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Saturday, August 25, 2012

Biogas (Methane) Production, Biogas Plant, Advantages


When methane is produced by the fermentation of animal dung the gaseous products are usually referred as biogas and the installations are called biogas plant or bioreactor. Biogas is a flammable mixture of 50-80% methane, 15-45% CO2, 5% water and some other trace gases.Biogas is produced by biomethenation and is self regulating symbiotic microbial process operating under anaerobic conditions and functions at temperature around 30oC.

Organisms involved are all found naturally in ruminant manures. In such system the animal dung is mixed with water and allowed to ferment in near anaerobic conditions, under ideal condition 10Kg of dry organic matter can produce 3m3 of biogas, which will provide 3hrs of cooking, 3hrs of lighting and 24hrs of refrigeration with suitable equipment.

Biogas Production Overview
biogas plant, biogas technology

Methane as an energy source may have economic value at local small scale production, but there is considerable doubt about the future of commercial large scale process for methane production.
  • An abundance of methane occurs in nature, particularly in natural gas fields and oil field overlays.
  • Methane production by gasification of coal is commercially more attractive.
  • Microbial production of methane is more expensive than natural gas.
  • Costs of storage, transport and distribution of gaseous fuels is not economical.
  • Methane cant be used in automobiles.
Production of Biogas

Production of Biogas

  • Biogas is a mixture of gases produced from the anaerobic digestion of waste materials such as animal & plant waste.
  • A biogas plant which uses only cow dung is called as the"gobar gas plant".
  • The gas is used as fuel for cooking or lighting.
  • Microorganisms involved in biogas production are a group of different Sps. which forms a consortium.
  • Bacteria involved in the initial stages are not strict anaerobes.
Anaerobic Digestion is accomplished by three stages
  1. Solubilization
  2. Acidogenesis
  3. Methanogenesis.
stages of biogas production process

  1. Anaerobic digestion of municipal industrial and agricultural wastes can have positive environmental values, since it can combine waste removal, stabilization and net yield of biogas formation.
  2. Solid or liquid residues can be used as fertilizer, soil conditioner or animal feed. hence biomass production continues to have high priority in alternative energy research

Tuesday, August 14, 2012

Strain Improvement - Methods and Applications

Several options are open to an industrial microbiology organization seeking to maximize its profits in the face of its competitors‘ race for the same market. The organization may undertake more aggressive marketing tactics, including more attractive packaging while leaving its technical procedures unchanged. It may use its human resources more efficiently and hence reduce costs, or it may adopt a more efficient extraction system for obtaining the material from the fermentation broth.

The operations in the fermentor may also be improved by its use of a more productive medium, better environmental conditions, better engineering control of the fermentor processes, or it may genetically improve the productivity of the microbial strain it is using. Of all the above options, strain improvement appears to be the one single factor with the greatest potential for contributing to  greater profitability. While realizing the
importance of strain improvement, it must be borne in mind that an improved strain could bring with it previously non-existent problems.

For example, amore highly yielding strain may require greater aeration or need more intensive foam control; the products may pose new extraction challenges, or may even require an entirely new fermentation medium. The use of a more productive strain must therefore be weighed against possible increased costs resulting from higher investments in extraction, richer media, more expensive fermentor operations and other hitherto non existent problems. This possibility not withstanding, strain improvement is usually part of the program of an industrial microbiology organization. To appreciate the basis of strain improvement it is important to remember that the ability of any organism to make any particular product is predicated on its capability for the secretion of a particular set of enzymes. The production of the enzymes, themselves  depends ultimately on the genetic make-up of the organisms. Improvement of strains can therefore be put down in simple term as follows:

  1. Regulating the activity of the enzymes secreted by the organisms.
  2. In the case of metabolites secreted extra-cellularly, increasing the permeability of the organism so that the microbial products can find these way more easily outside the cell.
  3. Selecting suitable producing strains from a natural population.
  4. Manipulation of the existing genetic apparatus in a producing organism.
  5. Introducing new genetic properties into the organism by recombinant DNA technology or genetic engineering. 

Genes are chemically the segments of DNA molecules except in some viruses, as some viruses are found to  contain RNA as genetic material.  They are normally transmitted with great exactness. But sometimes variations may be caused by physicalor chemical agents resulting in altered phenotype.  The heritable changes in the  genome of a cell are called mutations. Those mutations which occur in the somatic cells are called somatic mutations. These are not transmitted to next generation.
Mutations occurring in the germ cells are called germinal mutations. These mutations influence the gametes and are passed to next generation, generating new variability and contributing to the process of evolution.

Mutations have both advantages as well as disadvantages.Increasing the microbial mutation rates bring out the genetic changes which have been put to many important uses in the laboratory and industries. For example mutations in some plants like tulips producing colourful flowers. Mostly mutations effect the normal existence of cells.  For example in bacteria, auxotrophs are developed from wild type because of mutations.  In humans mutations leads to physiological abnormalities like sickle cell anemia.


Mutations are classified in different ways on the basis of one or the other criterion. They may be depending on their origin, depending on  the type of change in base composition, on the basis of type of the cell, on the basis of the nature of their effect,etc. Among all these classification criteria the significant one‘s are

(A) Depending on their origin. 
(B) Depending on the type of change in base composition.

