemical Industry|
|4. Alcohol||Lactobacillus sp.||Sake||Brewery|
Chemical Process of Fermentation of Glucose
The alcohol is produced by the fermentation of different substrates including carbohydrate-containing agricultural wastes with the help of different microbes. The cheapest and most common raw materials used are molasses from the sugar industry. The microorganism used is Saccharomyces cerevisiae. Other species involved are S. sake, S. ellipsoideus, and S. pireformis. The species grow vigorously and have a high tolerance for alcohol. The process involves the enzymatic activity of yeast that produces alcohol by the following reaction which is the overall chemical reaction for alcoholic fermentation.
The ability of yeast to convert sugars to alcohol has been used in large-scale production in industries. It requires a bioreactor or fermentation tank, the entire operation involves two processes.
It includes sterilization of the fermenter, and nutrient medium, and the preparation and growth of suitable strains of microorganisms.
Molasses obtained from sugar factories are diluted with water to bring the sugar concentration to 10-15% or slightly higher. The diluted molasses are then transferred to the fermenter. It directly acts as a nutrient medium. pH of the medium is adjusted to 4.5 by pouring the required amount of acid. Both the fermenter and medium are sterilized. Yeast does not possess enough diastase or amylase to break down starch present in the medium, so hydrolysis of starch is done in a separate tank at a high temperature of about 55°C for 30 minutes. This sweet medium thus formed is called Wort.
When the liquid medium (wort) is cooled down to the appropriate temperature, it is inoculated with an appropriate strain of yeast. The process soon becomes vigorous. A large quantity of CO2 is evolved during the process. Gas is collected as a by-product, purified, and used in various other industries. Fermentation occurs in two steps. The following chemical reactions summarize the fermentation of sucrose (C12H22O11) into ethanol (C2H5OH). Alcoholic fermentation converts one mole of sucrose into four moles of ethanol and four moles of carbon dioxide, producing two moles of ATP in the process.
Sucrose is a dimer of glucose and fructose molecules. In the first step of alcoholic fermentation, the enzyme invertase cleaves the glycosidic linkage between glucose and fructose and forms two glucose molecules.
Zymase secreted by the yeast cells converts glucose into ethyl alcohol.
It is a process in which some sugars (such as glucose) are converted into alcohol and carbon dioxide by the action of various yeasts, moulds, or bacteria on carbohydrate materials, some of which do not themselves undergo fermentation but can be hydrolyzed into fermentable substances. The process of fermentation can be carried out by the following methods:
- Batch Process: In this technique, the bioreactor used is very large in size with a capacity of upto 2,25000 liters. The inoculated medium is kept there until the maximum amount of alcohol is produced. It is called wash. The same is removed and the tank is prepared for the next batch.
- Continuous Process: In this technique, a portion of the fermented liquor is removed continuously with the addition of new nutrient media.
- Fed-Batch Process: The optimum activity of the microbe is utilized as nutrient media is fed in small quantities.
- Immobilized Yeast: Recently techniques have been developed for the production of alcohol by immobilized state in calcium alginate. This has proven to be 20 times more efficient.
The process involves the separation and purification of alcohol. The fermented medium contains alcohol and other volatile substances and unused components of molasses. Alcohol is purified by successive distillation. In the case of beer, the fermented liquor having an alcohol content of 3-6% is filtered, lightly hopped, and pasteurized. In the case of wines, 10-27% alcohol content is achieved through refinement and concentration. Fortification by direct addition of alcohol may also be carried out. Distillation is done in other cases, e.g., gin (40%), rum (40%), and brandy (60-70%). Rectified spirit is 95% alcohol. Absolute alcohol is 100% alcohol. By changing the feed material alcoholic beverages like beer, brandy, gin, rum, vodka, and whisky are obtained. For the production of beverages, the commonly used fermenter is a tubular tower fermenter.
Uses of Alcohol
- Alcohol is used as fuel to obtain light energy and power.
- It is used in the preparation of different drugs and medicinally used products.
- It is used in distilleries of alcoholic beverages, chemical manufacture vinegar manufacture plants, etc.
