User:Joan1912/sandbox

=Industrial Media= For the growth of microbes/eukaryotic cells/organisms in fermentation, they need a media designed specifically that suits the needs and fulfils the nutrient requirements of the organisms or cells to ensure the maximum efficiency of the fermentation which provides substrate for the synthesis of a single product or products synthesis in a fermentor. The formulation of a medium at a laboratory scale could be done by adding the respective carbon and nitrogen sources along with water and other necessary supplements in their undiluted form which ensures the proper growth of the organism at a non-industrial level. Ideally speaking the components of the fermentation medium should have the following standards to ensure maximum efficiency and it applies to all forms of industrial fermentation as this formulation is not a small scale one like that of the laboratory medium. The standards are as follows: •	Should ensure the maximum yield of the required product or maximum biomass for every gram of the substrate used up in the reaction. •	Enable the maximum concentration of the required product (Maximum titer concentration) •	The production of unwanted or unfavorable products should be minimized or reduced •	The product formed should be uniform, regular and stable with every repeating batch of the fermentation cycle. •	The medium designed should have components that are available at any given time and shouldn’t cause any complications with its preparation and handling. •	It should not hinder with any of the other processes of product formation such as extraction and purification to ensure minimal damage to the product formed. •	The addition of certain supplements, growth factors, vitamins, precursors, inducers, trace elements along with anti-foaming agents are added to facilitate easy microbe growth. The fermentation media used for the operation of fermentation processes in the industry are usually in the liquid phase or solid state. However, liquid media are mostly used in fermentation processes as they require less space compared to solid media and are cheaper to work with – since no additional cost of procuring agar or solid agents will be accrued. Liquid media are also more flexible to most genetic engineering processes than are solid media in fermentation processes. =Constituents Of Industrial Media For Its Formulation= Growth and product formation follow a simple equation Carbon and energy source + nitrogen + other components = biomass+ product+CO2+H2O+Heat

1.Carbon source
it is the basic source necessary for all biosynthetic reactions including fermentation (here industrial fermentation). The amount of carbon required is determined by calculation of Y (biomass yield coefficient). x = Yx/ s(S- Sr) x = biomass concentration (g/L) Yx/ s = yield coefficient (g biomass/g substrate utilised) S = initial substrate concentration (g/L) Sr = residual substrate concentration (g/L). Common carbon sources used are molasses, malted barley, starch and dextrins, sulphite and waste liquor, alkanes and alcohols and oils and fats, corn steep liquor etc.

2. Nitrogen source
Industrial microbes can make use of both organic and inorganic nitrogen sources. Salts of urea, ammonia, and nitrate is commonly used as nitrogen source. If the organisms used in fermentation are of non-proteolytic nature, they use the purified forms of urea, ammonia and nitrate as their source of nitrogen. For the organisms of proteolytic nature animal and plant raw material (Casein, cereal, peptones, yeast extract, soybean meal) is used.

3.Mineral elements
Sometime the reaction medium will need the presence of certain major mineral elements like calcium, magnesium and potassium to act as co-factors and help with the bio-reaction. Along with these elements some trace elements also make their way into the medium and affect the rate of the reaction as well as the product formed. Phosphorus is needed for production of phospholipids in cellular membranes and for the production of nucleic acids. The amount of phosphate which must be added depends upon the composition of the broth and the needs of the organism, as well as the objective of the fermentation. Penicillin’s primary and secondary metabolism is influenced by the presence of trace elements like iron, zinc and copper. Same is the case for citric acid production.

4.Chelators
Sometimes media preparation is not possible without precipitate formation during autoclaving. To overcome this some chelating agents are added to the media to form complexes with metal ions which are then utilised by the organism for the product formation. They are added separately after autoclaving is done. EDTA, Citric acid, and polyphosphates are some of the examples.

5.Growth Factors
Some of the microorganisms are not capable of synthesizing one or more growth factors such as vitamins. These growth factors are very expensive in pure form, hence crude sources are preferred. Yeast extract is a rich source of almost all growth factors. Generally, the substrates derived from plant or animal sources in a crude form are reasonably rich in mineral content.

