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FSN 411: INDUSTRRIAL FOOD MICROBIOLOGY AND BIOTECHNOLOGY

Industrial mcriobiology involves the use of microorganisms to produce commercially valuable products

Industrial microbiology includes many areas, including:

food production,

pharmaceuticals,

Biomass utilization

Bioremediation, Biotechnology involves the use of an organism’s biochemical and metabolic pathways for industrial production of commercially viable products

Fermentation in food processing typically is the conversion of carbohydrates to alcohols and carbon dioxide or organic acids using yeasts, bacteria, or a combination thereof, under anaerobic conditions. Fermentation usually implies that the action of microorganisms is desirable, and the process is used to produce alcoholic beverages such as wine, beer, and cider. Fermentation is also employed in the leavening of bread, and for preservation techniques to create lactic acid in sour foods such as sauerkraut, dry sausages,and yogurt, or vinegar (acetic acid) for use in pickling foods.

Food fermentation has been said to serve five main purposes

Enrichment of the diet through development of a diversity of flavors, aromas, and textures in food substrates

Preservation of substantial amounts of food through lactic acid, alcohol, acetic acid and alkaline fermentations

Biological enrichment of food substrates with protein, essential amino acids, essential fatty acids, and vitamins

Elimination of antinutrients A decrease in cooking times and fuel requirements

BIOCHEMISTRY

Fermentation is the process of deriving energy from the oxidation of organic compounds, such as carbohydrates, and using an endogenous electron acceptor, which is usually an organic compound. In contrast, respiration is where electrons are donated to an exogenous electron acceptor, such as oxygen, via an electron transport chain. Fermentation is important in anaerobic conditions when there is no oxidative phosphorylation to maintain the production of ATP

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(Adenosine triphosphate) by glycolysis. During fermentation, pyruvate is metabolised to various different compounds. Homolactic fermentation is the production of lactic acid from pyruvate; alcoholic fermentation is the conversion of pyruvate into ethanol and carbon dioxide; and heterolactic fermentation is the production of lactic acid as well as other acids and alcohols.

Sugars are the most common substrate of fermentation, and typical examples of fermentation products are ethanol, lactic acid, and hydrogen. However, more exotic compounds can be produced by fermentation, such as butyric acid and acetone. Yeast carries out fermentation in the production of ethanol in beers, wines and other alcoholic drinks, along with the production of large quantities of carbon dioxide. Fermentation occurs in mammalian muscle during periods of intense exercise where oxygen supply becomes limited, resulting in the creation of lactic acid.

Ethanol fermentation

Ethanol fermentation (performed by yeast and some types of bacteria) breaks the pyruvate down into ethanol and carbon dioxide. It is important in bread-making, brewing, and wine-making.

The chemical equation below summarizes the fermentation of glucose, whose chemical formula is C6H12O6. One glucose molecule is converted into two ethanol molecules and two carbon dioxide molecules:

C6H12O6 → 2C2H5OH + 2CO2

C2H5OH is the chemical formula for ethanol.

Before fermentation takes place, one glucose molecule is broken down into two pyruvate molecules. This is known as glycolysis.

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Lactic acid fermentation

Lactic acid fermentation is the simplest type of fermentation. Essentially, it is a redox reaction. In anaerobic conditions, the cell’s primary mechanism of ATP production is glycolysis. Glycolysis reduces – transfers electrons to – NAD+, forming NADH. However, there is only a limited supply of NAD+ available in a cell. For glycolysis to continue, NADH must be oxidized – have electrons taken away – to regenerate the NAD+. This is usually done through an electron transport chain in a process called oxidative phosphorylation; however, this mechanism is not available without oxygen.

Instead, the NADH donates its extra electrons to the pyruvate molecules formed during glycolysis. Since the NADH has lost electrons, NAD+ regenerates and is again available for glycolysis. Lactic acid, for which this process is named, is formed by the reduction of pyruvate.

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In heterolactic acid fermentation, one molecule of pyruvate is converted to lactate; the other is converted to ethanol and carbon dioxide. In homolactic acid fermentation, both molecules of pyruvate are converted to lactate. Homolactic acid fermentation is unique because it is one of the only respiration processes to not produce a gas as a byproduct.

Homolactic fermentation breaks down the pyruvate into lactate. It occurs in the muscles of animals when they need energy faster than the blood can supply oxygen. It also occurs in some kinds of bacteria (such as lactobacilli) and some fungi. It is this type of bacteria that converts lactose into lactic acid in yogurt, giving it its sour taste. These lactic acid bacteria can be classed as homofermentative, where the end product is mostly lactate, or heterofermentative, where some lactate is further metabolized and results in carbon dioxide, acetate or other metabolic products.

The process of lactic acid fermentation using glucose is summarized below. In homolactic fermentation, one molecule of glucose is converted to two molecules of lactic acid:

C6H12O6 → 2 CH3CHOHCOOH.

In heterolactic fermentation, the reaction proceeds as follows, with one molecule of glucose being converted to one molecule of lactic acid, one molecule of ethanol, and one molecule of carbon dioxide:

C6H12O6 → CH3CHOHCOOH + C2H5OH + CO2

FERMENTATION TECHNOLOGY

FERMENTATION DESIGN

The fermentation process requires the following:

a) a pure culture of the chosen organism, in sufficient quantity and in the correct physiological state;b) sterilised, carefully composed medium for growth of the organism;

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c) a seed fermenter, a mini-model of production fermenter to develop an inoculum to initiate the process in the main fermenter;d) a production fermenter, the functional large model; ande) equipment for i) drawing the culture medium in steady state, ii) cell separation, iii) collection of cell free supernatant, iv) product purification, and v) effluent treatment.Items a) to c) above constitute the upstream and e) constitutes the downstream, of the fermentation process,Fermenters/bioreactors are equipped with an aerator to supply oxygen in aerobic processes, a stirrer to keep the concentration of the medium uniform, and a thermostat to regulate temperature, a pH detector and similar

MICROBIAL CULTIVATION: INOCULUM DEVELOPMENT

The preparation of a population of microorganisms from a dormant stock culture to an active state of growth that is suitable for inoculation in the final production stage is called inoculum development. As a first step in inoculum development, inoculum is taken from a working stock culture to initiate growth in a suitable liquid medium.

Bacterial vegetative cells and spores are suspended, usually, in sterile tap water, which is then added to the broth. In case of nonsporulating fungi and actinomycetes the hyphae are fragmented and then transferred to the broth.

Inoculum development is generally done using flask cultures; flasks of 50 ml to 12 litres may be used and their number can be increased as per need. Where needed, small fermenters may be used. Inoculum development is usually done in a stepwise sequence to increase the volume to the desired level. At each step, inoculum is used at 0.5-5% of the medium volume; this allows a 20-200-fold increase in inoculum volume at each step. Typically, the inoculum used for production stage is about 5% of the medium volume.

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FERMENTION MEDIUM

Growth media are required for industrial fermentation, since any microbe requires water, (oxygen), an energy source, a carbon source, a nitrogen source and micronutrients for growth.

Carbon & energy source + nitrogen source + O2 + other requirements → Biomass + Product + byproducts + CO2 + H2O + heat

Nutrient Raw material

Carbon

Glucose corn sugar, starch, cellulose

Sucrose sugarcane, sugar beet molasses

glycerol

Starch

Maltodextrine

Lactose milk whey

fats vegetable oils

Hydrocarbons petroleum fractions

Nitrogen

Protein soybean meal, corn steep liquor, distillers' solubles

Ammoniapure ammonia or ammonium salts

urea

Nitrate nitrate salts

Phosphorus source phosphate salts

Vitamins and growth factors

Yeast, Yeast extract

Wheat germ meal, cotton seed meal

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Beef extract

Corn steep liquor

Trace elements: Fe, Zn, Cu, Mn, Mo, Co

Antifoaming agents : Esters, fatty acids, fats, silicones, sulphonates, polypropylene glycol

Buffers: Calcium carbonate, phosphates

Growth factors: Some microorganisms cannot synthesize the required cell components themselves and need to be supplemented, e.g. with thiamine, biotin

Inhibitors: To get the specific products: e.g. sodium barbital

Inducers: The majority of the enzymes used in industrial fermentation are inducible and are synthesized in response of inducers: e.g. starch for amylases, maltose for pollulanase, pectin for pectinase,olive oil and tween are also used at times.

Chelators: Chelators are the chemicals used to avoid the precipitation of metal ions. Chelators like EDTA, citric acid, polyphosphates are used in low concentrations.

BATCH PROCESSING OR CULTURE

At about the onset of the stationary phase, the culture is disbanded for the recovery of its biomass (cells, organism) or the compounds that accumulated in the medium (alcohol, amino acids), and a new batch is set up. This is batch processing or batch culture.

The best advantage of batch processing is the optimum levels of product recovery. The disadvantages are the wastage of unused nutrients, the peaked input of labour and the time lost between batches.

B. CONTINUOUS PROCESSING OR CULTURE

The culture medium may be designed such that growth is limited by the availability of one or two components of the medium. When the initial quantity of this component is exhausted, growth ceases and a steady state is reached, but growth is renewed by the addition of the limiting component. A certain amount of the whole culture medium (aliquot) can also be added periodically, at the time when steady state sets in. The addition of nutrients will increase the volume of the medium in the fermentation vessel. It is so arranged that the increased volume will drain off as an overflow, which is collected and used for recovery of products. At each step of addition of the medium, the medium becomes dilute both in terms of the concentration of the

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biomass and the products. New growth, stimulated by the added medium, will increase the biomass and the products, till another steady state sets in; and another aliquot of medium will reverse the process.

