Nitrogen Sources for Industrial Microbes

Nitrogen Sources

• Industrial microbes can utilize both inorganic and organic nitrogen sources.
• Inorganic nitrogen sources include ammonium salts (e.g., ammonium sulphate, diammonium hydrogen phosphate) or ammonia, which can also adjust the fermentation pH.
• Organic nitrogen sources include amino acids, proteins, and urea.
• Crude nitrogen sources such as corn steep liquor, yeast extracts, peptones, and soya meal are byproducts of other industries.
• Purified amino acids are used only in special situations as precursors for specific products.

Corn Steep Liquor

• Corn steep liquor is a byproduct of starch extraction from maize, first used in penicillin production in the 1940s.
• The composition of corn steep liquor varies depending on the maize quality and processing conditions.
• Concentrated extracts usually contain about 4% (w/v) nitrogen, including a range of amino acids, vitamins, and minerals.
• Any residual sugars are generally converted to lactic acid (9–20%, w/v) by contaminating bacteria.
• Corn steep liquor can be replaced by similar liquors from potato starch production.

Yeast Extracts

• Yeast extracts can be produced from waste baker’s and brewer’s yeast, or other S. cerevisiae strains.
• Alternate sources include Kluyveromyces marxianus grown on whey and Candida utilis cultivated using ethanol, or wastes from wood and paper processing.
• Extracts used in fermentation media are normally salt-free concentrates of soluble components of hydrolysed yeast cells.
• Yeast extracts with sodium chloride concentrations greater than 0.05% (w/v) cannot be used in fermentation processes due to potential corrosion problems.
• Yeast cell hydrolysis is often achieved by autolysis, using the cell’s endogenous enzymes.
• Autolysis can be initiated by temperature or osmotic shock, causing cell death without inactivating enzymes.
• Temperature and pH are controlled throughout autolysis to ensure an optimal and standardized process.
• Temperature control is particularly important to prevent vitamin loss.
• Autolysis is performed at 50–55°C for several hours before raising the temperature to 75°C to inactivate the enzymes.
• Finally, the cells are disrupted by plasmolysis or mechanical disruption.
• Cell wall materials and other debris are removed by filtration or centrifugation, and the resultant extract is rapidly concentrated.
• Extracts are available as liquids containing 50–65% solids, viscous pastes, or dry powders.
• They contain amino acids, peptides, water-soluble vitamins, and some glucose derived from the yeast storage carbohydrates (trehalose and glycogen).

Peptones

• Peptones are usually too expensive for large-scale industrial fermentations.
• Preparation is by acid or enzyme hydrolysis of high protein materials: meat, casein, gelatin, keratin, peanuts, soy meal, cotton seeds, etc.
• Amino acid compositions vary depending upon the original protein source.
• Gelatin-derived peptones are rich in proline and hydroxyproline, but almost devoid of sulphur-containing amino acids.
• Keratin peptone is rich in both proline and cystine but lacks lysine.
• Peptones from plant sources invariably contain relatively large quantities of carbohydrates.

Soya Bean Meal

• Residues remaining after soya beans have been processed to extract the bulk of their oil are composed of 50% protein, 8% non-protein nitrogenous compounds, 30% carbohydrates and 1% oil.
• This residual soya meal is often used in antibiotic fermentations because the components are only slowly metabolized, eliminating the possibility of repression of product formation.

Water

• All fermentation processes, except solid-substrate fermentations, require vast quantities of water.
• In many cases, water also provides trace mineral elements.
• Water is a major component of all media, and important for ancillary equipment and cleaning.
• A reliable source of large quantities of clean water, of consistent composition, is essential.
• Before use, removal of suspended solids, colloids and microorganisms is usually required.
• When the water supply is ‘hard’, it is treated to remove salts such as calcium carbonate.
• Iron and chlorine may also require removal.
• For some fermentations, notably plant and animal cell culture, the water must be highly purified.
• Water is becoming increasingly expensive, necessitating its recycle/reusage wherever possible.
• This minimizes water costs and reduces the volume requiring waste-water treatment.

Minerals

• Normally, sufficient quantities of cobalt, copper, iron, manganese, molybdenum, and zinc are present in the water supplies and as impurities in other media ingredients.
• Corn steep liquor contains a wide range of minerals that will usually satisfy the minor and trace mineral needs.
• Occasionally, levels of calcium, magnesium, phosphorus, potassium, sulphur and chloride ions are too low to fulfil requirements and these may be added as specific salts.

Vitamins and Growth Factors

• Many bacteria can synthesize all necessary vitamins from basic elements.
• For other bacteria, filamentous fungi and yeasts, they must be added as supplements to the fermentation medium.
• Most natural carbon and nitrogen sources also contain at least some of the required vitamins as minor contaminants.
• Other necessary growth factors, amino acids, nucleotides, fatty acids and sterols, are added either in pure form or as less expensive plant and animal extracts.

Precursors

• Some fermentations must be supplemented with specific precursors, notably for secondary metabolite production.
• When required, they are often added in controlled quantities and in a relatively pure form.
• Examples include phenylacetic acid or phenylacetamide added as side-chain precursors in penicillin production.
• D-threonine is used as a precursor in l-isoleucine production by Serratia marsescens, and anthranillic acid additions are made to fermentations of the yeast Hansenula anomaladuring l-tryptophan production.