(A) Depending on their origin : Mutations are of two types
  1. Spontaneous mutations
  2. Induced mutations.
Spontatneous mutations: Mutations that occur naturally are called spontaneous mutations. Their origin is indeterminate and unknown.They are generally assumed to be random changes in the nucleotide sequences of genes. Spontatneous mutations are linked to normal chemical processes in the organism that alter the structure or the sequences of genes. For example all the four common bases of DNA have unusual tautomeric forms. Which are,however, rare.Tautomers are the mutually interconvertable structural isomeric forms. Normally nitrogenous bases in DNA present in the keto form. As a result of tautomeric rearrangement they can be transformed into the enol form.

The tautomeric rearrangement changes the hydrogen bonding characteristics of bases. Normally AU and GT base pairs. The tautomeric changes during replication substitutes nitrogenous bases with others. If a purine for purine and pyrimidine for pyrimidine are substituted the type of mutation is called transition mutation. If a purine for pyrimidine and pyrimidine for purine substituted the mutation is called transversion. The transtition and transversion mutations are also termed point mutations. Spontaneous mutations also occurs by frame shifts of DNA.

Once an error is present in the genetic code, it may be reflected in the amino acid composition of the specified protein.  If the changed amino acid is present in a part of the molecule, determining the structure or biochemical activity, functional alteration can occur. Many spontaneous mutations are reported.  For example albinism and hares lip in man; a tobacco mutant producing seventy leaves all of a sudden in a normal progeny
producing an average of twenty leaves.

Induced mutations:  The mutations resulting from the influence of any artificial factor are considered to be induced mutations.  Muller subjected drosophila to powerful x-rays and obtained a number of mutations.  The chemicals or any other means that induce mu8tations are called as mutagens or mutagenic agents.The mutagens acts in different ways like incorporation of base analogs, specific mispairing and intercalation.

Base analogs are structurally similar to nitrogenous bases of DNA, and can be incorporated into the growing polynucleotide chain during replication. Specific mispairing is caused when a mutagen changes a bases structure, by that alters its base pairing characteristics.

The different types of mutations changing the nucleotide number or order of DNA are
  1. Frameshift mutations
  2. Chromosomal mutations
Frameshift mutations:  As pointed out in the third unit the genetic information in DNA is expressed first into mRNA by the transcription.  mRNA is translated to proteins on reading triplet code from a fixed starting codon.  If a single nucleotide is deleted or inserted in the normal sequence then the reading frame changes.
The mutations leading to the change in reading frame are called frame shift
mutation.  These are two types.
  1. Deletion mutations
  2. Insertion mutations
Deletion mutations:  The reading frame of mRNA does not have any punctuation. So if nucleotides deleted it changes the amino acid sequence of protein expressed by it.

Normal sequence :
 Protein  Phe  Arg  Trp  IIe  Ala  Asn

Addition mutation :
           DNA  AAA  GCT  ACC  ATA  TCG  GTT
        mRNA  UUU  CGA  TGG  TAT  AGC  CAA
        Protein  Phe   Arg  Trp  Tyr  Ser  Gin

Deletion mutation :
Protein  Phe  Arg  Gly  Stop

Deletion mutations are of variable length ranging in deletion of the number of nucleotides. Deletion of three successive nucleotides will not effect all the protein composition. It is with one amino acid less only, as the codon is triplet code.
Dyes like acridines can bring about deletion mutations. In heterozygous diploid eukaryotes, a deletion involving the dominant alleles amy result in the expression of the recessive phenotype.

Insertion mutations:  Inserting nucleotides into a normal gene results in a mRNA,
in which the reading frame is altered.  This type of mutations causing insertion
of nucleotides are called insertion mutations.
Chromosomal mutations:
 The mutations effecting the number, size, shape and gene
complements are chromosomal mutations.  These are of different types like a chromosomal segment may be lost by deletion, or it may undergo inversion or it may be translocated to a different site or may be duplicated to tandem repeats.

Mutations inducing agents are called mutagens.  They create mutations in different ways.  Depending on the nature of mutagens they are of two types
  • Physical mutagens
  • Chemical mutagens
Physical mutagens:  Mutations can be naturally or artificially induced by a variety of physical mutagens.  H.J.Muller, founder of genetics, demonstrated in 1927 that mutations can be artificially induced by treating flies with x-rays. Similarly L.J.Stadler in 1928 demonstrated and increase in the rate of mutations due to x-rays in barely and maize.  Besides x-rays gamma rays can also induce mutations.
The physical agents are broadly divided into two types :
  1. Ionizing radiation
  2. Non-Ionionizing radiation
Ionizing radiation: X-rays and gamma (Y) rays are ionizing radiations. They have short wavelength and high penetration power.  They can penetrate into deeper tissues causing ionization of the molecules along their way. When X-rays penetrate into cells, electrons are ejected from the atoms of molecules encountered by the radiation.  As a result the stable molecules and atoms change into free radicals and reactive irons.  The radicals and ions can initiate a variety of chemical reactions, which can affect the genetic material, resulting in point mutations. i.e., affecting only one base pair in a given location.  The rate of mutation increases with the increasing dose of X-rays administered.

Nonionizing radiation:  Ultra Violet (UV) rays are nonionizing radiations. They have long wavelength and low penetration power.  The purines and pyrimidines absorb UV radiation most intensely at about 260 nm.This property has been useful in the detection and analysis of nucleic acids.In 1934 it was discovered that UV radiation is mutagenic. The major effect of UV radiation is formation of pyrimidine dimmers, particularly between two thymines. Cytosine-cytosine and cytosine-thymine dimmers are less prevalent.
The dimers damage the DNA structure and effects normal replication.

