Presently, microorganisms represent a greater percentage of the biological agents used in Biotechnology. The reasons for the domineering roles of microorganisms in biotechnology include

1. They can metabolize very wide range of materials, degrading, de-toxifying or converting them to various useful materials.

2. Their relatively simple structure makes it easy to genetically modify them to perform various functions. For example, microorganisms have been genetically engineered to produce non-microbial products such as insulin, somatostatin, human growth hormones, virus vaccines, and interferon.

3. They can be modified to achieve shorter fermentation time, reduce production of undesired by-products, reduce oxygen demand, decrease foaming, use inexpensive substrates, and improve other growth and fermentation characteristics.

4. In comparison with plant or animal cells, they have very high growth rates. The specific growth rate can be as high as 0.8 h^-1. The high growth rates mean lower risk of contamination and also higher productivity. 

5. There are microorganisms that can grow in almost every environment. Examples include Psychrophiles, Thermophiles, Acidophiles, Alkalonophiles and Osmophiles.

6. In comparison with plant and animal cells, the media for most microorganisms are very cheap.

7. Vast range of metabolites are produced by microorganisms. The areas of application include food production, health care goods, mineral processing, and pest/disease control. The type of product depends on the type of media and the culture condition. In other words, a single microorganism can be used to produce different products. Even under the same culture condition, they can simultaneously produce various products.

8. They also have environmental applications. They can be used for bio-remediation of air, soil and water.

Selection of microorganisms for Biotechnological applications

It is known that only a very small percentage of microorganisms are used in Biotechnology. Many of them cannot yet be isolated and maintained in pure cultures, while a lot of those that are maintained in pure cultures are not used industrially because of their innate characteristics. Some criteria used to select microorganisms for Biotechnological applications include

1. Nutritional requirement: Those that require simple, cheap and readily available nutrients are preferred.
2. Growth characteristics: It is desirable to use microorganisms with high growth rates, that do not produce viscous culture broth, and do not produce excessive foaming.
3. Temperature characteristics: The required temperature characteristics depend on the purpose of cultivation. In some cases, thermophiles are required and in other cases, psychrophiles may be required. Yet, some processes required microorganisms that can grow well in wide range of growth temperatures so that the cost of controlling temperature is reduced.
4. Oxygen requirement: Microorganisms that can grow under atmospheric air but with low oxygen demand are preferred.
5. Phenotypic and genotypic stability: It is desirable to use microorganisms that are genotypically and phenotypically very stable so that they can be used for continuous processes or repeated batch processes for a very long period of time without problems of mutation.
6. Amenability to genetic manipulation: Typically, wild species of microorganisms have some innate limitations and require genetic manipulation for increased efficiencies. Thus, those microorganisms that can easily be genetically manipulated are preferred.
7. Productivity: Microorganisms with high productivity (rate of product formation) are desirable for economic reasons.
8. Product yield: For most biotechnological products, the cost of raw materials (substrate) represents a very high percentage of the final production cost. Thus, microorganisms with very high product yields (g-product/g-substrate) are needed for efficient conversion of the substrates into the products.
9. Product recovery: The cost of product separation and purification can be very high. Thus it is necessary to use microorganisms that produce relatively pure products (very low by-products and contaminants) and/or products that can easily be separated from the by-products.
10. Chemical composition and nutritional values: In the case of single cell protein production, it is necessary to select microorganisms with very high nutritional values.

Nutritional requirement of microorganisms

Introduction

Biological agents (microorganisms, plant and animal cells) require nutrients for growth, metabolism, maintence and product formation. All the necessary mutrients must be supplied in adequate amount in order to get the best out of the cells. The first step in developing an industrial process with any microorganism is therefore to understand its nutrient requirements. It is necessary to understand and supply the required nutrients in order to

1. Optimize growth of the cells
2. Maximize product formation
3. Maximize product/biomass yield from the supplied substrate(s).

It is important to note that the optimum medium (nutrients) and culture conditions for maximum cell growth may be different from those required for maximum product formation. For example, in cultivation of yeast cells, excess supply of nitrogen and adequate aeration are necessary to achieve high cell growth and biomass yield but production of ethanol is favored by reduced nitrogen sources and low dissolved oxygen concentrations.

A knowledge of the nutrient requirement of the microorganism brings about reduced cost of production because:

1. No nutrient is supplied in excess (no wastage of nutrients).
2. High growth and product formation rates mean that cultivation time is reduced, leading to reduced labour and space costs.
3. High product yield and product formation rate leads to high product concentration. This in turn leads to lower cost of down stream processing (separation and purification of the product).

