Media for industrial fermentation

INTRODUCTION

A detailed investigation is needed to establish the most suitable medium for an individual fermentation process, but
certain basic requirements must be met by any such medium. 

All microorganisms require water, sources of energy, carbon, nitrogen, mineral elements, and possibly vitamins plus
oxygen if aerobic. On a small scale it is relatively simple to devise a medium containing pure compounds, but the
resulting medium, although supporting satisfactory growth, may be unsuitable for use in a large-scale process.

For example, we have prepared a GYE medium for the production of alcohol by yeast at the lab level. 

So, in that case, suppose we are preparing 100 ml, 500 ml, 1L, 2L, 5L, or 10L of the medium.  

but the same medium when it has to be used to produce alcohol on large scale/ on an industrial scale, we require a
somewhat large quantity of media (in tonnes/litres). 

That is why it is needed to formulate a suitable medium for the industrial production of the product in such a way that it
should provide the maximum production of the product and it should be cheaper.

Selection criteria for medium and types of the media

On a large scale one must normally use sources of nutrients to create a medium which will meet as many as possible of
the following criteria:

1. It will produce the maximum yield of product or biomass per gram of substrate used.

2. It will produce the maximum concentration of product or biomass.

3. It will permit the maximum rate of product formation.

4. There will be the minimum yield of undesired products.

5. It will be of a consistent quality and be readily available throughout the year.

6. It will cause minimal problems during media making and sterilization.

7. It will cause minimal problems in other aspects of the production process particularly aeration and agitation, extraction,
purification, and waste treatment.

The choice of the medium is very critical for successful product formation. For industrial fermentation, the
microorganisms, in general, utilize a luxury metabolism. Therefore, good production yields are expected with an
abundant supply of carbon and nitrogen sources, besides requisite growth factors. The media used in fermentation
processes may be synthetic or crude.

Two major types of media are 

1. Synthetic media (defined media)

2. Complex media  (undefined media/ crude media)

1. Synthetic media: Media with all the requisite constituents in a pure form in the desired proportion represents synthetic
media. Use of this type of media in fermentation is not practicable.

A defined  medium will have known quantities of all ingredients. 

A defined medium (also known as chemically defined medium or synthetic medium) is a medium in which all
the chemicals used are known)

2. Complex media  (undefined media/ crude media):  An undefined medium has some complex ingredients, such as yeast
extract, which consists of a mixture of many, many chemical species in unknown proportions. 

Undefined media are sometimes chosen based on price and sometimes by necessity – some microorganisms have
never been cultured on defined media.

The non-synthetic media with naturally available sources are better suited for fermentation.

In practice, crude media with an addition of requisite synthetic constituents is ideal for good product yield in
fermentation.

There are many different types of media that can be used to grow specific microbes, and even promote certain cellular
processes; 

Eg: such as wort, the medium is the growth media for the yeast that makes beer. 

Without wort in certain conditions, fermentation cannot occur and the beer will not contain alcohol or be carbonated
(bubbly).

The media used for the growth of microorganisms in industrial fermentation must contain all the elements in a suitable
form for the synthesis of cellular substances as well as metabolic products. While designing a medium, several factors
must be taken into consideration. The most important among them is the ultimate product desired in fermentation.

In the laboratory, pure-defined chemicals may be used for culturing microorganisms. 

However, for industrial fermentation, undefined and complex substrates are frequently used for economic reasons.
Cheaper substrates are advantageous since they minimize the production cost of fermented products.

Wastes from agriculture and by-products of other industries are generally preferred, although they are highly variable in
composition. Raw materials used in fermentation largely depend on their cost at a particular time, since there are
seasonal variations.

MEDIUM FORMULATION

MEDIUM FORMULATION: Medium formulation is an essential stage in the design of successful laboratory

experiments, pilot-scale development, and manufacturing processes. 

The constituents of a medium must satisfy the elemental requirements for cell biomass and
metabolite production and there must be an adequate supply of energy for biosynthesis and cell maintenance. 