Chemical mutagens:  Charlotte Auerbach, author of Science of Genetics‖ was the first to find that mutatins can also be induced due to certain chemicals. Chemical mutagens can remove, replace or modify DNA bases.

Alkylating agents:  Alkylatin of nitrogenous bases by the alkylating agent either removes the base or modifies it.  Guanine residues can be alkylated by the methyl methane sulfonate and ethyl methane sulfonate. These agents alkylates guanine at N7 and weakens the purine-deoxyribose linkage.  This leads to deppurination creating ga at that site.  N-methyl-N1-nitro  –N  –nitrosoguanidine CH3-N(NOC(NH)-NH-NO2  is a powerful mutagen in E.coli.  Some alkylating agents change the GC positin ina nucleotide to AT.

Intercalating agents:  Intercalating agents produces frame shift mutation in bacteriophages like T4.  For example acridines are mutagenic to bacteriophphages but not to bacteria.  As the acridines are unable to enter bacterial cell.

Base analogs:  Base analogs are structurally similar to normal nitrogenous bases and can be incorporated into the growing polynucleotide chain during replication.  These analogs will have base pairing properties direrent from the bases they replace. One of  the first base analog formed to induce mutations in phage T2  is 5 bromouracil (BU) an analog of thymine.  In the normal keto form BU base pairs with adenine.But its tautomeric enol form pairs with guanine like cytosine.

Food Biotechnology Course material,by K V Anand Raj

Tuesday, August 7, 2012

Fermentation Media and its Ideal Characteristics

Characteristics of an Ideal Fermentation media

Fermentation media optimization

Chemical composition: the production medium must have a suitable chemical composition. Medium should contain a source of carbon, a source of Nitrogen, growth factors and mineral salts.

Precursors: In certain fermentation, the media should supply the required precursor for better yields of a desired product.

Buffering capacity: Maintenance of pH in the optimum range is necessary for making the process successful, since acidic and /or basic compounds depending on the nature of the fermentation process accumulate during the progress of the fermentation. To control the pH of the medium, buffers should be added to the medium (E.g. CaCO3). Media containing considerable quantities of proteins, peptides, and amino acids possess good buffering capacity in the pH range near neutrality. Additional buffering capacity in this pH range also provided by phosphates (Mono and di-hydrogen potassium or Sodium phosphates).

Avoidance of Foaming: Foaming is a serious problem in fermentation industry, Foaming can invite contamination the fermentation medium and also causes other problems for the fermentation. Hence defoamers (e.g. hard oil mixed with octadecanol for penicillin fermentation) should be used for controlling foam. These defoamers are added to the production medium before sterilization or incorporated after sterilization or added during the fermentation.

Toxicity: The ideal production medium free from any toxic effect on culture or product formation.

Consistency: In aerobic fermentation, it is necessary to supply sterile air into the medium. Under such circumstances, liquid media allow the diffusion of air throughout the medium under agitation. Fermentation media should not be viscous. Viscous nature of the medium creates difficulty in the penetration of the air interior of the medium. Air is not easily absorbed by the liquid medium.

Contamination: Certain conditions of the production medium are helpful to check the contamination. For example low pH values in citric acid production using Aspergillus niger avoids contamination.

Recovery: Recovery of the desired product is an important criteria. Components of the medium should be such that separation and extraction of the product becomes easy and cheaper.

Availability of raw materials: Raw materials required for designing of the production medium should be freely available in large quantities at a reasonable price.

Natural Media for Fermentation:
Different types of raw material are used in different types of industrial fermentation processes. Usually crude nutritive sources are preferred, since they are economical. Mostly agricultural products are utilized as a source of raw material in fermentation industries.

Carbon Source and Nitrogen Source
Carbon and Nitrogen sources are essential in fermentation media for the growth of microorganisms, These are certain carbon and nitrogen sources used in the fermentation media.

  • Saccharine Materials:
Sugar cane, sugar beets, molasses, and fruit juices may be included in this category.

Molasses: Molasses is a byproduct of the cane and beet sugar industry. It is recovered at any one of several stages in the sugar refining process. Chemical composition of sugarcane black strap is variable. It depends on the quality and variety of the cane but also on the process involved in the manufacture of sugar. About 95%of the total sugar in cane molasses is fermentable. It is particularly rich in biotin, pantothenic acid, thiamine, phosphorous and sulphur. The organic nitrogen content is less than beet molasses, since it does not contain betaine. But this substance is not assimilated by yeasts.

Beet molasses: Beet molasses are produced by the same process employed for cane molasses. Vitamins such as biotin, pyridoxine, thiamine, pantothenic acid and inositol are present in beet molasses also. Beet molasses have limited biotin. Therefore, in fermentation involving yeast culture, a small amount of cane black strap molasses or other biotin supplying material should be incorporated in the production medium. Because yeast require biotin for their growth. The largest utilization of cane black strap molasses in India is in the alcohol industry, which utilizes it for the manufacture of spirit, country liquors, rum, brandy, gin and whisky.

Fruit juices: Fruit juices contain soluble sugars. Grape juice contain glucose & fructose. Therefore, fruit juices can be used as a source of carbon in fermentation industries. Grapes are used in the production of wine.

Cheese whey: The straw coloured liquid produced as a byproduct of cheese making is called cheese whey. It is a major waste product for the cheese industry. It cannot be disposed of without proper treatment. Therefore, it is desirable to use it for useful products. It is also used as pig feed. For lactic acid production and SCP production it is served as raw material because it contains lactose, nitrogenous substances including vitamins (eg: vitamins) and inorganic salts.