- Alcohol is used in laboratories, and hospitals as a sterilizing liquid.
- It is also used as a preservative for animal and plant tissues.
3. Medicine Production
Antibiotics are natural chemotherapeutic agents produced by a group of microorganisms, which in very small quantities have an inhibitory effect on another group of microorganisms.
Antibiotics, also known as antibacterial substances, are types of medication that destroy or slow down the growth of bacteria. These are used to treat infections caused by bacteria or other microscopic pathogenic organisms. Some of which may cause serious illness. These are chemical substances secreted by some microorganisms which inhibit the growth and development of other microorganisms. Most of them are produced by actinomycetes, especially the genus Streptomyces, the filamentous bacteria. Selman Waksman used the general term antibiotics in 1942. The first antibiotic penicillin was discovered by Dr. Alexander Flemming from Penicillium notatum.
Types of Antibiotic:
- Broad-spectrum antibiotics are antibiotics that can kill or destroy a number of pathogens that belong to different groups with different structures and wall compositions.
- A specific antibiotic is an antibiotic that is effective only against one type of pathogen. Antibiotics have come to denote a broader range of antimicrobial compounds including antifungal and other compounds.
The main classes of antibiotics are
- Penicillius e.g., penicillin, amoxicillin.
- Cephalosporins such as cephalexin.
- Macrolides such as erythromycin, clarithromycin, and azithromycin.
- Fluoroquinolones such as ciprofloxacin, levofloxacin, ofloxacin.
- Sulponamides such as cotrimoxazole, trimethoprim.
- Tetracyclines such as tetracycline and doxycycline.
- Aminoglycosides such as gentamicin, and tobramycin.
Production of Antibiotics:
Antibiotics are produced industrially by a process of fermentation in a fermenter. In a fermenter selected strain of microorganisms is grown on a sterilized nutrient medium under optimum conditions of temperature, pH, aeration, and in the presence of an antifoaming agent and antibiotic precursor. At the initial stage rapid growth of the microorganism occurs. Antibiotics are secondary metabolites produced in considerable amounts in the next phase of growth. The antibiotic diffuses into the medium. After completion of the process, the microorganism is separated and the antibiotic is extracted from the medium by absorption, precipitation, and crystallization.
Mode of Action:
Antibiotics function either as bacteriocides or bacteriostatics. Their mode of action include
- Disruption of cell wall synthesis, e.g., penicillin, cephalosporin, bacitracin.
- Disruption of plasmalemma synthesis and repair, e.g., polymyxin, nystatin, amphotericin.
- Inhibition of 50S ribosome function, e.g., erythromycin.
- Inhibition of 30S ribosome function e.g., streptomycin.
- Inhibition of tRNA binding to ribosome, e.g., tetracycline.
- Inhibition of translation, e.g., chloramphenicol.
Characteristics of Good Antibiotics:
A good antibiotic should have the following properties:
- Harmless to host with no side effects.
- Harmless to normal microflora of the alimentary canal.
- Able to destroy the pathogen as well as broad spectrum.
- Effective against all strains of pathogen.
- Quick in action.
Resistance to Antibiotics:
It has been observed that the same antibiotic does not prove effective against a specific pathogen for long. This is due to the fact that pathogens acquire resistance to an antibiotic. Such resistance is produced due to the presence of extrachromosomal genes in the plasmids of bacteria. These genes confer resistance to the bacterium and also can pass from one bacterium to the other through the process of transformation and transduction. Such repeated transfer of plasmid has resulted in making certain strains of bacteria multiresistant, called superbugs, e.g., NDM-1. A few modifications that result in antibiotic resistance of bacteria are:
- Alternation of cell membrane so that antibiotic cannot recognize the pathogen.
- Development of a huge amount of mucilage.
- Alteration of cell membrane which prevents entry of antibiotics.
- Pathogen changes to L-form.
- Self-induced mutation in the pathogen.
- Production of enzymes by the pathogen which can modify the antibiotic.