This is continuous culture or processing. Since the growth of the organism is controlled by the availability of growth limiting chemical component of the medium, this system is called a chemostat. The rate at which aliquots are added is the dilution rate that is in effect the factor that dictates the rate of growth.

The events in a continuous culture are:

a) the growth rate of cells will be less than the dilution rate and they will be washed out of the vessel at a rate greater than they are being produced, resulting in a decrease of biomass concentration both within the vessel and in the overflow;

b) the substrate concentration in the vessel will rise because fewer cells are left in the vessel to consume it;

c) the increased substrate concentration in the vessel will result in the cells growing at a rate greater than the dilution rate and biomass concentration will increase; and

d) the steady state will be re-established.

Hence, a chemostat is a nutrient limited self-balancing culture system, which may be maintained in a steady state over a wide range of sub-maximum specific growth rates.

The continuous processing offers the most control over the growth of cells.

Commercial adaptation of continuous processing is confined to biomass production, and to a limited extent to the production of potable and industrial alcohol.

The steady state of continuous processing is advantageous as the system is far easier to control. During batch processing, heat output, acid or alkali production, and oxygen consumption will range from very low rates at the start to very high rates during the late exponential phase. The control of the environmental factors of the system becomes difficult. In the continuous processing, the rates of consumption of nutrients and those of the output chemicals are maintainable at optimal levels. Besides, the labour demand is also more uniform.

Continuous processing may suffer from contamination, both from within and outside. The fermenter design, along with strict operational control, should actually take care of this problem. The production of growth associated products like ethanol is more efficient in continuous processing, particularly for industrial use.

Continuous culturing is highly selective and favours the propagation of the best-adapted organism in culture.

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A commercial organism is highly mutated such that it will produce very high amounts of the desired product. But physiologically such strains are inefficient and give way in culture to inferior producers--a kind of contamination from within.

C. FED-BATCH CULTURE OR PROCESSING

In the fed-batch system, a fresh aliquot of the medium is continuously or periodically added, without the removal of the culture fluid. The fermenter is designed to accommodate the increasing volumes. The system is always at a quasi-steady state.

Fed-batch achieved some appreciable degree of process and product control.

A low but constantly replenished medium has the following advantages:

a) maintaining conditions in the culture within the aeration capacity of the fermenter;

b) removing the repressive effects of medium components such as rapidly used carbon and nitrogen sources and phosphate;

c) avoiding the toxic effects of a medium component; and

d) providing limiting level of a required nutrient for an auxotrophic strain.

Production of baker's yeast is mostly by fed-batch culture, where biomass is the desired product. Diluting the culture with a batch of fresh medium prevents the production of ethanol, at the expense of biomass; the moment traces of ethanol were detected in the exhaust gas.

STRAIN IMPROVEMENT

After an organism producing a valuable product is identified, it becomes necessary to increase the product yield from fermentation to minimise production costs. Product yields can be increased by (i) developing a suitable medium for fermentation,

(ii) refining the fermentation process and

(iii) improving the productivity of the strain.

Generally, major improvements arise from the last approach; therefore, all fermentation enterprises place a considerable emphasis on this activity. The techniques and approaches used to genetically modify strains, to increase the production of the desired product are called strain improvement or strain development.

Strain improvement is based on the following three approaches: (i)mutantselection,

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(ii)recombination, and(iii) recombinant DNA technology.

Approach Chief feature Example/Remark

A. Mutant Selection : Types  

The main approach to strain improvement; produces new alleles of existing genes

1. Spontaneous Mutations Occur without any treatment with a mutagen

Used in, the initial stages of strain improvement; also for maintenance of improved strains

2. Induced Mutations Induced by chemical (mainly) or physical mutagens

Mutagenesis followed by selection; several cycles employed

3. Major Mutations Affect the pattern of metabolite production

Production of 6-demethyl tetracycline in place of tetracycline by S. aureofaciens

4. Minor Mutations Affect the rate metabolite production

Small gains in each cycle of selection; substantial improvement after several cycles

B. Mutant Selection :Strategies    

1. Auxotrophic mutants Defective biosynthesis of a biochemical

Enhanced production of an amino acid, e.g., phe mutants accumulate tyrosine

2. Analogue resistant mutants

Feedback insensitive enzymes

Overproduction of metabolites, e.g., amino acids by C. glutamicus

3. Revertants of nonproducing mutants  

Some mutants are high producers, e.g., chlortetracycline by S. viridifaciens

4. Revertants of auxotrophic mutants   Some are high produces, e.g.

chlortetracycline by S. viridifaciens5. Resistance to the antibiotic produced by the organism itself

  Increased production, e.g., chlortetracycline by S. aureofaciens

C. Recombination   Produces new combinations of existing alleles

1. Sexual reproduction Conjugation; fusion of gametes

Some bacteria and Actinomycetes; fungi and yeast

2. Heterokaryosis Nuclear fusion followed, by mitotic recombination and mitotic reduction

Fungi

3. Protoplast fusion Protoplasts produced by lytic enzymes fusion by PEG,

Bacteria, Actinomycetes, fungi; quite; successful

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recombinant recovery

 

 BIOTRANSFORMATION

When an organic compound is modified by simple chemically defined reactions, catalyzed by enzymes present in cells, into a product that is recoverable, it is called biotransformation. Both the substrate and the product are not involved in the primary or secondary metabolism of the organism employed.

This is in contrast to the various metabolites, e.g., organic acids, amino acids, antibiotics etc., produced by the complex pathways of primary or secondary metabolism of the organism. Biotransformations are far more efficient and economical due to the rapid microbial growth and high metabolic rates of microorganisms.

BOICATALYSTS

Immobilization can be defined as the confinement of a biocatalyst inside a bioreaction system, with retention of its catalytic activity and stability. Cells can be immobilized

Immobilized Cell Bioreactors - Bioreactors of this type are based on immobilized cells. Cell immobilization is advantageous when

(i) the enzymes of interest are intracellular,(ii) extracted enzymes are unstable, (iii) the cells do not have interfering enzymes or such enzymes are easily inactivated/removed and(iv) the products are low molecular weight compounds released into the medium.

Under these conditions, immobilized cells offer the following advantages over enzyme immobilization:

(i) enzyme purification is not needed, (ii) high activity of even unstable enzymes,

(iii) high operational stability,(iv) lower cost and(v) possibility of application in multistep enzyme reactions. In addition, immobilization permits continuous operation of bioreactor, which reduces the reactor volume and, consequently, pollution problems. Obviously, immobilized cells are used for such biotransformations of compounds, which require action of a single enzyme.

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Cell immobilization may be achieved in one of the following ways.

(1) Cells may be directly bound to water insoluble carriers, e.g., cellulose, dextran, ion exchange resins, porous glass, brick, sand, etc., by adsorption, ionic bonds or covalent bonds.

(2) They can be cross linked to bi- or multifunctional reagents, e.g., glutaraldehyde, etc.(3) Polymer matrices may be used for entrapping cells; such matrices are polyacrylamide cell, ҝ-carrageenan (a polysaccharide isolated from a seaweed), calcium alginate (alginate is extracted from seaweed), poly glycol oligomers, etc.

Out of these approaches, calcium alginate immobilization is the most commonly used since it can be used for even very sensitive cells. Cell immobilization has been used for commercial production of amino acids, organic acids, etc.

 

BIOREACTORS

A bioreactor is a device in which a substrate of low value is utilized by living cells or enzymes to generate a product of higher value. A bioreactor is a device in which the microorganisms are cultivated and motivated to form the desired products by maintaining optimum conditions for growth and metabolic activity. A fermenter refers to the device used for the cultivation of prokaryotic cells e.g. bacteria, fungi etc. whereas a bioreactor is used for growing eukaryotic cells.

Bioreactors are extensively used for food processing, fermentation, waste treatment, etc. On the basis of the agent used, bioreactors are grouped into two broad classes:

(i) those based on living cells and, (ii) those employing enzymes.

But in terms of process requirements, they are of the following types:(i) aerobic, (ii) anaerobic, (iii) solid state, and(iv) immobilized cell bioreactors.

All bioreactors deal with heterogeneous systems having two or more phases, e.g., liquid, gas, solid. Therefore, optimal conditions for fermentation necessitate efficient transfer of mass, heat and momentum from one phase to the other.

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A bioreactor should provide for the following: (i) agitation (for mixing of cells and medium), (ii) aeration (aerobic fermenters; for O2 supply), (iii) regulation of factors like temperature, pH, pressure, aeration, nutrient feeding, liquid level, etc., (iv) sterilization and maintenance of sterility, and

(v) withdrawal of cells/medium (for continuous fermenters). Modem fermenters are usually integrated with computers for efficient process monitoring, data acquisition, etc.

The size of fermenters ranges from 1-21 laboratory fermenters to 500,000 1 or, occasionally, even more; fermenters of upto 1.2 million litres have been used. Generally, 20-25% of fermenter volume is left unfilled with medium as "head space" to allow for splashing, foaming and aeration. The fermenter design varies greatly depending on the type of fermentation for which it is used.