  • Starchy Material:
There are two main sources of commercial starches
  1. Cereals (Wheat, rice, maize)
  2. Roots & tubers (potatoes, tapioca e.t.c)
The moisture content of the grain is low where as that of roots and tubers are very high. Starch require pretreatment to bring about the conversion to fermentable sugars. This is done either by enzymatic or chemical agents.

Cellulosic Materials:

Cellulosic materials are complex carbohydrate materials. The cellulosic molecule is made up of the repeating units of beta-glucose. The formation of beta-cellobiose requires two molecules of beta-glucose, which are linked through alpha 1, 4-linkage. 1000 to 10,000 units of cellobiose are required to form a simple linear polymer called cellulose. Units of cellobiose are joined end to end through 1, 4-beta-glucosidic linkages. For this reason cellulosic materials require some sort of pretreatment. Cellulosic materials are Sulfite waste liquor, wood molasses and Rice straw.

Sulfite waste liquor:

In the manufacture of paper pulp, wood is subjected to hydrolysis which is brought about with the help of Calcium bisulfite under heat and pressure. This operation is called digestion process. At the end of this process, the spent liquid is left and it is referred to as sulfite waste liquor. It cannot be disposed of unless it is properly treated. Sulfite waste liquor contains 10 to 12 percent solids, of which sugars make up about 20%. It contains sugars in the form of hexoses and pentoses. It is used in the industrial production of ethyl alcohol using Saccharomyces cerevisiae and in the growth of Torula utilis cells for animal feed. Saccharomyces cerevisiae requires hexoses where as Torula utilis requires both hexoses and pentoses. Sulfite waste liquor can not used directly as fermentation medium. It contains free sulfurdioxide or sulfurous acid which is toxic to microorganisms. These toxicants are removed by steam stripping or precipitation with lime.

Wood molasses:
It is produced by acid hydrolysis of wood cellulose itself. This may produce 65-85% fermentable sugars. Sulphuric acid of about 0.5% concentration is used at a temperature range of 150 to 185oC. Using a continuous process a syrup may be obtained from saw dust. This syrup may contain 4 to 5% reducing sugars (a mixture of glucose and pentoses) with an overall yield of 45 to 55%. It may be subjected to concentration to give a kind of wood molasses.

Rice Straw:
Rice straw and related agricultural materials can serve as a good source of cellulose. It is a poor quality animal feed in its natural state because of its bulkiness, poor palatability, low protein content and low digestibility. Numerous microorganisms are capable of using cellulose for their growth. Rice straw has been used as a fermentation medium in the production of silage and single cell protein (SCP), mushroom cultivation etc.
  • Hydrocarbons & Vegetable Oils
Hydrocarbons used as fermentation substrates are usually mixtures of various hydrocarbon components. These fermentation raw materials are relatively cheap. However, purified hydrocarbon fractions or hydrocarbon compounds are more expensive.

Hydrocarbon substrates (e.g. gas oil and n-paraffins) are used to produce single cell protein (SCP) products. In this way biomass of yeasts (e.g. Candida lipolytica, Candida kofuensis, Candida tropicalis) can be produced on a significant scale under aerobic conditions.

Vegetable oils:
Oils obtained by deoiling of vegetable seeds are called vegetable oils.On the basis of their degree of unsaturation, they may be grouped into following three major classes:
  1. Oleic (or 'non drying' type): These include olive and groundnut oils.
  2. Linoleic (or 'semi drying' type): These have a higher content of the double unsaturated fatty acid found in maize, sunflower and cotton seed oils.
  3. Linolenic acid (or 'drying type): These include linseed and soya bean oils containing a fatty acid with three double bonds.
These oils may undergo drying if exposed to the atmosphere due to the oxidation of the unsaturated components. Commercial vegetable oils (e.g. maize oil) may be used in conjunction with surface active agent as anti foams or alone as a nutrient source of carbon.

  • Nitrogenous Materials:
Corn steep liquor(CSL): The used steep water results from the steeping of corn during the manufacture of starch, gluten and other corn products this by product is subjected to concentration to approximately 50% solids and this concentrate is called corn steep liquor. Corn steep liquor was originally found to be useful for penicillin production specifically. But, it is now recognized as valuable in many fungal antibiotic fermentation media. In addition to this, it is also used in the manufacture of food stuffs.

Soya bean oil: the material left after removing oil from the soya bean seeds are called as soya bean meal. Soya bean meal contains approximately 8% w/w nitrogen. This differs from corn steep liquor, since soya bean meal is a much more complex nitrogenous source than corn steep liquor, and therefore not readily available to microbes. This is used as a ingredient for fermentation media in the production of streptomycin.

Pharmamedia: Pharmamedia is a clean, yellow, finely ground powder prepared from the embryo of cotton seed. It contains 56% w/w protein, 24% carbohydrate, 5% oil, and 5% ash. Ash, in turn, contains calcium, iron, chloride, phosphorous and sulfate. It is used as an ingredient for production media (Eg: Tetracycline production).

Distillers Solubles: In the manufacture of alcohol using grain or maize, alcohol is distilled from fermented grain or maize, leaving the residue (containing 6 to 8% w/v total solids). The suspended solids from the residue are eliminated by screening, leaving the effluent. Thereafter effluent is subjected to concentration, until the solid content reaches 35% w/v giving 'evaporator syrup'. This syrup is then drum dried to yield 'distillers solubles'. This may be used as a production medium component, since it supplies nitrogen, together with many accessory food factors (e.g. vitamin B complex).

