Fermentation Medium preparation (Large scale Production)

FERMENTATION MEDIUM

Fermentation - a metabolic process of conversion of sugars into carbon-di-oxide and alcohol in the anaerobic environment.

·       Fermentation is an enzymatic process by bacteria, fungi, and algae.

·       Microbes require carbon, nitrogen, and mineral sources - medium for growth and metabolism.

Ex: Lactic acid fermentation = the pyruvic acid is reduced to lactic acid by lactate dehydrogenase.

        Alcoholic fermentation - Wine and beer.

 

Ø  New definition: a form of metabolism in which the end products could be further oxidized.

o   For example a yeast cell obtains 2 molecules of ATP per molecule of glucose when it ferments it into ethanol.

o   Fermentation takes place in the absence of oxygen when the electron transport chain is unusable.

o   It is used by the cell not to generate energy directly, but to recycle NADH into NAD+ so that glycolysis can continue, as long as glucose is present.

Ø following criteria need to be satisfied for the material to be treated as a medium at the industrial level.

§  It should give a maximum yield of product.

§  It should give a minimum yield of undesired products.

§  It should be consistently available throughout the year.

§  It should be cheap.

§  Must not affect the physiochemical parameters of the fermentation vessel such as temperature and aeration supply.

§  It should not interact negatively with other constituents of the fermentation medium.

§  Fermentation media must not affect the cleaning and sterilization process of the vessel.

Types of Media:

            1. Solid

            2. Liquid

            3. Semi-Solid

·       Depending on the materials used for medium formulation, fermentation media are classified as chemically defined or undefined media.

·       ­A defined medium is composed of known amounts of pure chemicals

·       A complex medium includes ingredients of natural origin, such as protein hydrolysates and extracts from plants or meat.

Complex undefined Media

Most standard microbial fermentation media contain hydrolysates and extracts from meat, casein, soy, and yeast with unknown chemical compositions.

1.      Peptones are protein hydrolysates - many complex media contain peptones as the source of nitrogen.

2.      Meat and plant extracts are commonly used as sources of nutrients and contain a mixture of amino acids, carbohydrates, minerals, and trace elements.

3.      Yeast extract is usually produced from baker’s or brewer’s yeast through autolysis and is a key component of complex media.

Cheap compounds support high productivity and are predominantly used in the commercial fermentation industry.

Requires extensive testing of raw materials used in industrial fermentations.

Defined media:

v  Commonly used to assure highly reproducible cultural conditions

v  Contain pure chemicals in known concentrations

o   Glucose, ammonia, minerals (PO₄, SO₄, Mg, K, and Cl), and trace elements are fulfilled.

v  Quite expensive

Advantages of defined media:

v  Improved cell performance

v  Favourable characteristics at large-scale fermentations

v  Better control and monitoring

v  Improved downstream processing

Sources of Media:

Carbon source:

v  Most common form of energy source.

v  Glucose is generally a preferred carbon source for microorganisms.

v  It is the most commonly used sugar in fermentation media and is efficiently converted into lipids by several yeast and fungi.

v  Monosaccharides, disaccharides, and polysaccharides promote good growth of different species.

Ø  Inhibitory effect of carbohydrates: glucose, fructose, sucrose, galactose, rhamnose, and ribose in media affected the production of the cell wall and CPS by strains of Lactobacillus rhamnosus.

Ø  Other sources of carbon:

o   Molasses, Malted barley, Starch and dextrins, sulfite waste liquor, alkanes and alcohols, n-alkanes, oils, and fats.

Ø  Amount of carbon sources used per liter is 100 to 200 g in citric acid fermentation for A.niger.

Nitrogen Sources

Ø  Two classes of nitrogen sources: inorganic and organic nitrogen sources.

Ø  Inorganic nitrogen sources are ammonium salts - ammonium sulfate and diammonium hydrogen phosphate, or ammonia.

Ø  Nitrogen is needed for the synthesis of amino acids, DNA, RNA, and ATP, among other molecules.

Ø  Depending on the organism, nitrogen, nitrates, ammonia, or organic nitrogen compounds can be used as a nitrogen source.

Ø  Organic nitrogen sources include amino acids, proteins, and urea.

o   soybean, peanut, and cottonseed meal, corn pulp, and steep

o   Corn Steep Liquor

o   Yeast Extracts

o   Peptones

Minerals

Ø  All microorganisms require certain mineral elements for growth and metabolism.

Ø  In many media magnesium, phosphorous, potassium, sulfur, calcium and chlorine are essential components and must be added.

Ø  Frequently added minerals are potassium salts, KH₂PO₄ and K₂HPO₄, or a mix of the two.

Ø  Others such as cobalt, copper, iron, manganese, molybdenum, and zinc are present in sufficient quantities

Chelators

Ø  Chelating agents are chemical organic compounds that react with metal ions to form a stable, water-soluble complex.

Ø is Also known as chelates, chelators, or sequestering agents.

Ø  Many media cannot be prepared without precipitation during autoclaving.

Ø  Hence some chelating agents are added to form complexes with metal ions which are gradually utilized by microorganisms.

Ø  Chelating agents have a ring-like center that forms at least two bonds with the metal ion allowing it to be excreted.

Ø  Specific chelating agents bind iron, lead, or copper in the blood and can be used to treat excessively high levels of these metals.

Ø  Examples of chelators: EDTA, citric acid, polyphosphates, Oxalic acid, Malic acid, Rubeanic acid, and Citric acid.

Ø  It is important to check the concentration of chelators otherwise it may inhibit the growth.

Ø  In many media these are added separately after autoclaving or yeast extract, peptone complex with these metal ions.