BIOREACTOR DESIGN

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A typical conventional bioreactor has cylindrical vessel with domed top and bottom generally made up of stainless steel. The reaction vessel which is surrounded by a jacket, is provided with a sparger at the bottom through which air (or other gases such as CO2 and NH3 (for pH maintenance) can be introduced. The reaction vessel also has side ports for pH, temperature and dissolved O2 sensors. The agitator shaft is connected to a motor at the bottom. Above the liquid level of the reaction vessel, connections for acid, alkali, antifoam chemicals and inoculum are located. The bioreactor is designed to work at very high temperatures (150-1800C), high pressure (377-412 kPa) and also to withstand vacuum which prevents its collapse while cooling.

 

Types of Bioreactors

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Depending on the design of the reactor, the bioreactors are of following types:

a) Continuous stirred tank bioreactors - These bioreactors have a cylindrical vessel with motor driven central shaft which gives support to one or more agitators (impellers). The shaft is fitted at the bottom of the bioreactor. The diameter of the impeller is usually one third of the vessel diameter. The impellers are available in different designs like- Rustom disc, concave bladded, marine propeller etc. In stirred tank reactors, the air is added to the culture medium under pressure through a device called sparger. The sparger along with the impellers (agitators) enables better and efficient gas distribution through out in the vessel. The advantages of using stirred tank reactors are: the efficient transfer of gas to growing cells which keeps the growth of cells in healthy limits, stirring ensures good mixing of the contents, the operating conditions are flexible and the bioreactors are easily available which makes them commercially viable products.

b) Bubble column bioreactors - In these bioreactors, the gas or air is introduced at the base of the column through perforated pipes or plates, or metal microporous spargers. The vessel used for bubble column bioreactors is usually cylindrical with an aspect ratio (height o diameter ratio) of 4-6. The rate of flow of gas affects the O2 transfer and mixing.

c) Airlift bioreactors - Airlift bioreactors are commonly used for aerobic bioprocessing technology. In the airlift bioreactors, the medium of the vessel is divided into two interconnected zones by means of baffle or draft tube. The air/gas is pumped into one of the two zones referred to as ‘riser’ and the other zone that receives no gas is known as ‘downcomer’. The dispersion flows up the riser zone while the down flow occurs in the downcomer. Further there are two types of bioreactors:

1) Internal loop bioreactor - These bioreactors have a single container with a central draft tube that creates interior liquid circulation channels which keeps the volume and circulation at a fixed rate for fermentation.

(2) External loop airlift bioreactor-These have an external loop to keep the liquid in circulation through separate independent channels. The modifications can be made in these bioreactors depending on the requirements of different fermentation processes.

(3) Two stage airlift bioreactors -These bioreactors have two bioreactors which are basically used for the temperature dependent formation of products. The growing cells from one bioreactor (maintained at temperature 300C are pumped into another bioreactor (at temperature 420C). This is done because it is very difficult to increase the temperature quickly from 300C to 420C in the same vessel. The cells are grown in the first bioreactor and with the help of the fitted valves and a transfer tube and pump, they are transferred into the second bioreactor, where the actual bioprocessing takes place.

(4) Tower bioreactors - In this type of bioreactor, a high hydrostatic pressure is generated at the bottom of the reactor which increases the solubility of O2 in the medium. Since the top is expanded, the pressure is reduced which helps in the expulsion of CO2. The cycle completes with the medium flowing back into the downcomer. The advantage with Tower bioreactor is that it has high aeration capacities without having moving parts.

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d) Fluidized bed bioreactors - These bioreactors are mainly suitable to carry out reactions involving fluid suspended biocatalysts such as immobilized enzymes, immobilized cells, microbial flocs etc. The design of the bioreactors is such that the top is extended and the reaction column is narrow which retains the solids in the reactor and allows the liquids to flow out. To maintain an efficient operation of fluidized beds, gas is sparged to create a suitable gas-liquid-solid fluid bed. The recycling of the liquid ensures continuous contact between the reaction contents and biocatalysts which increases the efficiency of bioprocessing.

e) Packed bed bioreactors- A packed bed bioreactor consists of a bed of solid particles, with biocatalysts on or within the matrix of solids, packed in a column. The solids are generally porous or non-porous gels which maybe compressible or rigid in nature. The nutrient broth continuously flows over the immobilized biocatalyst and the products are released into the fluid from where they are removed. However, due to poor mixing, it is difficult to control the pH of packed bioreactors by the addition of acid or alkali.

f) Photobioreactors - These bioreactors are specialized for fermentation that can be carried out either by exposing to sunlight or artificial illumination. The photobioreactors are made up of glass or transparent plastic which are the solar receivers. The cell cultures are circulated through the solar receivers by using centrifugal pumps or airlift pumps. These bioreactors work in the temperature which ranges from 25-400C. In these bioreactors, the microorganisms e.g. microalgae, cyanobacteria etc. grow during the day time while the products (e.g. beta-carotene, asthaxanthin) are produced during the night.

Solid substrate Fermentation (SSF)

In some biotechnological processes, the growth of the microorganisms is carried out on solid substrates more or less in the absence of free water. Only approximately 15% of moisture is present which is essential for solid-substrate fermentation. Cereal grains, wheat bran, sawdust, wood shavings etc are some of the commonly used solid substrates for SSF. The technique of SSF is used for the production of edible mushrooms, cheese, soy sauce, and many enzymes and organic acids. It is carried out as non-aspetic process and therefore saves sterilization costs. The bioreactors used in this type of fermentation process have simple designs with simple aeration process and effluent treatment. The yield of the products is very high, at low energy expenditure. However, in this process only microorganisms, that can tolerate only low moisture content can be used. It also difficult to monitor O2 and CO2 levels in SSF. The slow growth of microorganisms, also become a limiting factor for product formation.

Working of a bioreactor

In the operation of a bioreactor, there are a few steps that are followed.

a) Sterilization - The most important requirement is to maintain aseptic or sterile conditions for aseptic fermentation. In order to achieve this, the air supplied during fermentation, he growth medium and the bioreactor it self and all it’s accessories are sterilized. There are two methods of sterilization that are followed: (1) In situ sterilization- In this, the bioreactor is filled with the

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required medium followed by injection of pressurized steam into the jacket or coil surrounding the reaction vessel. The whole system is heated to about 1200C and maintained at this temperature for about 20 minutes. However, this method is not energy efficient and prolonged heating destroys the vitamins and precipitates the components of the medium. (2) Continuous heat sterilization - In this, the empty bioreactor is first sterilized by injecting pressurized steam and the medium is rapidly heated to 1400C for a short period again by injecting the pressurized steam. This is an energy efficient method and also does not precipitate the medium components.

b) Aeration - Oxygen is stored in compressed tanks and is introduced at the bottom of the bioreactor through a ‘sparger’. Aeration of the fermentation medium supplies oxygen to the production microorganisms and remove carbon dioxide from the bioreactor. The gases released during the fermentation accumulate in the headspace and then pass out through an air outlet. The headspace is a vacant space on the upper part of the bioreactor and is generally about 20% of it’s volume. The air-lift system of aeration involves sparging of air done at the bottom of the fermenter with an upward flow of air bubbles. The aeration capacity of the system depends on the air-flow rate and the internal pressure. The stirred system of aeration involves increasing the aeration capacity by stirring using impellers driven by motor. The aeration capacity of the fermenter depends on the rate of stirring, rate of air flow and internal pressure.

c) Inoculation and sampling - The sterilized bioreactors with growth medium are inoculated with the production organisms. The size of the inoculum is generally 1-10% of the total volume of the medium. During the fermentation process, the samples are regularly withdrawn to check contamination and to measure the amount and quantity of product formed.

d) Control systems - Various factors like- pH, temperature, dissolved oxygen, adequate mixing, concentration of the nutrients, foam formation etc are continuously monitored to maintain optimal growth environment in the bioreactor. Very sensitive sensors are available which carry out automated monitoring of these variables. The ideal pH range for optimal growth of microorganisms is between 5.5- 8.5. The pH changes due to the release of metabolites into the medium by the growing microorganisms. The required pH level is maintained by adding acid or alkali followed by thorough mixing of the medium components. The optimal temperature is maintained by using the heating and cooling systems fitted in the bioreactor. Continuous monitoring of dissolved oxygen concentration is also a must for the optimal bioreactions. The oxygen is sparingly soluble in water (0.0084g/l at 250C) and is introduced into the bioreactor as the air bubbles.

The concentration of the nutrients is also important because the limiting concentrations of nutrients helps in the optimal product formation and the high nutrient concentrations have inhibitory effect on the microbial growth. Another important factor to control is the “foam formation”. The protein rich media is used in industrial fermentation which leads to ‘froth’ or ‘foam formation’ on agitation during aeration. Some antifoam chemicals lower the surface tension of the medium and causes the foam bubbles to collapse. Mineral oils with silicone or vegetable oils are also used as antifoam agents. The bioreactors can also be fitted with mechanical foam control devices which break the foam bubbles and throw back into the fermentation medium. The proper and continuous mixing of the microbial culture is very

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important to maintain optimal levels of oxygen in the nutrient medium and to prevent the accumulation of toxic metabolic products.

e) Cleaning - After the completion of the fermentation process, the products are ‘harvested’ (removal of contents for processing) and the bioreactor is prepared for the next round of fermentation after cleaning technically referred to as ‘turn around’. The cleaning of the bioreactors is carried out by using high-pressure water jets from the nozzles fitted into the reaction vessel. In order to maintain the cost effectiveness of the bioreactor, the time taken for turn around which is known as ‘down time’, is kept as short as possible.