Precursors & Inducers:

Certain substances, which generally improves the yield or quality of the product. These substances are known as the precursors. These precursors are incorporated without any major change in to the molecule of the fermentation product. Eg: Phenyl acetic acid and Cobalt are being added for penicillin G and Vitamin B12. Corn steep liquor yields various pencillins but addition of phenyl acetic acid determines the penicillin G production. Proteases for various proteins, alpha-amylases for starch, cellualse for cellulose, pectinase for pectin and penicillin acylase for phenyl acetic acid.

Repressors: The substances which are being employed for the repression of the industrial cultures are known as repressors.
  1. Media allowing restricted growth provides high product yield as major portion of carbon and other components of the medium are shunted to product formation rather than to growth.
  2. Nitrogen sources such as soyabean meal and praline for streptomycin; production is probably due to their slow utilization, thus avoiding nitrogen metabolite repression.
  3. Aspergillus niger for gluconic acid production is first grown on a medium that supports a rich growth as well as product formation, then the mycelium is separated and placed in a fresh medium high in carbon substrate (sugar) but lacking combined nitrogen so that additional growth cannot occur.
Antifoams: Antifoams are surface active agents, reducing the surface tension in the foams and destabilizing protein films by hydrophobic bridges between two surface, displacement of absorbed protein and rapid spreading on the surface of the film. An ideal antifoam should have a fast action on the existing foam but should not be metabolized by the microorganisms. It should be cheap, heat sterilizable, non toxic, long acting and active at low concentrations.

Examples: Stearyl alcohol and octyl decanol, esters, fatty acids, cotton seed oil, linseed oil, castor oil, cod liver oil etc, silicones, sulphonates.

If the oxygen transfer rate is severely affected by antifoam addition, then mechanical foam breakers may have to be considered as a possible alternative.

Food Biotechnology Course material,by K V Anand Raj

Sunday, August 5, 2012

Different Types of Fermentors / Bioreactors

Different Types of Fermentors / Bioreactors

The heart of the fermentation or bioprocess technology is the Fermentor or Bioreactor. A bioreactor is basically a device in which the organisms are cultivated to form the desired products. it is a containment system designed to give right environment for optimal growth and metabolic activity of the organism.
A fermentor usually refers to the containment system for the cultivation of prokaryotic cells, while a bioreactor grows the eukaryotic cells (mammalian, insect cells, etc).

Types of Bioreactor

Types of Bioreactor
  1. Continuous Stirred Tank Bioreactor
  2. Airlift  Bioreactor
  3. Fluidized Bed  Bioreactor
  4. Packed Bed  Bioreactor
  5. Photobioreactor
  6. Membrane Bioreactor
Continous Stirred Tank Bioreactor 
In Continous Stirred Tank Bioreactor, the contents of the vessel no longer vary with time, this applies to the hold up of micro-organisms and the concentration of the components of the medium in the fermentor.Steady state conditions can be achieved by either Chemostatic or Turbidostatic principles.The former involves the adjustment of the flow rate of the fermentor to an appropriate and constant value and allowing the micro-organisms, substrates and biochemical product concentration to attain their natural levels. The turbidostat requires an experimental determination of the turbidity (ie, indirect measurement of microbial concentration). This thus used to control the flow rate. Both these methods have been employed in practice, though the former is obviously the simpler from every view point.
The most successful continuous systems to date have been those employing yeasts and bacteria, in which the desired products are the cells or primary metabolites, compounds that form the chemical 'inventory' of a microbe, (e.g. enzymes and amino acids), or some product clearly associated with growth or energy producing mechanisms (e.g. the production of alcohol).
The most widely used continous process based on CSTF (Continous Stirred Tank Fermentor) is the activated sludge process used in waste water treatment industry.
In continuous processing the autocatalytic (a reaction in which one of the products of the reaction increases the overall rate of a reaction) nature of microbiological reactions takes on a further significance. This is because the presence of one of the products, additional micro-organisms, enhances the overall rate of reaction. In the absence of micro-organisms no reaction can take place. Therefore, it is essential to retain at least a portion within the fermentor. It follows that if the flow rate is raised to a high value, then all the micro-organisms will be swept from the fermentor, and the conversion will cease. This phenomenon is commonly known as 'Wash-out'. if micro-organisms are fed to the fermentor simultaneously with the substrate feed, the problems associated with wash-out are abated, and the reaction proceeds normally.

Advantages of Stirred Tank Bioreactor
  1. Continuous operation
  2. Good temperature control
  3. Easily adapts to two phase runs
  4. Good control
  5. Simplicity of construction
  6. Low operating (labor) cost
  7. Easy to clean
Airlift Bioreactor

airlift bioreactor

This kind of fermenter works on the principle of an air lift pump. It is of two kinds: 
  1. Internal loop type
  2. External loop type.
The reactor's volume is determined by its capacity, kinetic data, and specific growth rate of the organism used. The rate of airflow of the reactor depends on the volumetric mass transfer coefficient in the reactor system. It is a uniform cylindrical cross type and has an internal loop or external loop riser configuration, 
diverging converging. The external loop riser configuration is adjustable and the change in the configuration improves the O2 transfer rate vis-a-vis mass transfer coefficient for a particular rate of airflow. This helps provide required particular dissolved O2 concentration for specific microbial system. This reactor reduces the operating cost for pumping air through the bioreactor.