The first step to consider is an equation based on stoichiometry for growth and product formation. 

Carbon and energy source + Nitrogen source  + 02 + other requirements ---> biomass products+  CO2 +  H2O
+ heat

This equation should be expressed in quantitative terms, which is important in the economical design of media
if component wastage is to be minimal. 

Thus, it should be possible to calculate the minimal quantities of nutrients which will be needed to produce a specific
amount of biomass.

Knowledge of the elemental composition of a process microorganism is required

for the solution of the elemental balance equation. 
Minimum quantities of N, S, P, Mg, and K should be included in an initial medium recipe. Trace elements (Fe, Zn, Cu, Mn,
Co, Mo, B) may also be needed in smaller quantities.
Raw Materials for fermentation Medium

 Water
 Carbon Source
 Nitrogen source
 Growth factors
 Minerals
 Inhibitors
 Inducers
 Precursors
 Anti-foam agents

WATER:

 It is needed to prepare media.

 It is also needed in many of the ancillary services  such as heating, cooling, cleaning and rinsing.
 It is important to consider pH, dissolved salts and effluent contamination of water.
 Eg:
 Hard waters containing high CaS04 concentrations are better for the English Burton bitter beers and Pilsen type
lagers, 
 while waters with a high carbonate content are better for the darker beers such as stouts.
 water may be treated by deionization or other techniques and salts added, or the pH adjusted, to favour different
beers so that breweries are not so dependent on the local water source.
 Water reuse is highly preferable in fermentation.

Substrates Used As Carbon Sources:

Carbohydrates constitute the most predominant source of energy in fermentation industry. Refined and pure
carbohydrates such as glucose or sucrose are rarely used for economic reasons.

1. Molasses:

 Molasses is a byproduct of sugar industry and is one of the cheapest sources of carbohydrates. 

 Sugar cane molasses (sucrose around 48%) and sugar beet molasses (sucrose around 33%) are commonly used.
Besides being rich in sugar, molasses also contain nitrogenous substances, vitamins and trace elements.
 Hydrol molasses, a byproduct in glucose production from corn is also used as a fermentation substrate. It contain
60% sugar but also high salt concentration.
 There occurs variation in the composition of the molasses which mostly depends on the climatic conditions and
production process. 

2.  Malt extract:

Barley grains  traditionally used as carbon source in brewing and formerly also in yeast production may be partially
germinated and heat treated to give the material known as malt, which contains a variety of sugars besides starch.

Malt extract, an aqueous extract of malted barley, contains about 80% carbohydrates (glucose, fructose, sucrose, and
maltose). Nitrogen compounds constitute around 4.5% (proteins, peptides, amino acids, purines, pyrimidine’s).

3. Starch, dextrin and cellulose:

The polysaccharides-starch, dextrin and cellulose can be metabolised by microorganisms. They are frequently used for
the industrial production of alcohol. Due to its wide availability and low cost, the use of cellulose for alcohol production is
extensively studied.

4. Whey:
Whey is a byproduct of dairy industry and is produced worldwide. Most of it is consumed by- humans and animals. Whey
is a reasonably good source of carbon for the production of alcohol, single-cell protein, vitamin B12, lactic acid and
gibberellic acid. Storage of whey is a limiting factor for its widespread use in fermentation industry.

5. Methanol and ethanol:

Some of the microorganisms are capable of utilizing methanol and/or ethanol as carbon source. 

Methanol is the cheapest substrate for fermentation. However, it can be utilized by only a few bacteria and
yeasts. Methanol is commonly used for the production of single-cell protein. 

Ethanol is rather expensive. However, at present it is used for the production of acetic acid.

Substrates Used As Nitrogen Sources

The nitrogen supply to the fermentation microorganisms may come from inorganic or organic sources.