 

Downstream processing (DSP)

The extraction and purification of a biotechnological product from fermentation is referred to as downstream processing. The methods adopted for downstream processing depends on the nature of the end products, it’s concentration, stability, and the degree of purification required. Both intracellular metabolites as well as extracellular metabolites are isolated by DSP. The intracellular metabolites are products located within the cells e.g. vitamins, enzymes etc. The extracellular metabolites are the products present outside the cells e.g. most antibiotics, amino acids, alcohol, citric acid, enzymes like amylases, proteases etc. some products like vitamin B12, flavomycin etc are present both as intracellular as well as extracellular products.

Downstream processing involves a number of steps which are as follows:

a) Solid-liquid separation - The first step is to separate whole cells and other insoluble substances from the culture broth. This is done by using several methods:

1) Flotation- The process of aeration involves the bubbling of gas in to the liquid broth. The cells and other solid particles get adsorbed on gas bubbles which form a foamy layer which is collected and removed.2) Flocculation - The cells or cellular debris form large aggregates and settle down which can be easily removed. Some flocculating agents like inorganic salt, organic poly-electrolyte, mineral hydrocolloid etc are often used to achieve appropriate flocculation.3) Filtration - this is the most commonly used technique to separate the biomass and culture filtrate. The rate of filtration depends on many factors such as the size of the organism, presence of other organisms, viscosity of the medium, and temperature. Several filters like depth filters, absolute filters, rotary drum vacuum filters, membrane filters etc. are used. There are three major types of filtration processes used depending on the size of the particles- microfiltration, ultrafiltration, and reverse osmosis.4) Centrifugation - The centrifugation is mostly used for separating solid particles from liquid phase. The technique of centrifugation is based on the principle of density differences between the particles to be separated and the medium. In the bioreactors, the continuous flow industrial centrifuges are used where there is a continuous feeding of the slurry and collection of clarified fluid. The solid deposits are intermittently removed. Various models of centrifuges used are:

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Tubular bowl centrifuge, Disc centrifuge, Multichamber centrifuge, Scroll centrifuge or decanter etc.

b) Release of intracellular products - The biotechnological products that are located with in the cells like vitamins, enzymes, etc are released in an active form for their further processing and isolation. The microorganisms or cells can de disintegrated or disrupted by physical, chemical, or enzymatic methods depending on the nature of the cells.

1) Physical methods of cell disruption are (i) Ultrasonication, (ii) Osmotic shock (used for releasing hydrolytic enzymes and binding proteins from gram-negative bacteria), (iii) Heat Shock treatment, (iv) High pressure homogenization, (v) Impingement which involves hitting a stationary surface or a second stream of suspended particles with a stream of suspended cells at high velocity and pressure. Microfluidizer is a device developed on the basis of the principle of impingement. (vi) Grinding with glass beads where the cells mixed with glass beads are subjected to a very high speed in a reaction vessel. The cells break as they are forced against the wall of the vessel by the beads2) Chemical methods- Treatment with alkalies, organic solvents, and detergents lyse the cells to release the content. Alkali treatment is used for the extraction of some bacterial proteins. The organic solvents like methanol, ethanol, isopropanol, butanol etc also disrupt the cells. The organic solvent, toluene, which is commonly used, dissolves membrane phospholipids and creates the membrane pores to release intracellular contents. The ionic detergents denature the membrane proteins and lyse the cells e.g. cationic-cetyl trimethyl ammonium bromide or anionic-sodium lauryl sulfate. Non-ionic detergents are less reactive and also affect the purification steps.3) Enzymatic methods - Lysozyme is the most commonly used enzyme which hydrolyses beta-1,4-glycosidic bonds of the mucopepide in bacterial cell walls e.g. gram positive bacteria. This enzyme is commercially available produced from hen egg white. For gram-negative bacteria, lysozymes are in use with EDTA to break the cells. When the cell wall gets digested by lysozyme, the periplasmic membrane breaks due to osmotic pressure, which releases the intracellular contents. Glucanase and mannanase along with proteases lyse the cell wall of yeast.

c) Concentration - The biological products are concentrated by getting rid of water which is present in the filtrate. Depending on the nature of the desired products and the cost effectiveness, the techniques used to concentrate the biotechnological products are evaporation, liquid-liquid extraction, membrane filtration, precipitation, and adsorption.

(i) Evaporation - The water from the broth is removed by the process of evaporation using evaporators. The evaporators use a heating device which supplies the steam. There is a unit for the separation of concentrated product and vapour and a condenser for condensing vapour. The commonly used evaporators are Plate evaporator, Falling film evaporator, Forced film evaporator, Centrifugal forced film evaporator. (ii) Liquid-Liquid extraction - In liquid-liquid extraction, the biological products are concentrated by transferring the desired product from one liquid phase to another liquid phase. The process of liquid-liquid extraction is categorized as extraction of low molecular weight products and extraction of high molecular weight products.(iii) Membrane filtration - This technique involves the use of a semipermeable membrane that

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selectively retains the particles/molecules that are bigger than the pore size while the smaller molecules pass through the membrane pores. The membranes are made up of polymeric materials such as polyethersulfone and polyvinyl difluoride. Now a days, microfilters and ultrafilters made up of ceramics and steel are being used which are easy to clean and sterilize. Pervaporation is a technique in which the volatile products are separated by a process of permeation through a membrane coupled with evaporation and is used to extract and concentrate volatile products. Perstraction- This technique is used to recover and concentrate hydrophobic compoundsvand is based on the principle of membrane filtration coupled with solvent extraction.(iv) Precipitation - This is the most common method used to concentrate proteins and polysaccharides in industrial processes. The alteration in temperature and in pH, neutral salts, organic solvents, high molecular weight polymers (ionic and non ionic) etc are used in precipitation. Neutral salts like commonly used ammonium sulphate increases the hydrophobic interactions between protein molecules which leads to their precipitation. Ethanol, acetone and propanol are the commonly used organic solvents for protein precipitation which reduce the dielectric constant of the medium and increase the electrostatic interaction between the protein molecules. The precipitation process is carried out below 00C because the proteins get denatured by organic solvents. Polyethylene glycol (PEG) is a high weight non-ionic polymer that also precipitates the proteins by reducing the quantity of water available for protein salvation. In the category of ionic polymers e.g. polyacrylic acid, polyethyleneimine are used which form complexes with oppositely charged protein molecules and neutralize the charges. This leads to the precipitation of proteins. Besides this, the physical factors like increase in temperature, increase in pH, etc also leads to the precipitation of proteins. Besides these, the affinity precipitation (affinity interaction e.g. between antigen and antibody)and precipitation using ligands is also used.

d) Purification – The products of fermentation are purified by using the technique of chromatography. Chromatography consists of a stationary phase and a mobile phase. The porous solid matrix packed in a column constitutes the stationary phase and the mixture of the compound to be separated is loaded on this. The compounds are eluted by a mobile phase. A large number of matrices are commercially available for the purification of proteins e.g. agarose, cellulose, porous silica, cross linked dextran etc. Some of the commonly used chromatography techniques used are: Gel-filtration chromatography- In this technique, the separation of molecules is based on the size, shape and molecular weight using sponge-like gel beads with pores serving as molecular sieves for separation of smaller and bigger molecules. The Ion-exchange chromatography involves the separation of molecules based on their surface charges. It is useful for the purification of antibiotics, besides the purification of proteins. In ion exchange chromatography, the pH of the medium is very crucial because the net charge varies with pH. The ionic bound molecules are eluted from the matrix by changing the pH of the eluant or by increasing the salt concentration. The ion-exchangers are of two types- Cation exchangers- having negatively charged groups like carboxymethyl and sulfonate e.g. Dowex, HCR, Amberlite IR etc. Anion exchangers have positively charged groups like DEAE (diethylaminoethyl) e.g. Dowex SAR, Amberlite IRA etc. The Affinity chromatography is based on an interaction of a protein with an immobilized ligand. The ligand can be a specific antibody, substrate, substrate analogue or an inhibitor. The protein bound to the ligand is eluted out by changing the pH of the buffer, altering the ionic strength or by using another free ligand molecule.

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e) Formulation- The maintenance of activity and stability of biotechnological products during storage and distribution is known as ‘formulation’. As proteins are highly sensitive and lose their biological activity, hence their formulations requires special care. In order to prolong their shelf life, certain stabilizing additives are added e.g. sugars (sucrose, lactose), salts (sodium chloride, ammonium sulphate), polymers (polyethylene glycol) and polyhydric alcohols (glycerol). Proteins are formulated in the form of solutions, suspensions or dry powder. For some small molecules (antibiotics, citric acid), formulation is done by crystallization using salts. ‘Drying’ is an important component of product formulation which involves the transfer of heat to a wet product for removal of moisture. The commercially available dryers in use are- contact, convection and radiation dryers. Spray drying is used for drying large volumes of liquids where small droplets of liquid containing the product are passed through a nozzle directing over a stream of hot gas. After the evaporation of water, the solid particles are left behind.