  • Simple design with no moving parts or agitator for less maintenance, less risk of defects.
  • Easier sterilization (no agitator shaft parts)
  • Low Energy requirement vs stirred tank : Obviously doesn’t need the energy for the moving parts (agitator shaft).
  • Greater heat-removal vs stirred tank: At the Airlift bioreactor it doesn’t need the heat plate to control the temperature, because the Draught-Tube which is inside the bioreactor can be designed to serve as internal heat exchanger. It is difference to the Stirred tank bioreactor that needs the heat coat or plate surrounding the tank to make warm bioreactor. It is clear enough that the Airlift bioreactor has greater heat-removal compare to Stirred tank.
  • Very low cost
Fluidized Bed  Bioreactor

This is a characteristic of beds of regular particles suspended in an up flowing liquid stream. 
If an additional gas phase is involved, there is a tendency for the particles in the bed to become less evenly distributed. 

There are two important features of the beds of mixed particle sizes: 
(i) The increase in porosity from the bottom to the top of the bed, and 
(ii) The decreased particle movement when compared with beds containing particles of constant size.

Since porosity or voidage is a measure of the local free space within a bed, it also represents a measure of the microbial hold-up when expressed as wet volume per unit bed volume. Thus, a variation in microbial hold-up is to be expected within a 'fluidised bed' fermentor  on fluidisation, the smaller particles rise relative to the larger particles, and produce a situation where the smaller particles are at the top and the larger 
particles are at the bottom of the bed.

As the smaller particles have the lowest settling velocity, the bed arranges itself, so that the smaller particles may be in the region of the highest porosity and the lowest linear velocity. The tower fermentor (developed for the continuous production of beer) is based upon these general principles (Ault et al, 1969). In this process yeast flocs are maintained in suspension by the upward movement of the nutrient medium. Moreover, any entrained particles are returned by means of a sedimentation device at the top of the tower. 
Essentially, the fermentor consists of a vertical cylinder with an aspect ratio (length to diameter) of approximately 10:1 At the top of the tower a separator is provided to induce the gas bubbles produced by 
th,e reaction, to coalesce and escape from the liquid phase.

Within the separator there is a quiescent lone, free of the rising gas, so that the yeast may settle and return to the main body of the tower, and clear beer can be removed. A flocculent yeast (i.e. a yeast capable of achieving  relatively large floc sizes) is essential for an alcoholic fermentation in a PBP at acceptable flow rates, otherwise a large proportion of the yeast would be washed out. As a result of this, an insufficient yeast concentration is maintained. A mean yeast concentration of 25 % by weight (expressed as centrifuged wet weight) is typical with values as high as 30-35% by weight at the bottom of the tower, and as low as 5-10% by weight at the top.

 A significant feature of the tower is the progressive and continuous fall in the specific gravity of the nutrient medium between the bottom and the top of the tower. There is an initial rapid fall at the bottom of the tower. It is followed by a slower fall over the middle and the top of the tower. This gradual fall in the specific gravity is due to the fermentation of the sugars.

Advantages of Fluidized Bed Reactor:
  • Uniform Particle Mixing: Due to the intrinsic fluid-like behavior of the solid material, fluidized beds do not experience poor mixing as in packed beds. This complete mixing allows for a uniform product that can often be hard to achieve in other reactor designs. The elimination of radial and axial concentration gradients also allows for better fluid-solid contact, which is essential for reaction efficiency and quality.
  • Uniform Temperature Gradients: Many chemical reactions require the addition or removal of heat. Local hot or cold spots within the reaction bed, often a problem in packed beds, are avoided in a fluidized situation such as an FBR. In other reactor types, these local temperature differences, especially hotspots, can result in product degradation. Thus FBRs are well suited to exothermic reactions. Researchers have also learned that the bed-to-surface heat transfer coefficients for FBRs are high.
  • Ability to Operate Reactor in Continuous State: The fluidized bed nature of these reactors allows for the ability to continuously withdraw product and introduce new reactants into the reaction vessel. Operating at a continuos process state allows manufacturers to produce their various products more efficiently due to the removal of startup conditions in batch process.


Advantages of Photobioreactor
  • Cultivation of algae is in controlled circumstances, hence potential for much higher productivity
  • Large surface-to-volume ratio. PBRs offer maximum efficiency in using light and therefore greatly improve productivity. Typically the culture density of algae produced is 10 to 20 times greater than bag culture in which algaeculture is done in bags – and can be even greater.
  • Better control of gas transfer.
  • Reduction in evaporation of growth medium.
  • More uniform temperature.
  • Better protection from outside contamination.
  • Space saving – Can be mounted vertically, horizontally or at an angle, indoors or outdoors.
  • Reduced Fouling – Recently available tube self cleaning mechanisms can dramatically reduce fouling.
Membrane Bioreactor

Membrane bioreactors successfully applied to various microbial bioconversions such as alcoholic fermentation, solvents, organic acid production, waste water treatment, etc.
In membrane bioreactor the soluble enzyme and substrate are introduced on one side of ultrafilter membrane by means of a pump. product is forced out through the membrane. membrane holds back the enzyme. good mixing in the reactor can be achieved by using a stirrer.
The most widely used membrane materials includes polysulfonte, polyamide and cellulose acetate.