 Inorganic nitrogen sources:

 Ammonium salts and free ammonia are cheap inorganic nitrogen sources, particularly in industrialized countries.
However, not all the microorganisms are capable of utilizing them, hence their use is limited.
 Inorganic nitrogen may be supplied as ammonia gas, ammonium salts or nitrates.
 Ammonia has been used for pH control and as the major nitrogen source in a defined medium for the commercial
production of human serum albumin by Saccharomyces cerevisiae.

Organic nitrogen sources:

Urea is fairly a good source of nitrogen. However, other cheaper organic forms of nitrogen sources are preferred.

Corn steep liquor:

This is formed during starch production from corn. Corn steep liquor is rich in nitrogen (about 4%) and is very efficiently
utilized by microorganisms. It is rich in several amino acids (alanine, valine, methionine, arginine, threonine, glutamate).

Yeast extracts:

They contain about 8% nitrogen and are rich in amino acids, peptides and vitamins. 

Glucose formed from glycogen and trehalose during yeast extraction is a good carbon source. 

Yeast extracts are produced from baker’s yeast through autolysis (at 50-55°C) or through plasmolysis (high
concentration of NaCI). Yeast extracts are very good sources for many industrially important microorganisms.

Soy meal:

After extracting the soy bean oil from the soy bean seeds, the left out residue is soy meal. It is rich in proteins (about
50%) as well as carbohydrates (about 30%) contents. Soy meal is often used in antibiotic production.

Peptone:

The protein hydrolysats are collectively referred to as peptone, and they are good sources for many microorganisms. 

The sources of peptone include meat, soy meal, peanut seeds, cotton seeds and sunflower seeds.

The proteins namely casein, gelatin and keratin can also be hydrolyzed to yield peptone.
In general, peptone derived from animal sources have more nitrogen content while those from plant sources have more
carbohydrate content. Peptones are relatively more expensive, hence not widely used in industries.

Minerals, chelators, Growth factors

Minerals:

 In many media, magnesium, phosphorus, potassium, sulphur, calcium and chlorine are essential components,
and because of the concentrations required, they must be added as distinct components.
 Others such as cobalt, copper, iron, manganese, molybdenum and zinc are also essential but are usually present
as impurities in other major ingredients.

The concentration of phosphate in a medium, particularly laboratory media in shake flasks, is often much higher than that
of other mineral components. Part of this phosphate is being used as a buffer to minimize pH changes when external
control of the pH is not being used.

The inorganic phosphate concentration also influences production of bacitracins, citric acid (surface culture), ergot,
monomycin, novobiocin, oxytetracycline, polyenes,  streptomycin etc.
An important function of calcium salts in fermentation media was to precipitate excess inorganic phosphates, and
suggested that the calcium indirectly improved the yield of streptomycin.

Chelators:

 Many media show formation of a visible precipitate of insoluble metal salts (salts of phosphates, sulphates etc.)
after  autoclave.
 The problem of insoluble metal phosphate(s) may eliminated by incorporating low concentrations chelating
agents such as ethylene diamine tetra acetic acid (EDTA), citric acid, polyphosphates, etc., into medium. These
chelating agents preferentially complexes with the metal ions in a medium.
 The metal ions then may be gradually utilized by the microorganism.

GROWTH  FACTORS:

 Some micro-organisms cannot synthesize a full complement of cell components and therefore require preformed
compounds called growth factors.
 The growth factors most commonly required are vitamins, but there may also be a need for specific amino acids,
fatty acids or sterols.
 It is important to remember that if only one vitamin is required it may be occasionally more economical to add the
pure vitamin, instead of using a larger bulk of a cheaper multiple vitamin source.
 Calcium pantothenate has been used in one medium formulation for vinegar production (Beaman, 1967).
 In processes used for the production of glutamic acid, limited concentrations of biotin must be present in the
medium Some production strains may also require thiamine.

Precursors, Inducers, elicitors, inhibitors

Some components of the medium help to regulate the production of the desired product rather than support the
growth of the micro organisms. 

This includes precursors, inhibitors, inducers and eliciters.