Freeze drying or lyophilization is the most preferred method for drying and formulation of a wide range of products- pharmaceuticals, food stuff, bacteria, viruses etc. Freeze drying does not cause loss of biological activity of the desired product. Lyophilization is caused due to the sublimation of a liquid from a frozen state. The product with the liquid is frozen and then freeze dried under vacuum. After releasing the vacuum, the product containing vials are sealed

BIOSENSORS

A biosensor is an analytical device for the detection of an analyte that combines a biological component with a physicochemical detector component.

It consists of 3 parts:

the sensitive biological element (biological material (e.g. tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc.), a biologically derived material or biomimic) The sensitive elements can be created by biological engineering.

the transducer or the detector element (works in a physicochemical way; optical, piezoelectric, electrochemical, etc.) that transforms the signal resulting from the interaction of the analyte with the biological element into another signal (i.e., transducers) that can be more easily measured and quantified;

associated electronics or signal processors that are primarily responsible for the display of the results in a user-friendly way. This sometimes accounts for the most expensive part of the sensor device, however it is possible to generate a user friendly display that includes

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transducer and sensitive element.

A common example of a commercial biosensor is the glucose biosensor, which uses the enzyme glucose oxidase to break blood glucose down. In doing so it first oxidizes glucose and uses two electrons to reduce the FAD (a component of the enzyme) to FADH2. This in turn is oxidized by the electrode (accepting two electrons from the electrode) in a number of steps. The resulting current is a measure of the concentration of glucose. In this case, the electrode is the transducer and the enzyme is the biologically active component.

There are many potential applications of biosensors of various types. The main requirements for a biosensor approach to be valuable in terms of research and commercial applications are the identification of a target molecule, availability of a suitable biological recognition element, and the potential for disposable portable detection systems to be preferred to sensitive laboratory-based techniques in some situations.

FERMENTATION PRODUCTS

Since olden times people are using fermented products in their daily food consumption. There are many advantages of fermented foods such as they easily digestible and have improved flavour, texture and nutritive value. Some of the most commonly used fermented products are cheese, bread, yoghurt, sausages, soy sauce etc. With the advances made in microbiology and biotechnology, food and beverage fermentation and production has become a major industry. The food biotechnology has helped in improving the quality, nutrition value, safety and preservation of foods which in turn has helped in making these foods available through out the year.

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YOGHURT

Two species of bacteria Lactobacillus bulgaricusand Lactococcus thermophilus in approximately equal proportions, are used to make yoghurt. Commercial producers pasteurize and homogenize the milk before adding the starter. After stirring, the mixture is then incubated for 3-6 hours at 40-450C. At this temperature the two bacteria have a mutually stimulating effect on one another. Proteolytic enzymes from L. bulgaricus break down milk proteins into peptides. These stimulate the growth of L. thermophilus which, in turn, produce formic acid and carbon dioxide, growth stimulants for L. bulgaricus. As the incubation proceeds, L. bulgaricus converts the lactose to lactic acid and the pH falls to 4.2-4.4 which leads to the coagulation of proteins by lactic acid and the thickening of the yoghurt. Further processing involves the addition of flavour, colour, fruit pulp and heat treatment to kill off any bacteria. Typical products

(a) Sweetened and flavored yoghurt

To offset its natural sourness, yoghurt can be sold sweetened, flavored or in containers with fruit or fruit jam

(b) Strained yoghurts

Strained yoghurts are types of yoghurt which are strained through a paper or cloth filter, traditionally made of muslin, to remove the whey, giving a much thicker consistency and a distinctive, slightly tangy taste.

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Metabolic products of bacteria used in yoghurt making

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Stages in the manufacture of processed yoghurt

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WINE PRODUCTION

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The process of fermentation in wine is the catalyst function that turns grape juice into an alcoholic beverage. During fermentation yeast interact with sugars in the juice to create ethanol, commonly known as ethyl alcohol, and carbon dioxide (as a by-product. Fermentation may be done in stainless steel tanks, which is common with many white wines, in an open wooden vat, inside a wine barrel and inside the wine bottle itself as in the production of many sparkling wines.

Process

The most common species of wine grape is Vitis vinifera, which includes nearly all varieties of European origin. Red wine is made from the must (pulp) of red or black grapes that undergo fermentation together with the grape skins. White wine is made by fermenting juice which is made by pressing crushed grapes to extract a juice; the skins are removed and play no further role. To start primary fermentation yeast is added to the must for red wine or juice for white wine. Cultured yeasts most commonly used in winemaking belong to the Saccharomyces cerevisiae (also known as "sugar yeast") species. Within this species are several hundred

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different strains of yeast that can be used during fermentation to affect the heat or vigor of the process and enhance or suppress certain flavor characteristics of the varietal. During this fermentation, which often takes between one and two weeks, the yeast converts most of the sugars in the grape juice into ethanol (alcohol) and carbon dioxide. . Upon the introduction of active yeasts to the grape must, phosphates are attached to the sugar and the six-carbon sugar molecules begin to be split into three-carbon pieces and go through a series of rearrangement reactions. During this process the carboxylic carbon atom is released in the form of carbon dioxide with the remaining components becoming acetaldehyde. The absence of oxygen in this anaerobic process allows the acetaldehyde to be eventually converted, by reduction, to ethanol. During the conversion of acetaldehyde a small amount is converted, by oxidation, to acetic acid which, in excess, can contribute to the wine fault known as volatile acidity (vinegar taint). After the yeast has exhausted its life cycle, they fall to the bottom of the fermentation tank as sediment. The carbon dioxide is lost to the atmosphere.

After the primary fermentation of red grapes the free run wine is pumped off into tanks and the skins are pressed to extract the remaining juice and wine, the press wine blended with the free run wine at the wine makers discretion. The wine is kept warm and the remaining sugars are converted into alcohol and carbon dioxide. The next process in the making of red wine is secondary fermentation. This is a bacterial fermentation which converts malic acid to lactic acid. This process decreases the acid in the wine and softens the taste of the wine. Red wine is sometimes transferred to oak barrels to mature for a period of weeks or months, this practice imparts oak aromas to the wine. The wine must be settled or clarified and adjustments made prior to filtration and bottling.

Conditions

During fermentation there are several factors that winemakers take into consideration. The most notable is that of the internal temperature of the must. Typically white wine is fermented between 18-20 °C though a wine maker may choose to use a higher temperature to bring out some of the complexity of the wine. Red wine is typically fermented at higher temperatures up to 29 °C. For every gram of sugar that is converted, about half a gram of alcohol is produced, so to achieve a 12% alcohol concentration, the must should contain about 24% sugars. Sulfur dioxide has two primary actions, firstly it is an anti microbial agent and secondly an anti oxidant. In the making of white wine it can be added prior to fermentation and immediately after alcoholic fermentation is complete. If added after alcoholic ferment it will have the effect of preventing or stopping malolactic fermentation, bacterial spoilage and help protect against the damaging effects of oxygen.

Clarification

The clarification and stabilization of wine in winemaking involves removing insoluble and suspended materials that may cause a wine to become cloudy, gassy, form unwanted sediment deposit or tartaric crystals, deteriorate quicker or develop assorted wine faults due to physical,

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chemical or microbiological instability. These processes may include fining, filtration, centrifugation, flotation, refrigeration, barrel maturation, pasteurization and racking. Most of these processes will occur after the primary fermentation and before the wine is bottled. The exception is for white wine production which will usually have the must separated from some of the grape skins and particles prior to fermentation so as to avoid any unwanted maceration. Some of the materials that are removed from the must during this stage of winemaking include dead yeast cells (lees), bacteria, tartrates, proteins, pectins, various tannins and other phenolic compounds, and pieces of grape skins, pulp, stems and gums.

Stabilization

Tartaric acid is the most prominent acid in wine with the majority of the concentration present as potassium acid salt. The crystallization of these tartrates can happen at unpredictable times if the wine is exposed to low temperature. To prevent this from happening after the wine has been bottled, winemakers stabilize the wine by putting it through a cold stabilization process where it is exposed to temperatures below freezing to encourage the tartrates to crystallize and precipitate out of the wine. During the cold stabilizing process after fermentation, the temperature of the wine is dropped to close to freezing for 1–2 weeks. This will cause the crystals to separate from the wine and stick to the sides of the holding vessel. When the wine is drained from the vessels, the tartrates are left behind

Filtration

Filtration in winemaking is used to accomplish two objectives, clarification and microbial stabilization. While fining clarifies wine by binding to suspended particles and precipitating out as larger particles, filtration works by passing the wine through a filter medium that captures particles that are larger than the hole size of the medium. Most filtration in a winery can be classified as either depth filtration or surface filtration.

Depth filtration is often the first type of filtration a wine sees after fermentation when the wine is pushed through a thick layer of pads made from cellulose fibers, diatomaceous earth or perlite which traps the particles and can be removed from the wine. Surface filtration involves running the wine along a thin film of polymer material filled with holes tinier than the particles that are being filtered out.