Elements required by Microorganisms

Nutrients required by microorganisms can be classified into i) Macro or major elements, ii) Micro or trace elements, and iii) Growth factors. There are about 109 elements in the periodic table and it has been demonstrated that 35 to 40 of them are required by microorganisms. However, the number and amount required may vary even among species within the same genera. Also for an individual strain, the required types and amounts of these elements vary depending on the purpose of the culture and even the growth phase of the microorganism.

Macro elements

Six non-metals (C, O, H, N, P, S) and two metals (K and Mg) comprise an average of 98% of dry weight of bacteria and fungi. These are collectively called macro or major elements. They are required in concentrations more than 10^-4 moles/L of the medium. The total weight of microorganisms comprises 80 to 90% water while C, O, H, and N, which are the main constituents of cellular materials comprise 90 to 95% of the dry weight. These figures vary depending on the microorganism, the growth phase (age) and the culture conditions.

Micro elements

These are also called trace elements or micro-nutrients. As the same suggests, these elements are required in very minute quantities by microorganisms for growth. The required concentration is usually less than 10^-9 mol/L of the culture media. Some important microelements required by many microorganisms include Mn, Zn, Fe, Cu, Co, Ca, Na, Cl, Ni, and Se. Other micro-elements such as B, Al, Si, Cr, As, V, Sn, Be, F, Sc, Ti, Ga, Ge, Br, Zr, W, Li, and I are rarely required by many microorganisms.

  Although lack of an essential micro-element can completely prevent growth, in most cases, deficiency is manifested in a batch culture by an increase in the lag phase and subsequent decrease in the specific growth rate, decrease in the biomass (dX/dS) and/or decrease in the product (dP/dS) yield coefficients.

Growth factors

Some complements of cell components cannot be synthesized by some microorganisms from the simple nutrients. Thus some microorganisms have absolute requirement for preformed organic molecules, which form building blocks of cell components. Such compounds are called growth factors. They stimulate the growth of microorganisms by decreasing the synthetic load on the cell. The growth factors can be classified into i) vitamins, ii) Amino acids, and iii) Miscellaneous growth factors.

Culture preservation and cell cultivation

Introduction

Success of any microbial process depends to a great extent on the characteristics (capabilities) of the microbial strain. Strain development (isolation, selection, and strain improvement using various techniques) can be very expensive. It is therefore very important to preserve the strain. As cells grow and multiply, they tend to degenerate (change their characteristics) in successive generations. The aims of microbial strain preservation include the following:

i) To maintain the cell viability.

ii) To maintain the growth characteristics of the cells.

iii) To maintain the capacity for product formation.

In other words, strain preservation is aimed at maintaining the genetic constituents (genotype) of the strain. One method of achieving this is to store the cells under a condition where they will remain viable for a long period of time without cell division. However, during storage, the viable cell number continues to decrease gradually and there is thus a need to transfer the cells to a new medium. The cells again grow to a desirable extend and are again stored. The longer the cells can be stored before transferring to a new medium (sub-culturing), the better is the preservation condition.

In terms of preservation, the cells can be divided into “master” strain and “working” strain. Master strain is the main stock strain preserved over a long period of time. They are used to generate a working strain when there is a problem with the working strain. It is a “reference” strain to which the characteristics of the working strain are occasionally compared. It is also the stock used for further genetic improvement. They are transferred into a new culture only once in a few years. On the other hand, working strains are used for routine production and are thus stored and transferred to new medium once in a few weeks.

Preservation of the working strain

The working strain are usually stored in a refrigerator at temperatures of 2 to 6 ℃. They can be kept as stab or slant cultures in agar, or in liquid cultures. The strains are transferred to new cultures once in 8 to 16 weeks, depending on the strain. Under this condition, growth is not completely stopped and contamination risk is relatively high. There is thus a need to occasionally check the sterility, growth characteristics and their capacity for product formation. Some cells can even be stored in sterile distilled water for some weeks without any problem.

Preservation of master strain

The master strains are preserved by freezing at –18℃ or –80℃ in deep freezers, or at –196℃ under liquid nitrogen. In the case of –196℃, it is necessary to reduce the temperature gradually in order to avoid rapid decline in cell viability. Typically, the temperature is reduced at a rate of 1℃/min. Furthermore, protecting agents such as glycerol are added to avoid crystal formation. Although these types of cultures can be kept for several years, a high percentage of the cells die during freezing and subsequent thawing. Another effective method of preserving master strain is lyophilization (freeze drying). As in the case of freezing, protective agents such as skim milk and sucrose are added to prevent rapid decrease in cell viability.

Fundamentals of Cell cultivation

Cell cultivation means to grow the cells. The cells are inoculated into an appropriate medium and allowed to grow under desired conditions of temperature, pH, oxygen tension, agitation etc.  Cultivation of cells may involve three various stages of cell activation, seed culture and main cultivation.