6.Buffers
Maintenance of pH is crucial getting an optimal yield in fermentation. Some organisms are active only at a certain ph. Any fluctuation in these values will lead to low yield or no yield at all. In a fermentation vat, the concentration of CO2 in the solution is higher than normal due to the fermentation activity. Much of this excess CO2 bubbles off making the solution more acidic due to the formation of carbonic acid. however, because dissolved CO2 combines with water to create carbonic acid. If the solution became too acidic, it could inhibit for example the growth of yeast as they prefer a range between 4-6 for their growth. For example, protein, peptides, amino-acids act as good buffers at neutral pH. Sometimes inorganic buffers like K2HPO4, KH2PO4, and CaCO3 etc, can be added as required. Generally, during the fermentation process, pH changes to acidic or alkaline pH. The cheapest and easily available buffer is CaCO3.

7.Precussors
Precursors are added in the fermentation media at time of fermentation as it gets incorporated in the molecules of product without bringing any kind of change to the final product. This helps in improving yield and quality of product. Sometimes, precursors are added in pure form depending upon the need of product. For example, cobalt chloride is used in minute quantities in the fermentation of vitamin B12.

8.Inhibitors/Inducers
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 forpectinase. Inhibitors: Are used to obtain specific products by reducing or increasing the production of the final product, reduce or increase the viability of the microorganism or completely stop the fermentation process. For example, ethanol the major product of fermentation can sometimes act as an inhibitor. Other examples include carboxylic acids, furans and phenolic compounds.

9.Water
Fermentation sometimes requires a solid media or a liquid media and this is achieved by using water. This provides a uniform medium for mixing of the reaction mixture as well as in product formation. Most fermentation requires liquid media or broth.

10.Oxygen
The industrial fermentation is usually carried out in oxygen presence in order to obtain the maximum cellular metabolic performance of the biological system used. The majority of the fermentations are of aerobic nature and require oxygen. A microbial culture is supplied with oxygen during its growth cycle at a rate that is adequate to satisfy the organisms need. This is done by aeration and agitation of the fermentation media.

11.Anti-foam
When the aeration/agitation of the media is done there is a whirlpool of a small column of air created within the reaction chamber and this gives rise to the formation of foam between the media and the top of the bioreactor. This then affects the product formation by hindering with the hydrogen ion concentration, probe efficiency and increases the chances of contamination. An antifoaming agent such as silicon oil or vegetable oil is used. These agents are compatible with the microbial culture used in the bioreactor. High concentration of these Anti-foam agents is not used as they have adverse effects on the bioreaction. =Types of media=

Synthetic media
The variation in the level and concentration of nutrients in this type of media can be predetermined as each and every component is chemically defined and regulated. The effects of nutrients of the growth and product formation can be easily analyzed as it is monitored in a lab environment. It is also used to determine the type of metabolic pathway followed in the product formation. With the help of radio-isotope labelling technique the main ingredients needed for the required product formation can be determined as well as optimized. However, this media lacks sources of protein and peptides due to which there is no foam formation and the chances of contamination is less and the product recovered is in its pure form. One main disadvantage of this media is that it is very expensive and at an industrial level it is not economic and profitable. This is only used for experimentation in a lab in small quantities. Sometimes this is used in the analysis of novel cultures whose chemical composition is sometimes unknown.

Semi-synthetic Media
It is a chemically defined media in which one of the components not defined but its composition is controlled. This type of media is mostly used for research and small-scale lab related works for which sometimes plant, animal, fish and microbial extracts are used as a stock to supply necessary growth factors and vitamins. For example, yeast extract, beef tract.

Crude Media
This type of media is mainly composed of substance that are of plant/animal origin which have a defined as well as a varied composition. This type of media preparation varies from every batch and its composition is affected as it is highly guided by the location of its origin, production methodology, weather conditions etc.

Enrichment Media
It is a liquid based media that allows for the microbial culture to multiply with the essential nutrients and growth factors required for it. It rarely contains inhibitory substances that prevents the growth of normal competitors. It basically prevents the growth of non-pathogenic bacteria by the over growth of pathogenic bacteria. For example, Selenite F broth favors the growth of Salmonella also prevents the growth of normal competitors like E. coli. E.coli does not die in the medium but they do not flourish like Salmonella does.

