B: Questions

Question 1

Advantages of crude media

Answer 1

Advantages of crude media: (a) Higher product yields; (b) Relatively cheap.

Question 2

Disadvantages of crude media

Answer 2

Disadvantages: (a) Foaming; (b) Product recovery more complex; (c) Variable availability to microbes (may need pre-treatment); (d) Unwanted components; (e) Batch-to-batch variation in some components.

Question 3

List two carbon sources (negative cost raw materials are commonly used) and two nitrogen sources commonly used in industrial fermentation processes

Answer 3

Nitrogen sources (corn steep liquor, yeast extract, peptones, soymeal) and Carbon sources (molasses, malt extract, starch, sulfite waste liquor, cellulose, whey, fats)

Question 4

What is the Maillard reaction and what is the significance of this for microbiological culture media?

Answer 4

The Maillard reaction is a non-enzymatic browning reaction caused by heating sugars and proteinaceous material in acid solution. Heating solutions of carbohydrates may cause hydrolysis of complex sugars or the formation of oxidation products (common with solutions of low pH, relatively low water content, and containing a high proportion of reducing sugars). This can cause some amino acids to be unavailable for metabolism (especially lysine).

Question 5

What is the impact of the Maillard reaction on fermentation processes and how may it be avoided/reduced?

Answer 5

The impact of the Maillard reaction is that it reduces the nutritive content of the media. This can be avoided by sterilising the sugars and proteins separately

Question 6

What is a precursor?

Answer 6

Precursors are substances 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.

Question 7

Requirements of a buffering agent in a culture medium/fermentation growth medium?

Answer 7

Should be very soluble in water and non-volatile; medium concentration, temperature and ionic strength should have negligible effect on buffer capacity; the buffer should not absorb light in the UV-Vis region of the spectrum; buffer components should be non-toxic, free of inhibitors and non-metabolisable.

Question 8

Water in medium

Answer 8

Water is an important component of the medium (representing >70% and often >90% of composition); clean water of consistent composition is vital. Water may also supply trace minerals (Cu, Mo, Zn and Bo). Water quality can be critical (especially re the flavour of a beverage product) and can even influence the location of production facilities such as breweries.

Question 9

Water purification

Answer 9

Purified water is generated from potable water, and purification schemes are designed to take into account the potential impurities in this source.
1. Potable water is subjected to depth filtration (either sand or multi-media filters)
2. Passed through activated charcoal (which removes chlorine and soluble organics – prolongs the lifetime of the anion ion exchange)
3. Run through an anion- cation exchange resin (cations are exchanged for H+ and anions exchanged for OH-)
4. Deionization step monitored by measuring the resistivity
4. Filters are then deployed to remove microbes (0.22 and 0.45 μm).
5. These steps yield purified water.

Water-for–Injection (WFI) is the highest purity and is used in aspects of downstream processing and final product finishing. WFI is generated by further purification steps: either reverse osmosis or distillation

Question 10

Highly Purified Water

Answer 10

HPW may be prepared by combinations of methods such as reverse osmosis, ultrafiltration and deionization. Water-treatment methods are not considered to be as reliable as distillation

Question 11

Carbon functions

Answer 11

The central element of all organic life forms, as well as the major energy source for chemoheterotrophic-type microbes

Question 12

Buffers

Answer 12

Retard gross changes in pH values due to an acidic or an alkaline fermentation product.

pH changes during growth are due to:
1. breakdown of proteins and nitrogeneous compounds to form ammonia or similar alkaline products
2. uptake of certain anions and cations;
3. formation of organic acids from utilization of carbon substrates;
4. the use of nitrate or sulphate as terminal electron acceptors;
5. the use of NH + or sulphate as an energy source.

Main effects of adverse pH on the cell include:
1. altering cell wall permeability and influencing the rates of reactions associated with the enzymes bound to the cell wall
2. may also affect the type of product formed; optimum pH for product formation is usually different from that required for growth.

Question 13

Buffer example

Answer 13

Phosphate buffer is commonly used (KH2PO4; pH range 6.5 – 8.0)

Question 14

Foaming

Answer 14

Foaming reduces the working volume of the fermentor and can cause incorrect monitoring and control.

Question 15

Antifoam/defoaming agents

Answer 15

Antifoam/defoaming compounds are surface active agents, containing insoluble particles that spread across the surface of the air-liquid interface

Reduce the viscosity and surface tension in the foam lamellae

Destabilize protein films by penetrating the bubble wall, spreading the liquid-gas interface and allowing the bubble to drain

Bubble becomes unstable and collapses

Antifoam agents may be inert (silicone-based or polypropylene glycol, block copolymers) or crude-organic (oils - lard oil, soy oil, corn oil, palm oil).

Question 16

Inducers

Answer 16

induced enzymes are synthesized only in response to the presence in the environment of an inducer (substrate). Most of the enzymes of industrial interest are inducible.

Question 17

Trace elements/minerals

Answer 17

essential for growth when present at low concentration, but toxic at high concentration (in g/100 g dry biomass: Ca, 0.1; Fe, 0.015; Mn, 0.005; Zn, 0.005; Cu, 0.001 and Mo, 0.001). Toxic concentrations are in the order of 10-4 M.

Question 18

Growth factors

Answer 18

organic nutrients that cannot be synthesized by the cell and must be provided in the medium (B group vitamins, certain amino acids, fatty acids or sterols). Yeast products such as cell hydrolysates or autolysates are a good source of growth factors.

Question 19

Medium Aspect 1 - Substrates

Answer 19

Provide substrates for the production of new cell material and extracellular products, economically. The latter encompasses providing energy necessary for driving synthetic reactions and also maintaining concentrations of materials inside cells which are different to those of the environment.

Question 20

Medium Aspect - YOP

Answer 20

Medium must maximize the yield of product or biomass per gram of substrate used and also the rate of product or biomass formation:
If biomass or a growth-associated product is the objective of the fermentation, then the medium must permit maximum growth potential throughout the process.

Compound - not growth limited (such as Abs), a medium developed which after initial growth phase, becomes deficient in one or more nutrients.

Limitation of P, N, carbohydrate or trace metals have all been used successfully.

Question 21

Medium aspect - cheap

Answer 21

The medium must be as cheap as possible (raw materials can account for 60 – 80% of the variable costs of fermentation), be of consistent quality and be available all year round (commodity market prices fluctuate with seasonal and other variables).

Question 22

Medium Aspect - not interfere

Answer 22

Medium components should not interfere with other aspects of the production process (such as the product purification steps). The medium should preferably be low in viscosity, provide for easy cell separation and be low in residual compounds that could adversely affect the final product specification.