Some Common Antibiotics Obtained from Bacteria, Actinomycetes, and Fungi are Listed as Follows:
|Antibiotic||Source||Mode of Action|
|Antibiotics from Bacteria|
|Polymyxin-B||Bacillus polymyxa||Deteriorates plasma membrane.|
|Bacitracin||Bacillus subtilis||Inhibits cell wall synthesis.|
|Gramicidin||Bacillus brevis||Deteriorates plasma membrane.|
|Antibiotics from Streptomyces sp.|
|Streptomycin||Streptomyces griseus||Inhibits protein synthesis.|
|Chloramphenicol||Streptomyces venezuelae||Interferes with protein synthesis.|
|Tetracyclines||Streptomyces aureofaciens||Inhibits binding of aminoacyl tRNA to ribosome.|
|Oxytetracycline||Streptomyces rimosus||Interferes with protein synthesis.|
|Erythromycin||Streptomyces erythreus||Induces abnormal protein synthesis.|
|Neomycin||Streptomyces fradiae||Inhibit the translation process.|
|Mitomycin||Streptomyces antibiotics||Inhibit carcinoma of the stomach or pancreas.|
|Antibiotics from Fungi|
|Penicillin||Penicillium nototum, P. chrysogenum||Inhibits cell wall synthesis of gram-positive bacteria.|
|Cephalosporin||Cephalosporium acremonium||Inhibits cell wall synthesis.|
|Griseofulvin||Penicillium griseofulvum||Inhibits fungal growth, especially for ringworms.|
Use of Antibiotics:
- Used as medicines for the treatment of a number of pathogenic or infectious diseases.
- Used as preservatives of food substances, pasteurized and canned foods.
- Used as a feed supplement for animals, and poultry birds because they enhance growth.
Sometimes steroids include drugs used to relieve many diseases. Steroid production in artificial methods is very expensive and complex. However, the production of steroids by microorganisms in culture media is cheap.
Some microorganism involved in steroid production is mentioned below:
|Previous Compound → Steroid||Micro-organisms Involved/Fungi|
|1. Cortisol → Prednisolone, Cortisone → Prednisone||Corynebacterium Simplex|
|2. 19-nor testosterone → Estradiol + Estrone||Pseudomonas Testosteroni|
|3. Progesterone → 11 α hydroxy-progesterone||Rhizopus nigricans|
|4. Androstenidione → Testosterone||Saccharomyces|
It is an antigenic substance prepared from the causative agent of a disease or a synthetic substitute, used to provide immunity against one or several diseases. Some living, nonliving, or toxins from microorganisms are toxoids and are used for vaccine production. Some micro-organisms used for vaccine production are mentioned below.
|1. Cholera||Part of a Cell of Vibrio cholerae|
|2. Tuberculosis||Inhibit the Production of Disease by Mycobacterium Tuberculosis|
|3. Plague||Dead Yersinia Pestis|
|4. Pertussis (whooping cough)||Dead Bordetella Pertussis|
|5. Haemophilus meningitis||Polysaccharide obtained from Haemophilus influenzae|
(iv) Production of Vitamins
Some vitamins are produced from bacterial fermentation in industry.
|1. Riboflavin||Clostridium Acetobutylicum, Mycobacterium smegmatis|
|2. Vitamin-B12 or Cyanocobalamin||Propionibacterium shermanii, Pseudomonas denitrificans|
|3. Vitamin C or Ascorbic Acid||Gluconobacter oxydans|
(v) Organic Acids
A number of organic acids are being manufactured with the help of microorganisms. Important examples are acetic acid, citric acid, lactic acid, butyric acid, etc. Lactic acid was the first organic acid produced through microbial fermentation.
(a) Acetic Acid:
The production of acetic acid takes place in two steps. The first step involves the initial anaerobic fermentation of the carbohydrate foodstuffs into alcohol by Saccharomyces cerevisiae, followed by the second step, the secondary transformation of alcohol to acetic acid by Acetobacter and Gluconobacter. This process is called acetification.
Substances used in the commercial production of vinegar (acetic acid) are fruits like oranges, grapes, apples, pears, etc. Vegetables like potatoes, malted cereals (barley, wheat, rye, etc.), and many other substances. Acetic acid is employed in pharmaceuticals, colouring agents, flavouring, solvents, insecticides, plastics, etc. Vinegar is a condiment, souring agent, and preservative.