Fining

In winemaking, fining is the process where a substance (fining agent) is added to the wine to create an adsorbent, enzymatic or ionic bond with the suspended particles, making them a larger molecule that can precipitate out of the wine easier and quicker. The most common organic compounds used include egg whites, casein derived from milk, gelatin and isinglass obtained from the bladders of fish. Pulverized minerals and solid materials can also be used as fining agents with bentonite clay being one of the most common fining agent used due to its effectiveness in absorbing proteins and some bacteria. Activated carbon derived from charcoal is used to remove some phenols that contribute to browning colors as well as some particles that

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produce "off-odors" in the wine.[5] In a process known as blue fining, potassium ferrocyanide is used to remove copper and iron particles that may have entered the wine through the use of metal winery and vineyard equipment, vineyard sprays such as the bordeaux mixture, and the use of bentonite as a fining agent

Bottling

A final dose of sulfite is added to help preserve the wine and prevent unwanted fermentation in the bottle. The wine bottles then are traditionally sealed with a cork, although alternative wine closures such as synthetic corks and screwcaps, which are less subject to cork taint, are becoming increasingly popular. The final step is adding a capsule to the top of the bottle which is then heated for a tight seal.

There are four main methods of sparkling wine production. The first is simple injection of carbon dioxide (CO2), the process used in soft drinks, but this produces big bubbles that dissipate quickly in the glass. The second is the Metodo Italiano - Charmat process, in which the wine undergoes a secondary fermentation in bulk tanks, and is bottled under pressure. The third method is the traditional method or méthode champenoise. With this method the bubbles for more complex wines are produced by secondary fermentation in the bottle. As the name suggests, this is used for the production of Champagne and other quality sparkling wines, but is slightly more expensive than the Charmat process. The fourth method is the "transfer method". This method will take the cuvee to bottle for secondary fermentation, which allows for the additional complexity, but then will transfer the wine out of the individual bottles into a larger tank after it has spent the desired amount of time on yeast.

VINEGAR PRODUCTION

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Oxidative fermentation

Vinegar is produced by a second fermentation of beer, cider, or wine. The second fermentation is aerobic and dependent on a mixed culture of Acetobacter , including A. scheutzenbachii, A. curium, and A. orleanse. In the presence of oxygen, these bacteria convert alcohol to acetic acid by the following route:

Alcohol (ethanol)-------› acetaldehyde------› acetic acid

Given sufficient oxygen, these bacteria can produce vinegar from a variety of alcoholic foodstuffs. Commonly used feeds include apple cider, wine, and fermented grain, malt, rice, or potato mashes. The overall chemical reaction facilitated by these bacteria is:

C2H5OH + O2 → CH3COOH + H2O

One of the first modern commercial processes was the "fast method" or "German method", first practised in Germany in 1823. In this process, fermentation takes place in a tower packed with wood shavings or charcoal. The alcohol-containing feed is trickled into the top of the tower, and fresh air supplied from the bottom by either natural or forced convection. The improved air supply in this process cut the time to prepare vinegar from months to weeks.

Nowadays, most vinegar is made in submerged tank culture, first described in 1949 by Otto Hromatka and Heinrich Ebner. In this method, alcohol is fermented to vinegar in a continuously stirred tank, and oxygen is supplied by bubbling air through the solution. Using modern applications of this method, vinegar of 15% acetic acid can be prepared in only 24 hours in batch process, even 20% in 60-hour fed-batch process.

Anaerobic fermentation

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Species of anaerobic bacteria, including members of the genus Clostridium, can convert sugars to acetic acid directly, without using ethanol as an intermediate. The overall chemical reaction conducted by these bacteria may be represented as:

C6H12O6 → 3 CH3COOH

It is interesting to note that, from the point of view of an industrial chemist, these acetogenic bacteria can produce acetic acid from one-carbon compounds, including methanol, carbon monoxide, or a mixture of carbon dioxide and hydrogen:

2 CO2 + 4 H2 → CH3COOH + 2 H2O

This ability of Clostridium to utilize sugars directly, or to produce acetic acid from less costly inputs, means that these bacteria could potentially produce acetic acid more efficiently than ethanol-oxidizers like Acetobacter. However, Clostridium bacteria are less acid-tolerant than Acetobacter. Even the most acid-tolerant Clostridium strains can produce vinegar of only a few per cent acetic acid, compared to Acetobacter strains that can produce vinegar of up to 20% acetic acid. At present, it remains more cost-effective to produce vinegar using Acetobacter than to produce it using Clostridium and then concentrate it

Vinegar

In the form of vinegar, acetic acid solutions (typically 4% to 18% acetic acid, with the percentage usually calculated by mass) are used directly as a condiment( A condiment is sauce, or seasoning added to food to impart a particular flavor or to complement the dish. Often pungent in flavour and therefore added in fairly small quantities), and also in the pickling of vegetables and other foods. Table vinegar tends to be more diluted (4% to 8% acetic acid), while commercial food pickling, in general, employs more concentrated solutions. The amount of acetic acid used as vinegar on a worldwide scale is not large, but is by far the oldest and best-known application.

BEER

The basic ingredients of beer are water; a starch source, such as malted barley, able to be fermented (converted into alcohol); a brewer's yeast to produce the fermentation; and a flavouring such as hops. A mixture of starch sources may be used, with a secondary starch source, such as maize (corn), rice or sugar, often being termed an adjunct, especially when used as a lower-cost substitute for malted barley. Less widely used starch sources include millet, sorghum and cassava root

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PROCESS

The purpose of brewing is to convert the starch source into a sugary liquid called wort and to convert the wort into the alcoholic beverage known as beer in a fermentation process effected by yeast.

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All beers are brewed using a process based on a simple formula. Key to the process is malted grain— mainly barley, though other cereals, such as wheat or rice, may be added. Malt is made by allowing a grain to germinate, after which it is then dried in a kiln and sometimes roasted. The germination process creates a number of enzymes, notably α-amylase and β-amylase, which convert the starch in the grain into sugar. Depending on the amount of roasting, the malt will take on a dark colour and strongly influence the colour and flavour of the beer. The malt is crushed to break apart the grain kernels, expose the cotyledon which contains the majority of the carbohydrates and sugars, increase their surface area, and separate the smaller pieces from the husks.

Malting is the process where the barley grain is made ready for brewing. Malting is broken down into three steps, which help to release the starches in the barley. First, during steeping, the grain is added to a vat with water and allowed to soak for approximately 40 hours. During germination, the grain is spread out on the floor of the germination room for around 5 days. The goal of germination is to allow the starches in the barley grain to breakdown into shorter lengths. When this step is complete, the grain is referred to as green malt. The final part of malting is kilning. Here, the green malt goes through a very high temperature drying in a kiln. The temperature change is gradual so as not to disturb or damage the enzymes in the grain. When kilning is complete, there is a finished malt as a product.

The next step in the brewing process is milling. This is when the grains that are going to be used in a batch of beer are cracked. Milling the grains makes it easier for them to absorb the water that they are mixed with and which extracts sugars from the malt. Milling can also influence the general characteristics of a beer.

Mashing is the next step in the process. This process converts the starches released during the malting stage, into sugars that can be fermented. The milled grain is dropped into hot water in a

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large vessel known as a mash tun. In this vessel, the grain and water are mixed together to create a cereal mash. The leftover sugar rich water is then strained through the bottom of the mash in a process known as Prior to lautering, the mash temperature may be raised to about 75 °C (known as a mashout) to deactivate enzymes. Additional water may be sprinkled on the grains to extract additional sugars (a process known as sparging).

At this point the liquid is known as wort. The wort is moved into a large tank known as a "copper" or kettle where it is boiled with hops and sometimes other ingredients such as herbs or sugars. The boiling process serves to terminate enzymatic processes, precipitate proteins, isomerize hop resins, concentrate and sterilize the wort.

Hops are added during boiling as a source of bitterness, flavour and aroma. The flower of the hop vine is used as a flavouring and preservative agent in nearly all beer made today. The flowers themselves are often called "hops". Hops may be added at more than one point during the boil. The longer the hops are boiled, the more bitterness they contribute, but the less hop flavour and aroma remains in the beer.

After boiling, the hopped wort is now cooled, ready for the yeast. In some breweries, the hopped wort may pass through a hopback, which is a small vat filled with hops, to add aromatic hop flavouring and to act as a filter; but usually the hopped wort is simply cooled for the fermenter, where the yeast is added . A type of yeast is selected and added, or "pitched", to the fermentation tank. Yeast metabolises the sugars extracted from grains, which produces alcohol and carbon dioxide, and thereby turns wort into beer. In addition to fermenting the beer, yeast influences the character and flavour. The dominant types of yeast used to make beer are ale yeast (Saccharomyces cerevisiae) ‘top fermenting yeast’ and lager yeast (Saccharomyces uvarum) An example of bottom cropping yeast is Saccharomyces pastorianus, formerly known as Saccharomyces carlsbergensis. The most common method of categorising beer is by the behaviour of the yeast used in the fermentation process. Beers using a fast acting warm fermentation which leaves behind residual sugars are termed "ales", while beers using a slower-acting cool fermentation, with a yeast which removes most of the sugars, producing a clean, dry beer, are termed "lagers". Ale is typically fermented at temperatures between 15 and 24°C. Lager yeast is a cool bottom-fermenting yeast (Saccharomyces pastorianus) and typically undergoes primary fermentation at 7–12 °C (the fermentation phase), and then is given a long secondary fermentation at 0–4 °C (the lagering phase). During the secondary stage, the lager clears and mellows. The cooler conditions also inhibit the natural production of esters and other byproducts, resulting in a "cleaner"-tasting beer.

