Biology

Learninsta presents the core concepts of Microbiology with high-quality research papers and topical review articles.

Immobilization of Industrail Microbiology

It is technique used for the physical or chemical fixation of plant, animal cells, organelles, enzymes or other proteins (monoclonal antibodies) onto a solid matrix or retained by a membrane, in order to increase their stability and make possible their repeated or continued use.

The immobilized enzyme is defined as the enzyme physically confined or localized in a certain defined region of space with retention of its catalytic activity which can be used repeatedly and continuously.

The selection of appropriate carrier and immobilization procedure is very essential procedure is very essential for the immobilization technique.

Various types of materials like cellulose, dextran, agarose, gelatin, albumin polystyrene, Calcium alginate polyacrylamide, collagen carrageenan and polyurethane, inorganic materials (brick, rand, glass, and ceramics, magnetic) are used for immobilization.

The linkage is mediated by ionic bonds, physical absorption or bio specific binding.

The immobilization methods can be classified into four categories:-

1. Carrier–binding
2. Cross–linking
3. Entrapping
4. Combining

Among all these methods entrapping is discussed in brief.

Entrapping

The enzymes, cells are not directly attached to the support surface, but simply trapped inside the polymer matrix. Entrapping is carried out by mixing the biocatalyst into a monomer solution followed by a polymerization. It is done by change in temperature or by chemical reactions.

Advantages of immobilization

1. Immobilized growing cells serve as self proliferating and self regenerating bio catalyst
2. They are stable
3. They are used either repeatedly in a series of batch wise reactions or continuously in flow systems.

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Industrial Production of Citric Acid

Citric acid is obtained from citrus fruits; pineapple etc., and after the development of microbial fermentation, citric acid production becomes very cheap, easy and cost effective. 70% of citric acid produced is used in food and beverage industry. Many microbial strains such as fungi Aspergillus flavus, Aspergillus niger and Trichoderma viridae, yeast Hansenulla polymorpha and Candida lipolytica are generally are involved in the production of citric acid.

Citric acid production can be carried out in the following three methods.

• Koji process or solid state fermentation
• Liquid surface culture
• Submerged fermentation

Media used in citric acid production

Citric acid production is carried out by using carbohydrates and n-alkenes. Generally beet molasses, cane molasses, sucrose, commercial glucose and starch hydrolysate are used as carbohydrate sources. The carbohydrate material is diluted and mixed with a nitrogen source (ammonium salts or urea) and the pH and temperature are adjusted according to the process.

Inoculum development

Fungal strains that are used for production are stored in soil or silica gel in the form of spores. Spores are suspended in a freshly prepared sterile water containing Tween 80 and after a period of growth, it can be used as inoculum for large scale production.

Steps involved in citric acid production
Production Medium

Sucrose, beet molasses, used as carbon source need pretreatment, as it contains excessive amount of trace metals. So ferrocyanide or ferricyanide is added to the production medium before sterilization. Inorganic salts, carbon, hydrogen, oxygen trace metals. Nitrogen, potassium, phosphorus,sulphur and magnesium
are taken in Aluminum or stainless steel shallow pans or tray (5-20 cm deep).
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Inoculated with spores of A. niger by blowing over the strains of Aspergillus niger for fermentation
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The medium is kept at 28-30ºC with relative humidity 40-60% and aerated with purified air for 8-12 days
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Citric acid produced is determined by checking the pH or the total acid content of the broth.
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Fermented liquid is drained off and processed further for the recovery of citric acid

Recovery

The mycelial mat is pressed.
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Milk of lime (calcium carbonate) is added so calcium citrate is formed.
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Again sulphuric acid is added, so calcium sulphate is formed.
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The remaining citric acid solution is filtered and washed. Finally the impure solution of citric acid subjected to treatment with activated carbon and finally pure form of citric acid is collected.

Uses

It is used as a Acidulant in food, (Jams, Preserved fruits, Fruit drinks) and pharmaceutical industries.

1. It is mainly used in food and beverage industry (Jams, preserved fruits, fruit drinks)
2. It is used is pharmaceuticals, and other industrial processes
3. Citrate and citrate esters are used as plasticizers
4. It is used as a chelating and sequestering agent (Tanning of animal skins)

Generally citric acid obtained from citrus fruits, pineapple etc., After the development of microbial fermentation, citric acid production becomes very cheap and easy cost effective.

Learninsta presents the core concepts of Microbiology with high-quality research papers and topical review articles.

Industrial Production of Single Cell Protein

Single cell protein refers to the microbial cells or total protein extracted from pure microbial cell culture (monoculture) which can be used as protein supplement for humans or animals. During ancient times, the tribes in the Central African Republic used a spiral shaped Cyanobacterium named Spirulina platensis as food.