Selective Media
A media that tolerates the growth of certain (necessary) types of organisms while inhibiting the growth of other organisms by the addition of certain inhibitors like antibiotics, dyes, alteration of hydrogen ion concentration/temperature or a combination of all these methods that doesn’t affect the required microorganisms but inhibits the growth of other organism. Examples of commonly used selective media includes: PALCAM agar medium or Mac conkey agar medium. =Industrial Media Optimization= Optimization of the fermentation media is an essential step for metabolite production. Optimization techniques help in reducing the overall product cost. Media optimization has become more vibrant, effective, efficient, economical and robust in giving the results. For designing a production medium, the most suitable fermentation conditions (e.g., pH, temperature, agitation speed, etc.) and the appropriate medium components (e.g., carbon, nitrogen, etc.) must be identified and optimized accordingly.

One-Factor-at-a-Time (OFAT)
In the classical medium optimization technique, one-factor-at-a-time (OFAT) experiments, only one factor or variable is varied at a time while keeping other variables constant. The concentrations of the selected medium components are changed over a desired range. In this methodology, sometimes the optimum point may be missed completely, thus it requires a large number of experiments to determine the optimum level, which becomes laborious, time consuming, and uneconomical most of the time.

Removal experiments
In this type of experiment, all the medium components are removed from the production medium one-by-one, followed by incubation, their effects on the production of secondary metabolite or the product of interest is observed in terms of suitable parameters.

Replacement experiments
For medium formulation, carbon/nitrogen sources showing enhancement effect on the desired metabolite production in supplementation experiments are generally tried to be used as a whole carbon/nitrogen source.

Supplementation experiments
Supplementation experiments are generally performed to weigh up the effects of various carbon and nitrogen supplements on metabolite production.

Statistical Medium Optimization
With the advancement of statistical techniques, medium optimization has found new dimensions, as these techniques improve the efficiency of the process, reduces the time required in the process and labor cost etc., thus contributing toward the overall economics of the process. By using experimental design, the amount of experiments required to obtain a for reliable process optimization can be reduced.

Plakett Burman design
Plackett–Burman design (PBD), is a two-level design, which is very useful for economically detecting the main effects and assuming all the other interactions are negligible when comparing the some important major effects, i.e., when there are no interactions, the observed effect of a factor can be superior or under estimated by other factors.

Taguchi design
This method designed by Dr.Genichi Taguchi gives us an idea of how different parameters affect the yield in a small number of experiments instead of testing all the possible combinations, like, the factorial design. It helps in determination of the factors affecting the product production with the least number of experiments thereby saving time and resources. Analysis of variance (ANOVA) on the collected data from the Taguchi Experiment can be used to select the new parameter values to optimize the performance characteristic.

Response surface methodology (RSM)
This method uses factorial designs which affects the optimization of the production of the desired metabolites by the application of mathematical approach,  experimental designs and multiple analysis to seek the optimal formulation under a set of well-defined equations. RSM is used to determine the factor levels which can simultaneously satisfy a set of desired specifications. This method helps to determine, how a specific response is affected by changes in the level of the factors over the specified levels of interest and to achieve a quantitative understanding of the system behavior over the test region.

Artificial neural network
An artificial neural network (ANN) is a mathematical or computational model that is influenced by the structural and/or functional aspects of the biological neural networks which is applied in the estimation and prediction of problems and is also used as an adjusting factors for the parameter of a process instead of a conventional controller(here inhibitor or its analogues). ANN mimics the brain and consists of inputs which are compared to a synapse and are then multiplied by weights of the signal followed by computation by a mathematical function.

Genetic algorithm (GA)
This optimisation method mimics the process of mutation and is based upon the principle “survival of the fittest” and is based on the biological process of evolution that is natural selection. The GA follows mainly three types of rules at each step to create the next generation from the current population: Selection rule selects the individuals, known as parents that contribute to the population of the next generation. Crossover rule combines two parents to form children for the next generation. Mutation rule applies random changes to individual parents to form children. By using ANN coupled GA method, designed an optimized the media for actinomycin V production by using a newly isolated strain of Streptomyces triostinicus and reported 4-folds (yield 452 mg/l) higher yield in optimized media in comparison to the normal production medium (yield 110 mg/l).

Nelder–Mead simplex
NM simplex method is based on a real-parameter black-box optimization method which works well with irregular objective functions. The “term” simplex denotes a regular-sided figure in n + 1 dimension. For two dimensions, the simplex should be an equiangular triangle and for three dimensions, it should be a tetrahedron. NM simplex method for the function of n parameters compares the objective function at the n + 1 vertices of a simplex and gives the worst vertex through stepwise simplification search. During optimization process to maximize lipid production, full factorial and multiple linear regressions were used to fit the polynomials to the data obtained. =Reference=