Precursors: 

Precursors are defined as “substances added prior to or simultaneously with the fermentation which are

incorporated without any major change into the molecule of the fermentation product and which generally serve to
increase the yield or improve the quality of the product”.
E.g.: Corn-steep liquor was found to contain phenylethylamine which was preferentially incorporated into the
penicillin molecule to yield benzyl penicillin (Penicillin G).

Phenylacetic acid is still the most widely used precursor in penicillin production.

Inhibitors:

Inhibitors are added to redirect metabolism towards the target product and reduce formation of other
metabolic intermediates. They may also favour the accumulation of a metabolic intermediate by inhibiting its
further metabolism.

When certain inhibitors are added to fermentation process, more of a specific product may be produced. 

Eg: Microbial production of Glycerol is favoured by removing Acetaldehyde from ethanol fermentation. 

Sodium bisulphite is added to the broth resulting into formation of Acetaldehyde bisulphite. 
An acetaldehyde is not available, so NADH2 is reoxidized by dihydroxy acetone phosphate producing glycerol-
3-phosphate which is converted to glycerol.

Inducers and elicitors:

Inducers and elicitors are those specific chemical compounds, when incorporated in media they induce or
provoke formulation of desired industrial fermentation products.

They are added into the culture medium or are added at a specific point during the fermentation process.

The majority of enzymes which are of industrial interest are inducible. Induced enzymes are synthesized only in response
to the presence in the environment of an inducer.

Inducers are often substrates such as starch or dextrins for amylases, maltose for pullulanase and pectin for pectinases.

One unusual application of an inducer is the use of yeast mannan in streptomycin production.

In plant cell culture, the production of secondary metabolites such as flavonoids and Terpenoids can be triggered by
adding elicitors which may be obtained from various microorganisms especially from plant pathogens.

Antifoam Agents and oxygen requirements

ANTIFOAMS:

 In most microbiological processes, foaming is a problem.

 It may be due to a component in the medium or Some factor produced by the micro-organism.
 The most common cause of foaming is due to proteins in the medium, such as corn-steep liquor, Pharmamedia,
peanut  meal, soybean meal, yeast extract or meat extract.

There are three ways of approaching the problem:

1. To try and avoid foam formation by using a defined medium and a modification of some of the physical parameters
(pH, temperature, aeration and agitation). This assumes that the foam is due to a component in the medium and not a
metabolite.

2. The foam is unavoidable and antifoam should be used. This is the more standard approach.

3. To use a mechanical foam breaker.

The following compound highly used as antifoam agents:

1. Alcohols; stearyl and octyl decanol.

2. Esters.

3. Fatty acids and derivatives, particularly glycerides which include cottonseed oil, linseed oil say-bean oil, olive oil, castor
oil, sunflower oil rapeseed oil and cod liver oil.

4. Silicones.

5. Sulphonates.

6. Miscellaneous; Alkaterge C, oxazaline, polypropylene glycol.

Oxygen requirement:

It is sometimes forgotten that oxygen, although not added to an initial medium as such, is nevertheless a very important
component of the medium in many processes, and its availability can be extremely important in controlling the growth
rate and metabolite production.

The medium may influence the oxygen availability in a number of ways including the following:

1. Fast metabolism. The culture may become oxygen limited because sufficient oxygen cannot be made available in the
fermenter if certain substrates, such as rapidly metabolized sugars are added.  It lead to a high oxygen demand, when
available in high concentrations.

Eg: Penicillium chrysogenum  will utilize glucose more rapidly than lactose or sucrose, and it therefore has a higher
specific oxygen uptake rate when glucose is the main carbon source.

2. Rheology. The individual components of the medium can influence the viscosity of the final medium and its
subsequent behavior with respect to aeration and agitation.

Eg: Polymers in solution, particularly starch and other polysaccharides, may contribute to the rheological behavior of the
fermentation broth.

3. Antifoams. Many of the antifoams in use will act as surface active agents and reduce the oxygen transfer rate.