They collected it as mats from the bottom of seasonally dried up ponds and shallow waters around Lake Chad and dried them in the sun and made small cakes called “Dihe”.

During the World war II, when there were shortage in proteins and vitamins in the diet, the Germans produced yeasts and a mould named Geotrichum candidum was used as food.

The term Single Cell Protein was coined by C.L Wilson (1966) at Massachusetts Institute of Technology (MIT), to represent the cells of algae, bacteria, yeasts and fungi, grown for their protein contents. The name was introduced by Prof. Scrimshow of MIT in 1967.

The organisms like Pseudomonas facilis, P. flava, Chlorella, Anabaena, Spirulina, Chlamydomonas, and Agaricus are commonly used for SCP production. Large scale production of SCP is shown in the Figure 6.11

There are several methods available for SCP production. In the Japanese method, flat tray is used with artificial sunlight algae are cultivated in shallow ponds with mechanical stirrers or in deeper ponds (not more than 20-30 cm deep) with circulation pumps. Optimum, light is an important parameter for maximum
growth of SCP. Scenedesmus sp. grows 20 times faster in optimum light than in natural conditions.

Optimum temperature and optimum pH is varied according to the strain and intensity of light. Example: Spirulina is cultivated at 25-35ºC with pH 9.5. Table 6.6 shows different types of microorganisms and substrates used for SCP production.

List of microorganisms and substrates used for SCP production

Steps involved in SCP production

Provision of carbon source with added nitrogen, CO₂, ammonia, trace minerals for growth
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Prevention of contamination by using sterilized medium and fermentation equipments
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Selected microorganism is inoculated in a pure form
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Adequate aeration and cooling is provided
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Microbial biomass is harvested and recovered by flocculation or centrifugation flocculants
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Harvested algae are dewatered and dried on open sand beds
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Processing biomass and enhancing it for use and storage

Advantages of using microorganisms for SCP production:

1. Microorganisms grow at a very rapid rate under optimal culture conditions.
2. The quality and quantity of protein content in microorganisms is better compared to higher plants and animals.
3. A wide range of raw materials which are otherwise wasted, can be fruitfully used for SCP production
4. The culture conditions and the fermentation processes are very simple.
5. Microorganisms can be easily handled and subjected to genetic manipulations.

During the cultivation of SCP, care must be taken to prevent and control the contamination by other micro organisms, which produce mycoxins or cyanotoxins. This is controlled by using the fungus Scytaliclium acidophilum which grows at a low PH. It allows the hydrolysation of paper wastes to a sugar medium and also creates aseptic condition at low cost.

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Industrial Production of Wine

An alcoholic distilled beverage is produced by concentrating alcohol from fermentation by distillation. Beer or ale is produced by the fermentation of malted grains. Wine is prepared from grapes belonging to species Vitis vinefera. It is also produced from other fruits like peach, pear, dandelion and honey.

Generally wine contains 16% of alcohol. Wine production from crushed grapes is called enology. The various forms of wine are listed below in the table 6.5.

Shows diffrent varieties of wine

Red wine is extracted from the skin of red grapes containing red pigment (anthocyanin). During the preparation of red wine, all the anthocyanin pigments are solubilized by the extract. Pink wine is obtained from either pink grapes or red grapes in which fermentation last for only 12 to 36 hour and only less amount of anthocyanin pigments are solubilized. White wine is prepared from the white grapes or from the red grapes in which pigment involved in colouring is removed.

Generally yeasts are the natural microbiota of grapes

Both wild yeast and cultivated yeast are involved in the wine fermentation. Natural yeast is not potable because they do not produce much wine and are less alcohol tolerant and produce undesirable compounds, affecting the quality of the wine.

The cultivated wine yeast, Saccharomyces ellipsoideus, is used for commercial production. Figure 6.10 shows steps involved in wine production.

Steps involved in Wine production Grapes are stemmed, cleaned and crushed
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Sodium or Potassium Meta – bisulphate is added to check the undesirable microorganism
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Must (crushed grapes) is treated with Sulfur dioxide to kill the wild yeasts and bacteria or sometimes pasteurized to destroy the natural microbiota
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Must is inoculated with Saccharomyces ellipsoideus (2.5%) and selected fermentation is carried from 50 to 50000 gallons at 20 to 24°C
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Oak, cement, stone glass lined metal are used as fermentor
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Temperature and time required for fermentation White wine: 10 – 21°C, 7 – 12 days; Red wine: 24 – 27°C, 3 – 5 days
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In red wine production, after three to five days of fermentation, sufficient tanin and colour is extracted from the pomace and the wine is drawn off for further fermentation
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Racking improves flavour and aroma, where wine is separated from the sediment containing yeast cells as precipitate form
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The wine is subjected to aging at lower temperature. Ageing process is typically much longer for red wine than white wine
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Wines are clarified in a process called fining. Fining is done by filtration through casein, tannin, diatomaceous earth or bentonite clay, asbestos, membrane filters or centrifugation
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The wine produced is placed in casks, tank and bottles

After wine production, cork should be used for preventing the entry of air into the bottles. The presence of air allows the growth of vinegar bacteria that convert the ethanol to acetic acid. The final alcohol content of wine varies depending upon the sugar content of the grapes, length of the fermentation and type of strain used.