(b) Citric Acid:
It is obtained through the fermentation carried out by Aspergillus niger, Penicillium citrinum, Mucor pyriformis, Torulopsis sp., etc., on sugary syrups. A variety of carbohydrates such as beet molasses, cane molasses, sucrose, commercial glucose, starch hydrolysate, etc. used in the fermentation medium. Citric acid is recovered through precipitation with Ca(OH)2 when calcium citrate is formed. It is regenerated by sulphuric acid.
Citric acid is employed in dyeing, engraving, medicines, inks, flavouring, and preservation of food and candies. Citric acid is a crystalline chemical that is added to soft drinks, jams, jellies, and frozen fruits as a preservative.
(c) Lactic Acid:
Lactic acid was the first organic acid to be produced from the microbial fermentation. Lactic acid is produced by several microorganisms like Streptococcus lactis, Lactobacillus sp., Rhizopus oryzae, etc.
The culture medium contains semirefined sugar, molasses, whey starch, maltose, lactose, sucrose, and calcium carbonate with ammonium hydrogen phosphate. Lactic acid is used in confectionery, fruit juices, essences, pickles, curing of meat, lemonades, canned vegetables, and fish products. It is employed in the leather industry for the delining of hides, in the textile industry, and in pharmaceuticals. The pure form of lactic acid is used in the plastic industry.
(d) Gluconic Acid:
Gluconic acid is obtained through the fermentation activity of Aspergillus niger and Penicillium sp. Calcium gluconate is used in the preparation of pharmaceuticals, as a source of calcium for infants, cows, and lactating mothers.
Other Industrial Production:
|A. Organic Acid|
|1. Lactic acid||Lactobacillus delbrueckii||Vegetables, preservation of meat and fish, dye industry, leather tanning.|
|2. Citric acid||Aspergillus niger, Candida lipolytica||Fruit preservation, jam, jelly, chocolate, cosmetic production.|
|3. Acetic acid (Vinegar)||Acetobacter aceti||Meat and vegetable preservation, Medicine industry.|
|4. Gluconic acid||Aspergillus niger||Ca and Fe deficiency treatment, refining of metal.|
|B. Amino Acid|
|1. Glutamine||Corynebacterium glutanicum||Perfume production.|
|2. Lysine||Bravibacterium flavum||Add nutrients to food.|
Enzymes are large biological molecules responsible for the thousands of chemical interconversions that sustain life. They are highly selective catalysts, greatly accelerating both the rate and specificity of metabolic reactions from the digestion of food to the synthesis of DNA. Most enzymes are proteins, although some catalytic RNA molecules have been identified. Enzymes adopt a specific three-dimensional structure and may employ organic cofactors to assist in catalysis.
In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all the chemical reactions in a biological cell need enzymes in order to occur at rates sufficient for life. Since enzymes are selective for their substrates and speed up only a few reactions among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.
Enzymes are proteinaceous substances produced by living cells that act as biological catalysts that change the rate of chemical reactions and remain unchanged at the end of the reaction. A number of enzymes are produced commercially for industrial and chemical uses.
These are enzymes that act on peptide linkages of proteins. Proteases are commercially available from organisms like Bacillus licheniformis and different species of Bacillus and Aspergillus. The enzymes are heat stable and are used in modern detergent formulations and they are used on a large scale in modern laundry for removal of spots of blood, milk, tea, etc. The proteases are used in the bakery to reduce the mixing time and improve the quality of the loaf. They are used in the leather industry for bating of hides and as a meat tenderizer. Proteases are also used in beverage industries in the clarification and maturing of malt beverages. It also helps in the digestion of fish liver to release fish oil.
Amylases break the starch into glucose. They are of various types α, β, and glucoamylases. They are obtained from Aspergillus niger, A. oryzae, Rhizopus sp., and bacteria like Bacillus subtilis and B. diasticus. The amylases are used in
- Removal of spots in clothes.
- Softening and sweetening of bread.
- Production of alcoholic beverages (e.g., beer, whiskey) from starchy materials.