Advantages of Membrane Bioreactor
  1. The loss of enzyme is reduced.
  2. Enzyme lost by denaturation can be make up by periodic addition of enzyme.
  3. Substrate and enzyme can be easily replaced.
Food Biotechnology Course material,by K V Anand Raj

Production and Estimation of Pectinase by Aspergillus niger

Purified pectinase is a multicomponent preparation highly effective in de-polymerising plant pectins with varying degree of esterification. Important enzymatic activities include pectin lyase and activities on polygalacturonic acid. Pectinase are utilized to eliminate pectin and pectin like colloids in fruit juices to clarify the juice and a means to prevent jelling of the juices during concentration steps of fruit juice processing. pectinase is also used in plant protoplast studies, pectinase is produced by various fungi although commercial production utilizes the species of aspergillus and pencillium.

Principle of Pectinase Production
Pectinase production by fungi is stimulated by the presence of pectin containing compounds in the fermentation medium, the enzyme is recovered from both the sources. Thus at harvest mycelium is dried and ground. The commercial preparation contains at-least two types of pectinase differing in the extent to which they degrade pectin.
Pectinase yield is more in solid state fermentation than in the submerged acidity plays an important role in the production of solid state fermentation.A low pH of 5.0 also maintains asepsis during culture. wheat bran, coffee pulp, citrus waste & apple pomace are few substrate used for pectinase production by solid state fermentation.

Pectinase Production Medium
Weight in gms
1000 ml
Wheat Bran / Coffee Pulp

Solid State Fermentation of Aspergillus niger

  1. Aspergillus niger was inoculated on an agar slant of pectin medium and allowed to grow for 4days at 25oC.
  2. 25.0 gm of wheat bran was mixed with phosphate buffer at pH 5.0 to get required consistency & was autoclaved.
  3. The spores from agar slant was scraped and suspended in 5.0ml of buffer and this was added to the wheat bran. the flask was incubated at 25oC for 5 days.
  4. 50ml of phosphate buffer was added to the flask and kept in a shaker 37oC to extract the enzyme.
  5. The extract was centrifuged at 5000 rpm & supernatant was used for the assay of the enzyme.
Assay of Pectinase / Estimation of Pectinase
One unit of pectinase liberates 1 micro-mole of D-galactouronic acid from polygalactouronic acid per minute at 37oC, pH 5.0.

Reagents Required:
  1. 0.1M  citric acid or phosphate buffer, pH 5.0.
  2. Colour Reagent A: Dissolve 40g of anhydrous sodium carbonate in 600ml distilled water. add 16g of glycine & stir until it dissolves, make up the volume to 1L using distilled water.
  3. Colour Reagent B: Dissolve 12g of neoprene hclin 1L distilled water and store at 4oC in brown bottle.
  4. D-galactouronic acid standard - 1mg/ml
  5. 0.5% polygalactouronic acid substrate: Heat 500ml assay buffer on a hot plate, heat and still well until it dissolves. it will be slightly viscous and opaque. don't boil, cool and make up the volume. if required store at 4oC.
Estimation of Pectinase Procedure
  1. Take 6ml of the substrate in a tube and add 1ml of the enzyme solution.
  2. Incubate the tube at 37oC for 1 hour.
  3. Similarly, prepare a blank by denaturing the enzyme by boiling for 10min, After incubation transfer the tube to ice.
  4. Take 100µl of the sample & blank in two separate tubes& add 2ml of colour reagent A and colour reagent B.
  5. Mix well and keep in a boiling water bath for 15mins.
  6. Add 2ml of water and note the absorbance at 450nm.
  7. Prepare a standard D-galactouronic acid using 10µg - 125µg of it.
  8. Plot graph of absorbance against the amount of standard & find the amount of product releassed by  the enzyme.

Saturday, August 4, 2012

Citric Acid Production by Aspergillus niger, Estimation of Citric acid

Citric acid is an important organic acid and it was initially being extracted from citrus fruits. Nowadays it is largely produced by microbial fermentation. Citric acid is commercially used in foods, soft drinks, pharmaceuticals, leather tanning, electroplating etc. Aspergillus niger is the most commonly used species for the production of citric acid.
Most strains of  Aspergillus niger which are mutants cannot oxidize citric acid and hence accumulate in culture medium. The composition of the culture medium is critical for obtaining high yield of citric acid. Its is essential to limit the growth of the fungus, so that high yield of citric acid accumulates in the medium. this can be accomplished by keeping trace metal deficiency in the medium. Acid is added to achieve low pH of 3.5. Sucrose serves as a carbon source for the production of citric acid. Ammonium nitrate is used to prevent the fermentation of oxalic acid glutamic acid.Fermentation is aerobic and can be carried out by submerged culture method.

Citric acid is an important organic acid and it was initially being extracted from citrus fruits. Nowadays it is largely produced by microbial fermentation. Citric acid is commercially used in foods, soft drinks, pharmaceuticals, leather tanning, electroplating etc. Aspergillus niger is the most commonly used species for the production of citric acid.

Citric Acid Production Principle
Most strains of  Aspergillus niger which are mutants cannot oxidize citric acid and hence accumulate in culture medium. The composition of the culture medium is critical for obtaining high yield of citric acid. Its is essential to limit the growth of the fungus, so that high yield of citric acid accumulates in the medium. this can be accomplished by keeping trace metal deficiency in the medium. Acid is added to achieve low pH of 3.5. Sucrose serves as a carbon source for the production of citric acid. Ammonium nitrate is used to prevent the fermentation of oxalic acid & glutamic acid. Fermentation is aerobic and can be carried out by submerged culture method.