The second to last stage in the brewing process is called racking. This is when the brewer racks the beer into a new tank, called a conditioning tank. Conditioning of the beer is the process in which the beer ages, the flavour becomes smoother, and flavours that are unwanted dissipate. Fermentation is sometimes carried out in two stages, primary and secondary. Once most of the alcohol has been produced during primary fermentation, the beer is transferred to a new vessel and allowed a period of secondary fermentation. Secondary fermentation is used when the beer requires long storage before packaging or greater clarity.

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After one to three weeks, the fresh (or "green") beer is run off into conditioning tanks. After conditioning for a week to several months, the beer enters the finishing stage. Here, beers that require filtration are filtered, and given their natural polish and colour. Filtration also helps to stabilize the flavour of the beer. Filtering the beer stabilizes the flavour, and gives beer its polished shine and brilliance. Some brewers add one or more clarifying agents to beer, which typically precipitate (collect as a solid) out of the beer along with protein solids and are found only in trace amounts in the finished product. This process makes the beer appear bright and clean. Examples of clarifying agents include isinglass, obtained from swimbladders of fish; Irish moss, a seaweed; kappa carrageenan, from the seaweed Kappaphycus cottonii; Polyclar (artificial); and gelatin. After the beer is filtered, it undergoes carbonation, and is then moved to a holding tank until bottling.

Some beers undergo a fermentation in the bottle, giving natural carbonation. When the beer has fermented, it is packaged either into casks for cask ale or kegs, aluminium cans, or bottles for other sorts of beer

Cheese Production

Cheese is made from the casein of milk that is produced after separating the whey –the liquid portion of the milk. The bacteria used in cheese making are either gas producers or acid producers. Gas producers release carbon dioxide, while the acid producers form lactic acid from lactose. It is the gas producers that determine the texture of a cheese and the acid producers determine the flavour. The cheese production involves the following steps:a) Acidification of milkb) Coagulum formationc) Separation of curd from wheyd) Ripening of cheese.

Cheddar cheese is made from milk sterilized at 720C for 15 secs. A starter consisting of Streptococcus lactis is added and the milk is left to ripen for an hour. During the “ripening” the lactic acid content rises after which the milk is subjected to ‘renneting’. Rennet is a mixture of

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chymosin and pepsin from the stomach of a calf which coagulates the casein, the principal milk protein. There are several sources of rennet for cheese production. These include calves, adult cows, pigs, and fungal sources. Using genetic engineering, some workers have cloned the genes of animal chymosin and transfer the same into microorganisms. After renneting, a semi solid mass or ‘coagulum’ is formed consisting of water, fat and solutes trapped in a casein matrix.The coagulum is cut into pea-sized pieces to separate it into small, creamy particles of curd suspended in a watery whey. ‘Scalding’ the mixture at 30-390C for 45 minutes is done to expel more whey and to change the texture of the curd. After the scalding, the curd is allowed to settle under gravity or ‘pitch’ and the whey is run off. After the formation of blocks of curd, the blocks are cut, stacked, drained and turned in a process called ‘cheddaring’. Following the cheddaring, the pH falls to 5.2 and the curd is ‘milled’ in to small pieces. In the final stages of preparation, salt is added which helps to preserve the finished cheese and bring out it’s flavour. ‘Ripening’ consists of storing the cheese under appropriate conditions so that bacteria and other microorganisms can cause chemical changes in the curd, improving and enhancing its flavour.

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An outline of various stages in the manufacture of Cheddar cheese

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YEAST PRODUCTION

Yeasts can grow in the presence or absence of air. Anaerobic growth, growth in the absence of oxygen, is quite slow and inefficient. For instance, in bread dough, yeast grow very little. Instead, the sugar that can sustain either fermentation or growth is used mainly to produce alcohol and carbon dioxide. Only a small portion of the sugar is used for cell maintenance and growth. In contrast, under aerobic conditions, in the presence of a sufficient quantity of dissolved oxygen, yeast grow by using most of the available sugar for growth and producing only negligible quantities of alcohol. This means that the baker who is interested in the leavening action of carbon dioxide works under conditions that minimize the presence of dissolved oxygen. On the other hand, a yeast manufacturer that wants to produce more yeast cell mass, works under aerobic conditions by bubbling air through the solution in which the yeast is grown.

The problem posed to the yeast manufacturer, however, is not as simple as just adding air during the fermentation process. If the concentration of sugar in the fermentation growth media is greater than a very small amount, the yeast will produce some alcohol even if the supply of oxygen is adequate or even in abundance. This problem can be solved by adding the sugar solution slowly to the yeast throughout the fermentation process. The rate of addition of the sugar solution must be such that the yeast uses the sugar fast enough so that the sugar concentration at any one time is practically zero. This type of fermentation is referred to as a fed-batch fermentation.

The baker’s yeast production process flow chart can be divide into four basic steps. In order these steps are, molasses and other raw material preparation, culture or seed yeast preparation, The baker’s yeast production process flow chart can be fermentation and harvesting and filtration and packaging. The process takes approximately five days from start to finish.

The basic carbon and energy source for yeast growth are sugars. Starch can not be used because yeast does not contain the appropriate enzymes to hydrolyze this substrate to fermentable sugars. Beet and cane molasses are commonly used as raw material because the sugars present in molasses, a mixture of sucrose, fructose and glucose, are readily fermentable. In addition to sugar, yeast also require certain minerals, vitamins and salts for growth. Some of these can be added to the blend of beet and cane molasses prior to flash sterilization while others are fed separately to the fermentation. Alternatively, a separate nutrient feed tank can be used to mix and deliver some of the necessary vitamins and minerals. Required nitrogen is supplied in the form of ammonia and phosphate is supplied in the form of phosphoric acid. Each of these nutrients is fed separately to the fermentation to permit better pH control of the process. The sterilized molasses, commonly referred to as mash or wort, is stored in a separate stainless steel tank. The mash

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stored in this tank is then used to feed sugar and other nutrients to the appropriate fermentation

Baker’s yeast production starts with a pure culture tube or frozen vial of the appropriate yeast strain. This yeast serves as the inoculum for the pre-pure culture tank, a small pressure vessel where seed is grown in medium under strict sterile conditions. Following growth, the contents of this vessel are transferred to a larger pure culture fermentor where propagation is carried out with some aeration, again under sterile conditions. These early stages are conducted as set-batch fermentations. In a set-batch fermentation all the growth media and nutrients are introduced to the tank prior to inoculation.

From the pure culture vessel, the grown cells are transferred to a series of progressively larger seed and semi-seed fermentors. These later stages are conducted as fed-batch fermentations. During a fed-batch fermentation, molasses, phosphoric acid, ammonia and minerals are fed to the yeast at a controlled rate. This rate is designed to feed just enough sugar and nutrients to the yeast to maximize multiplication and prevent the production of alcohol. In addition, these fed-batch fermentations are not completely sterile. It is not economical to use pressurized tanks to guarantee sterility of the large volumes of air required in these fermentors or to achieve sterile conditions during all the transfers through the many pipes, pumps and centrifuges. Extensive cleaning of the equipment, steaming of pipes and tanks and filtering of the air is practiced to insure as aseptic conditions as possible.

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At the end of the semi-seed fermentation, the contents of the vessel are pumped to a series of separators that separate the yeast from the spent molasses. The yeast is then washed with cold water and pumped to a semi-seed yeast storage tank where the yeast cream is held at 34 degrees Fahrenheit until it is used to inoculate the commercial fermentation tanks. These commercial fermentors are the final step in the fermentation process and are often referred to as the final or trade fermentation. Commercial fermentations are carried out in large fermentors with working volumes up to 50,000 gallons. To start the commercial fermentation, a volume of water, referred to as set water, is pumped into the fermentor. Next, in a process referred to as pitching, semi-seed yeast from the storage tank is transferred into the fermentor. Following addition of the seed yeast, aeration, cooling and nutrient additions are started to begin the 15-20 hour fermentation. At the start of the fermentation, the liquid seed yeast and additional water may occupy only about one-third to one-half of the fermentor volume. Constant additions of nutrients during the course of fermentation bring the fermentor to its final volume. The rate of nutrient addition increases throughout the fermentation because more nutrients have to be supplied to support growth of the increasing cell population. The number of yeast cells increase about five- to eight-fold during this fermentation.

Air is provided to the fermentor through a series of perforated tubes located at the bottom of the vessel. The rate of airflow is about one volume of air per fermentor volume per minute. A large amount of heat is generated during yeast growth and cooling is accomplished by internal cooling coils or by pumping the fermentation liquid, also known as broth, through an external heat exchanger. The addition of nutrients and regulation of pH, temperature and airflow are carefully monitored and controlled by computer systems during the entire production process. Throughout the fermentation, the temperature is kept at approximately 86 degrees Fahrenheit and the pH in the range of 4.5-5.5.

At the end of fermentation, the fermentor broth is separated by nozzle-type centrifuges, washed with water and re-centrifuged to yield a yeast cream with a solids concentration of approximately 18%. The yeast cream is cooled to about 45 degrees Fahrenheit and stored in a separate, refrigerated stainless steel cream tank. Cream yeast can be loaded directly into tanker trucks and delivered to customers equipped with an appropriate cream yeast handling system. Alternatively, the yeast cream can be pumped to a plate and frame filter press and dewatered to a cake-like consistency with a 30-32% yeast solids content. This press cake yeast is crumbled into pieces and packed into 50-pound bags that are stacked on a pallet. The yeast heats up during the pressing and packaging operations and the bags of crumbled yeast must be cooled in a refrigerator for a period of time with adequate ventilation and placement of pallets to permit free access to the cooling air. Palletized bags of crumbled yeast are then distributed to customers in refrigerated trucks.