Learninsta presents the core concepts of Microbiology with high-quality research papers and topical review articles.

Industrial Production of Penicillin

Penicillin is a broad spectrum antibiotic. Penicillin is first obtained from the mould, Penicillium notatum (Figure 6.8).

Penicillium chrysogenum is a high yielding strain, used for the commercial production of penicillin. This strain is highly unstable, so the spore suspensions are maintained in a dormant state to prevent contamination. Most penicillin form filamentous broth and hence is difficult to mix and it hinders
oxygen transfer due to their high viscosity.

This is avoided by using bubble columns air lift reactors which agitates the medium providing even oxygen distribution. Penicillin has a basic structure 6-amino penicillanic acid β-(APA). It consists of a thiazolidine ring with a condensed β-lactum ring. It carries a variable side chain in position 6. Natural penicillins are produced in a fermentation process without adding any side chain precursors.

If a side chain precursor is added to the broth, desired penicillin is produced and it is called biosynthetic penicillin. Semi synthetic penicillin is one in which, both fermentation and chemical approach are used to produce useful pencillins. It can be taken orally and active against gram negative bacteria. (eg) Amphicilin. Nowadays, semi synthetic pencillins makeup the bulk of the penicillin market.

The initial strain of Penicillium chrysogenum (NRRL, 1951) was low yielding strain and so it is was treated with mutagenic agents such as X-rays, UV right and some other repeated methods to get a high yielding strain Q-176.

Production methods

Penicillin production is done by one of the following.

1. Surface culture
2. Submerged fermentation process

Inoculum Production

Inoculation methods

To inoculate fermentation medium one of the following methods can be employed.

1. Using dry spores to seed the fermentation medium.
2. Making suspension along with non toxic wetting agent like Sodium lauryl sulphate and inoculating germinated organism
3. Using pellet inocula obtained by the germination of spores

The lyophilized spores (or) spores in well sporulated frozen agar slant are suspended in water or in a dilute solution of a nontoxic wetting agent.

(1: 10,000 sodium lauryl sulphonate)
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Spores are then added to a bottles containing wheat bran solution It is incubated for 5-7 days at 24°C for heavy sporulation.
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The resulting spores are then transferred to production tank
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The micro organism in the inoculum tank is checked for contamination.

Production process

The production tanks are inoculated with a mycelial growth.
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Production medium contains following medium components.

Carbon source as Lactose, Nitrogen source as Ammonium sulphate, Acetate or Lactate (Corn steep liquor is the cheap and easy source of nitrogen)

Mineral sources as K, P (Potassium di hydrogen phosphate), Mg, S (Magnesium sulphate), Zn, Cu(Copper sulphate) (Corn steep liquor supply some of these minerals)

Precursor (Example: phenyl acetic acid) is added to the medium
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Antifoam agent (Example: corn or soybean oil) is added before sterilization.

The sufficient aeration and agitation is given and are incubated at 25°C to 26°C for 3 to 5 days at PH range of 7 to 7.5

Penicillin Production

Process of penicillin production occurs in three phases:

First phase:

Growth of mycelium occurs in this phase where the yield of antibiotic is low. The pH increases due to the release of NH₃.

Second phase:

In this phase, intense synthesis of penicillin occurs due to rapid consumption of Lactose and Ammonium nitrogen. The mycelial mass increases and the pH remain unchanged (Figure 6.10).

Third phase:

In this phase, the concentration of antibiotics decreases in the medium. Autolysis of mycelium starts, liberating Ammonia leading to slight rise in pH.

Recovery

After penicillin fermentation, the broth is filtered on rotary vacuum filter
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Mycelium is separated
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To the both sulphuric acid or phosphoric acid is added
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Pencilin is converted into anionic form
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It is extracted in counter current solvent extractor, by using organic solvent, amyl acetate, methyl isobutyl (ketone)
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It is then back extracted with water from the organic solvent by adding potassium or sodium hydroxide
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Shifts between water and solvent aid in the potassium or sodium hydroxide
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Shifts between water and solvent aid in the purification of pencilin
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The resulting sodium or potassium pencillin is then crystallized
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Then it is washed, dried and used for commercial purpose.