Citric Acid Production Medium
Sucrose - 150 gm
Ammonium nitrate - 2.5 gm
Potassium Dihydrogen Orthophosphate - 1.0 gm
Magnesium sulphate heptahydrate - 0.25 gm
Distilled water - 1L
pH - 3.5

Culturing Aspergilus niger
  1. prepare the citric acid medium & dispense about 50ml in 250ml conical flask.
  2. Autoclave and allow it cool.
  3. Inoculate the medium with spores of Aspergillus niger & incubate it on a shaker water bath at 25oC with gentle shaking for 3-5 days.
  4. After Incubation, filter the mycelium using double layered muslin cloth & measure the amount of citric acid in the filtrate by colorimetric and titrimetric methods.
Estimation Citric Acid by Titrimetric Method
The filtrate obtained is titrated against an alkali of known strength using phenolphthalein as indicator.The end point is the formation of pale pink colour. The volume of alkali used for neutralization is used to find the normality and the percentage of acid in the sample.

Estimation of Citric acid using phenolphthalein as indicator
  1. 100ml of the filtrate is pipetted into a conical flask and 2-3 drops of phenolphthalein indicator is added to it.
  2. This is titrated against 0.1N NaOH taken in the burette till a pale pink colour is formed.
  3. The titration is repeated till concordant values are obtained.
Example of Titration Results
Initial Burette Reading
Final Burette Reading
Vol of NaOH used

Normality of Citric acid  =  [N(NaOH) * V(NaOH)] / V(Citric acid)
                                      = [0.1 * 8.1]/ 10
                                      = 0.081

% of Citric acid = [Normality * Equivalent wt of citric acid * 100] / Volume of filtrate
                        = [0.081*96*100] / 10
                        = 77.76%

N - Normality, V - Volume.
Equivalent wt of Citric acid - 96.

Yoghurt / Yogurt Production: Procedure, Role of bacteria in yoghurt & Therapeutic Value of yoghurt

Yoghurt / Yogurt is a fermented milk with clustered like consistancy, Flavoured or Non-Flavoured product fermented by Lactobacillus bulgaricus Streptococcus thermophilus. Yoghurt has higher nutritive value compared to other fermented products of milk because of its higher milk solid contents.

Production of yoghurt
Yoghurt / Yogurt

Yoghurt / Yogurt is produced by the controlled fermentation of milk by the two species of bacteria. Bacillus Sps.& Streptococcus Sps. The sugar, Lactose in milk is fermented to lactic acid and that it causes the formation of curd. This acid also restricts the growth of food poisoning bacteria & some other bacteria that causes the spoilage of food. So milk is potential source and yoghurt is safer and can be kept for up-to ten days under proper storage conditions.
                                         The correct balance between the two bacteria's are important for good quality of yoghurt. In practice a dried culture can be obtained and this can be grown on pasteurized milk & can be kept in a refrigerator. A part of the master culture can be used everyday for a week & the last part can be re-inoculated into milk to form new starter culture. This method can be continued for several months provided good hygiene. But eventually undesirable bacteria will contaminate the culture & it must be replaced.
                                        If a pure culture is not available, it is possible to add one or two spoonful of commercial yoghurt as starter culture. This can be done everyday. Finally it is possible to add part of yoghurt production to a new batch of milk the following day, there is a greater risk of contamination using this method. Yoghurt can be either stirred or set. Yoghurt is fermented in bulk, stirred and then dispensed into pot. Fruits and nuts can be added into each type but care should be taken to avoid contamination. In some countries layers of fruit syrup is added on the set yoghurt.

yoghurt fermentation
Large Scale Production of Yoghurt
                                                              Image Source
Yoghurt Production Procedure
Yoghurt can be easily produced in small scale, the procedure is as follows:
  1. Collect milk carefully in a cleaned covered vessel.
  2. Pasteurize the milk at 80oC for 15 to 20 mins.
  3. Cool the milk to 40 to 45oC as soon as possible.
  4. Added starter culture and mix well.
  5. Keep the milk in an incubator at 40 - 45oC for 3 - 4 hrs.
  6. Specific coloures and flavours can be added if required.
Process of yoghurt production

Role of Bacterial Culture in Yoghurt / Yogurt Production
In the production of yoghurt, the two yoghurt cultures excerts synergetic action during the initial incubation of Streptococcus thermophilus grows luxuriously because of the liberation of numerous aminoacids from the casein produced by Lactobacillus bulgaricus which stimulate growth of Streptococcus thermophilus.
It is demonstrated that acid production by L.bulgaricus is enhanced by formate & CO2 produced in yoghurt during acidification has been proposed. S.thermophilus grows faster during early part of incubation due to stimulatory effect of amino acids liberated by L.bulgaricus there by removing oxygen and producing the CO2 and formate in milk. After the growth of S.thermophilus it is slowed by increasing concentration of lactic acid. The more acid tolerant L.bulgaricus increases the effect of compounds generated by Streptococci.

Therapeutic Value of Yoghurt / Yogurt
  1. Yoghurt has been used as a biomedicine in treating the patients suffering from gastrointestinal disturbances.
  2. The yoghurt bacteria stops the anaerobic spore formation in the large intestine thus prevents petrification process taking place in alimentary canal.
  3. It is used as refreshing beverage.
  4. Yoghurt is rich in amino acids, Vitamins & Minerals.
  5. Improves Lactose intolerance.
  6. Boost immune system.
  7. High in Calcium, Potassium and Protein.