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Types of baker's yeast

Baker's yeast is available in a number of different forms, the main differences being the moisture contents. Though each version has certain advantages over the others, the choice of which form to use is largely a question of the requirements of the recipe at hand and the training of the cook preparing it. Dry yeast forms are good choices for longer-term storage, often lasting several months at room temperatures without significant loss of viability.With occasional allowances for liquid content and temperature, the different forms of commercial yeast are generally considered interchangeable.

Cream yeast is essentially a suspension of yeast cells in liquid, siphoned off from the growth medium. Its primary use is in industrial bakeries with special high-volume dispensing and mixing equipment, and it is not readily available to small bakeries or home cooks.

Compressed yeast is essentially cream yeast with most of the liquid removed. It is a soft solid, beige in color, and arguably best known in the consumer form as small, foil-wrapped cubes of cake yeast. It is also available in larger-block form for bulk usage. It is highly perishable; though formerly widely available for the consumer market, it has become less common in supermarkets in some countries due to its poor keeping properties, having been superseded in some such markets by active dry and instant yeast. It is still widely available for commercial use, and is somewhat more tolerant of low temperatures than other forms of commercial yeast; however, even there, instant yeast has made significant market inroads

Active dry yeast is the form of yeast most commonly available to noncommercial bakers. It consists of coarse oblong granules of yeast, with live yeast cells encapsulated in a thick jacket of dry, dead cells with some growth medium. Under most conditions, active dry yeast must first be proofed or rehydrated. It can be stored at room temperature for a

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year, or frozen for more than a decade, which means that it has better keeping qualities than other forms, but it is generally considered more sensitive than other forms to thermal shock when actually used in recipes.

Instant yeast appears similar to active dry yeast, but has smaller granules with substantially higher percentages of live cells per comparable unit volumes. It is more perishable than active dry yeast, but also does not require rehydration, and can usually be added directly to all but the driest doughs. Instant yeast generally has a small amount of ascorbic acid added as a preservative.

Rapid-rise yeast is a variety of dried yeast (usually a form of instant yeast) that is of a smaller granular size, thus it dissolves faster in dough, and it provides greater carbon dioxide output to allow faster rising

CITRIC ACID

Citric acid is the product of fermentation of Aspegillus niger. The acid is one of the principal organic acids produced in the citric acid cycle. During the production of CA, the activity of the condensing enzyme (operating in the condensation of acetyl CoA and oxaloacetic acid to citric acid) is increased, while the activities of the isocitrate dehydrogense and acotinase disappear. The enzyme acotinase is responsible for the control of biosynthesis of isocitric acid from citric acid and in turn isocitrate dehydrogenase mediates in the hydrogen removal which yield axalosuccinic acid from isocitric acid. Inactivity of these enzymes is the reason for CA accumulation.

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Culture conditions which also lead to accumulation of CA are:

1. Sugar type and concentration- only sugars which are rapidly catabolised by the fungus such as sucrose, maltose or glucose, allow high yields as well as high rates of acid accumulation

2. Trace metal ions- while all usual trace metal ions are essential for A. niger, some of them particularly Mn2+, Fe2+, Zn2+ have to be present in the medium at growth limiting concentrations to give high citric acid yields

3. pH –citric acid accumulation has been to accumulate in significant amounts only when the pH is below 2.5. pKa for CA is 1.8

4. Dissolved oxygen tension higher than that required for vegetative growth is essential and even sparging with pure oxygen can be useful

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5. Nitrogen- nitrogen sources used in medium for Ca production include ammonium salts, nitrates and the potential ammonia source, urea under special conditions. In continuous culture the N must be limiting for attaining highest CA yields

6. Phosphates- not very critical but appropriate balance is essential

PRODUCTION OF CITRIC ACID

There are two different types of fermentations carried out for the production of citric acid:

1. The surface process2. The submerged process

1.The surface process

The fermentation is carried out in aluminum trays, filled with nutrient medium to a depth of between 50-200 cm. Spores are distributed over the surface of the trays and sterile air is passed over them. The mycelium grows over a period of 7-15 days.

2.Submerged method

A niger may be cultured as submerged mycelium in aerated vats i.e. CSTR. The mycelium germinates to form stubby. Forked and bulbous hyphae, which aggregate to small pellets, which have a firm, smooth surface and sediment quickly when harvested.

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Beet molasses have been widely used in the pan techniques, although pure sucrose and syrups have been use in submerged cultures in vats. Metallic cations can be removed from the carbohydrates using cation-exchange process. The carbohydrate is diluted to a concentration of about 20% to 25%. This high sugar concentration inhibits formation of acids other than citric acid. The manufacture of citric acids needs a low pH and HCl as added to reduce the medium to range of pH 2-5 when fungal spore in the inoculum germinate. In the vat, pH is kept below 3.5. The prepared medium is added with spores from stock cultures. Highly aerobic conditions are needed and submerged cultures are aerated with sterile air and agitated.

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The temperature must be maintained in the range of 20-30oC during the incubation period (7-10d). Lime is added during harvesting, to the culture medium to precipitate any oxalic acid which formed and the mycelium and calcium citrate are filtered off. The recovery of CA from the broth is accompanied by three steps

1. Precipitation- a slurry of calcium citrate, which precipitates out is filtered and the treated with sulphuric acid which precipitates the calcium

2. Extraction- solvent extraction, which makes the use of various aliphatic alcohols, ketones, amines, etc

3. Ion exchange- the dilute CA is purified by treatment with Carbon and is demineralised by successive passage through cation and anion exchange resins

This purified CA is the evaporated; crystallization is performed in the crystallizers. Citric acid monohydrate (CAM) is formed below 36oC and citric acid anhydride is formed at higher temperature

L-GLUTAMATE PRODUCTION

FERMENTATION METHOD

The fermentation method is a production process in which a specific amino acid is synthesized in large amounts by a specially selected microorganism in culture. The selected microorganism is cultured with carbohydrates and ammonia and releases the l-form of the amino acid into the culture medium. The cell produces glutamate from 2-oxo-glutarate (2-oxo-pentanedioic acid) by reductive ammonia fixation that uses the enzyme glutamate dehydrogenase, a normal cellular constituent. Many bacteria useful in glutamate production have been isolated, including Corynebacterium glutamicum, Brevibacterium lactofermentum, and Brevibacterium flavum. These glutamate-producing bacteria are all coryneform bacteria, which are gram positive, non-spore-forming, and nonmotile and require biotin for growth. Glutamate accumulation in the medium occurs only under biotin-limiting conditions. The requirement for biotin limitation prevented the use of standard raw materials such as sugar molasses because they contained biotin. Significant efforts were thus expended to overcome this difficulty. Ultimately, methods were discovered, such as

(i) the addition of a surfactant or of penicillin (ii) the use of microorganisms auxotrophic for glycerol or oleate, that allowed the

bacteria to produce large amounts of glutamate without biotin limitation. (iii) Addition of local anasthetics

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The industrial production of MSG using fermentation technology continues to improve in terms of conversion yield from sugar to glutamate and in production speed of the fermentation. Fermentation allows the isolation of l-glutamate to be a simple process because the cells produce the l-isomer. To improve MSG purity, a new method for purifying l-glutamic acid crystals was developed, which uses recrystallization of the β-form and subsequent conversion to MSG. The mother liquor of the crystallization process is then concentrated and used as a liquid fertilizer (after pH adjustment with ammonia). The invention of the fermentation method has dramatically improved glutamate production methods and allowed production to keep up with demand for the product.

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PRODUCTION OF EDIBLE MUSHROOMS

Mushrooms are fungi (class- basidiomycetes and ascomycetes) which can be cultivated on large scale for human consumption. Out of 4000 species, 200 are edible out of which only 12 species are cultivated on large scale. Some of the common edible mushroom varieties are- Agaricus bisporus (button mushroom), Pleurotus ostreatus (Oyster mushroom), Volvariella volvacea (Chinese mushroom). The edible mushrooms contain 35-45% protein, 7-10% fats and free fatty acids, 5-15% carbohydrates and minerals in good concentration. Therefore, edible mushrooms are also called vegetable meat. Edible mushroom cultivation has become one of the examples where microbial culture is being used for direct human consumption. Cultivation of edible mushrooms has become one of the most important and profitable biotechnological industry all around the world. Mushrooms have a short life 8-12 hours and should be therefore stored at low temperatures of 2-50C. Some of the mushrooms are cultivated in the summer and rainy season such as Volirariella sp. Agaricus bisporus and Pleurotus sp grow well in winters.

Process of mushroom production

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The production of mushroom involves solid-substrate fermentation which is basically carried out by using substrates like straw, sawdust, compost, wooden logs etc. The compost with desired formulation is prepared and sterilized and then spread into trays which are then transferred to production room. This is followed by inoculation with spawn. ‘Spawn’ is the term used for the mushroom inoculums containing spores and/or small pieces of fruiting body. The culture is maintained at optimal growth conditions of temperature 150C and pH of 7.0. The humidity is maintained at 70-80% by watering the trays regularly. A single crop of mushroom is ready in 7-10 days. It is possible to have 3-4 crops before terminating the production process.

The outline of edible mushroom production

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