Inducers and Elicitors

• If product formation depends on a specific inducer compound or structural analogue, it must be incorporated into the culture medium or added at a specific point during fermentation.
• In plant cell culture, the production of secondary metabolites, such as flavonoids and terpenoids, can be triggered by adding elicitors, which may be isolated from various microorganisms, particularly plant pathogens.
• Inducers are often necessary in fermentations of genetically modified microorganisms (GMMs).
• This is because the growth of GMMs can be impaired when the cloned genes are ‘switched on’, due to the very high levels of their transcription and translation.
• Consequently, inducible systems for the cloned genes are incorporated that allow initial maximization of growth to establish high biomass density, whereupon the cloned gene can then be ‘switched on’ by the addition of the specific chemical inducer.

Inhibitors

• Inhibitors are used to redirect metabolism towards the target product and reduce the formation of other metabolic intermediates, or to halt a pathway at a certain point to prevent further metabolism of the target product.
• An example of an inhibitor specifically employed to redirect metabolism is sodium bisulphite, which is used in the production of glycerol by S. cerevisiae.
• Some GMMs contain plasmids bearing an antibiotic resistance gene, as well as the heterologous gene(s).
• The incorporation of this antibiotic into the medium used for the production of the heterologous product selectively inhibits any plasmid-free cells that may arise.

Cell Permeability Modifiers

• These compounds increase cell permeability by modifying cell walls and/or membranes, promoting the release of intracellular products into the fermentation medium.
• Compounds used for this purpose include penicillins and surfactants.
• They are frequently added to amino acid fermentations, including processes for producing l-glutamic acid using members of the genera Corynebacterium and Brevibacterium.

Oxygen

• Depending on the amount of oxygen required by the organism, it may be supplied in the form of air containing about 21% (v/v) oxygen, or occasionally as pure oxygen when requirements are particularly high.
• The organism’s oxygen requirements may vary widely depending upon the carbon source.
• For most fermentations the air or oxygen supply is filter sterilized prior to being injected into the fermenter.

Antifoams

• Antifoams are necessary to reduce foam formation during fermentation.
• Foaming is largely due to media proteins that become attached to the air–broth interface where they denature to form a stable foam.
• Uncontrolled foam may block air filters, resulting in the loss of aseptic conditions, and the release of microorganisms into the environment.
• Minimizing foaming allows increased throughputs.
• Three possible approaches to controlling foam production: modification of medium composition, use of mechanical foam breakers and addition of chemical antifoams.
• Chemical antifoams are surface-active agents which reduce the surface tension that binds the foam together.
• The ideal antifoam should be readily and rapidly dispersed with rapid action, have high activity at low concentrations, prolonged action, non-toxic to fermentation microorganisms, humans or animals, low cost, thermostability, compatibility with other media components and the process, i.e. having no effect on oxygen transfer rates or downstream processing operations.
• Natural antifoams include plant oils (e.g. from soya, sunflower and rapeseed), deodorized fish oil, mineral oils and tallow.
• The synthetic antifoams are mostly silicon oils, poly alcohols and alkylated glycols.
• Some of these may adversely affect downstream processing steps, especially membrane filtration.

Animal Cell Culture Media

• Animal cell culture media are normally based on complex basal media, such as Eagle’s cell culture medium, which contains glucose, mineral salts, vitamins and amino acids.
• For mammalian cells a serum is usually added, such as fetal calf serum, calf serum, newborn calf serum or horse serum.
• Sera provide a source of essential growth factors, including initiation and attachment factors, and binding proteins.
• They also supply hormones, trace elements and protease inhibitors.
• The highly complex composition of sera makes substitution with lower cost ingredients very difficult.
• Sterilization of formulated animal culture media and media constituents is also more problematic as many components are thermolabile, requiring filter sterilization.
• Normally, sera constitute 5–10% (v/v) of the medium, but attempts have been made to reduce and ultimately eliminate its use.
• This is necessary due to its high cost and the fact that it is a potential source of prions and viruses.
• In some circumstances levels have now been lowered to 1–2% (v/v) and some cell lines have been developed that grow in serum-free media.

Plant Cell Culture Media

• In contrast to most animal cell culture media, those used for plant cell culture are usually chemically defined.
• They contain an organic carbon source, a nitrogen source, mineral salts and growth hormones.
• Sucrose is frequently incorporated as the carbon source, particularly for secondary metabolite production, but glucose, fructose, maltose and even lactose have been used.
• Nitrate is the usual nitrogen source, often supplemented with ammonium salts.
• However, some species may require organic nitrogen, normally in the form of amino acids.
• The combination and concentration of plant hormones provided depend upon the specific fermentation.
• Auxins are usually supplied, along with cytokinins to promote cell division.
• A two-phase culture has often proved to be useful in increasing productivity, particularly for producing secondary metabolites such as shikonin.
• The first phase uses a medium optimized for growth, the second promotes product formation.

Culture Maintenance Media

• These media are used for the storage and subculturing of key industrial strains.
• They are designed to retain good cell viability and minimize the possible development of genetic variation.
• In particular, they must reduce the production of toxic metabolites that can have strain-destabilizing effects.
• If strains are naturally unstable, they should be maintained on media selective for the specific characteristic that must be retained.