- Sizing agent in the textile industry.
- In the syrup industry, the conversion of corn starch to fructose, to form corn syrup.
They break the ester bond (i.e., 1st bond and remove CH3 group) and 1, 4-glycosidic bond. A number of commercial firms produce fungal pectinases using Aspergillus niger, and Byssochlamys fulva in pectin-containing media. Pectinases are used in
- Clearing of fruit juices.
- Retting of fibers.
- Fermentation of green coffee.
- Enhancing juice extraction from fruits.
They are lipid-digesting enzymes that are obtained by growing, Candida lipolytica, Aspergillus niger, Geotrichum candidate etc. Lipases are used in
- Flavouring cheese.
- Hydrolyzing oils for manufacturing soap.
- Detergent formulations for removing oily stains from laundry.
(e) Streptokinase (Tissue Plasminogen Activator – TPA):
It is an enzyme obtained from cultures of some hemolytic bacterium Streptococcus. It has a fibrinolytic effect. This is utilized in the treatment of thrombotic disorders. In some cases of coronary thrombosis, streptokinase is injected to dissolve the clot inside the blood vessels.
(f) Protein Modifying Enzymes:
Certain enzymes can modify proteins from other sources into desired proteins. Pig insulin has been converted into human insulin or ‘humulin’ through this process.
They are enzymes that can convert lactose (milk sugar) into lactic acid. Lactic acid coagulates milk protein casein. Lactases can be obtained by the fermentation activity of Saccharomyces fragilis and Torula cremaris. These enzymes can prevent crystal formation in dairy preparations like ice cream and processed cheese.
An extract from the stomach of a calf that contains milk curdling enzyme rennin is called rennet. It is now being produced using strains of Mucor. It is used in the preparation of cheese in the dairy industry.
Application of Enzymes in Food Processing:
In food processing, the application of enzymes plays an important role. Amylase from fungi and plants is used in the production of sugars from starch, such as in making high-fructose corn syrup. In the baking industry, it catalyzes the breakdown of starch in the flour into sugar. Yeast fermentation of sugar produces the carbon dioxide that raises the dough.
Enzymes from barley are released during the mashing stage of beer production. They degrade starch and proteins to produce simple sugars, amino acids, and peptides that are used by yeast for fermentation. Industrially produced barley enzymes are widely used in the brewing process to substitute for the natural enzymes found in barley. Amylase, glucanases, and proteases split polysaccharides and proteins in the malt. Betaglucanases and arabinoxylanases improve the wort and beer filtration characteristics. Proteases remove cloudiness produced during the storage of beers. Acetolactate carboxylase (ALDC) increases fermentation efficiency by reducing diacetyl formation. Cellulases and pectinases clarify fruit juices.
In the Dairy industry, rennin, derived from the stomachs of young ruminant animals (like calves and lambs) is used in the manufacture of cheese, used to hydrolyze protein. Microbially produced enzymes are now finding increasing use in the dairy industry. Lipases are implemented during the production of Roquefort cheese to enhance the ripening of the blue mold cheese. Lactases break down lactose into glucose and galactose. Meat tenderizers like papain are used to soften meat for cooking.
Amylases, amyloglucosidases, and glucoamylases convert starch into glucose and various syrups. Glucose isomerase converts glucose into fructose in the production of high-fructose syrups from starchy materials. These syrups have enhanced sweetening properties and have lower calorific values than sucrose for the same level of sweetness. Restriction enzymes, DNA ligase, and polymerases are used to manipulate DNA in genetic engineering and are important in pharmacology, agriculture, and medicine. Essential for restriction digestion and the polymerase chain reaction.
It is an important bioactive compound produced by the fungus Trichoderma polysporum. This chemical is used as an immunosuppressive agent in organ transplantation. It has a membered cyclic oligopeptide which inhibits activation of T cells and thus prevents rejection against the transplant.
It is produced by the yeast Monascus purpureus. It is used as a blood cholesterol-lowering agent by acting as a competitive inhibitor of enzymes for cholesterol synthesis, e.g., pravastatin, lovastatin, and simvastatin.