Cell activation.

During preservation, the cells loose viability and most of the metabolic activities are arrested. Before inoculating such cultures into a seed or main culture, it is necessary to activate them by transferring into a fresh medium under optimal conditions. During this period, the cells regain their viability (activity) through activation/induction of various metabolic enzymes. Cells preserved by freezing or lyophilizing require several days to be activated but those stored in refrigerator can be activated within a few hours. However, the activation period depends on the strain. For example, fungi and Actinomycetes require several days even for refrigerated cultures.

Seed cultures

Seed culture (also called pre-culture) is the culture that is used to inoculate the main (production) culture. The activated cells are grown to log phase and used to inoculate the main culture. There is an optimum amount of inoculum for each culture. Depending on the strain and process, the percentage inoculum ranges from 0.5 to 10%. If very low percentage inoculum is used, the lag phase is prolonged and the subsequent grow and product formation may not be satisfactory. When the volume of the main culture is large, the seed culture has to be done in stages. For example, assuming an inoculum percentage of 10%, a 100L main culture requires 10L seed culture. This can be done in 4 stages. The activated cells are used to inoculate 100 mL of the seed medium, it is cultivated to log phase and used to inoculate 1.0L bioreactor. This is also cultivated to log phase and used to inoculate 10.0L bioreactor and this is subsequently used to inoculate the 100L seed culture. If small volume of activated culture is used to directly inoculate lare volume of the main or seed culture, the lag phase will be unnecessarily long and subsequent cell growth may not be satisfactory. Another important point about the seed culture is that the medium and culture conditions for the main culture are considered in choosing the medium and conditions for the seed culture. It is also important to state that extra care is required to ensure that both the activation and seed cultures are not contaminated. Contamination at these stages of the culture will in most cases result in total process failure.

Main cultures

Depending on the processes, the main culture can be as large as 500m³. The appropriate volume of the seed culture is used to inoculate the main culture. The culture is then maintained at optima conditions of temperature, dissolved oxygen concentration, pH, pressure, agitation, and other conditions depending on the strain and process (see factors that affect cell growth). It is important to note that the optima conditions for cell growth may not be the optima conditions for product formation. In such cases, a compromise has to be made between cell growth and product formation. Alternatively, the cultivation is done in stages. During the initial stage, the conditions are controlled to favour cell growth and after obtaining the desired cell concentration, the conditions are changed to favour product formation. For flask cultures, the temperatures are controlled by cultivating in temperature-controlled incubators or culture rooms. However, bioreactors are equipped with heating and cooling devices for temperature control. Oxygen is supplied by aeration. The required dissolved oxygen concentration is achieved by increasing the aeration rate, increasing the agitation speed or increasing the oxygen partial pressure in the aeration gas (by using pure oxygen gas or mixing oxygen and air in various proportions). The culture pressure can be increased to between 0.2 and 0.5 bar in order to reduce the risk of contamination. Increasing the pressure also increases gas solubility in the culture broth. However, high pressure has adverse effects on the growth of some target microorganisms. Stirring is also desired to break up air bubbles and thus increase oxygen transfer, keep the culture homogeneous, and prevent cell sedimentation. However, many cells, especially cells without cell wall and filamentous cells are very sensitive to hydrodynamic stress. Also depending on the strain and process, it may be necessary to control the substrate and product concentrations to avoid substrate or product inhibition and substrate limitation.

Microbial cell culture systems

Microbial cell culture systems can be classified into liquid surface culture, solid state culture and submerged cultures.

Liquid surface culture

This is perhaps the oldest culture system. Here microorganisms are inoculated into un-aerated non agitated culture medium in shallow trays. The microorganism grows on top of the culture broth, forming a mart.  The microorganisms obtain oxygen from the air space, and produce metabolites which are secreted into the culture broth. In its simplest form, the culture is carried out under ambient temperature and there is no deliberate attempt to control any of the culture parameters. However, the modern systems are conducted in temperature controlled chambers with good air circulation to control the oxygen partial pressure in the head space. Depending on the strain of microorganisms and target products, the humidity inside the chambers is also controlled. In order to maximize space, the trays are stacked, forming cascade system (Figure 6-1).

Figure A cascade system for liquid surface culture

  Liquid surface culture has been used for industrial production of many enzymes and antibiotics because it is very simple and requires minimal energy input. However, it has several disadvantages which include low biomass concentration and thus low productivity, increased risk of contamination, and requirement for large surface area per volume of the culture broth. Although it is suitable for cultivation of many species of filamentous fungi, it is not suitable for unicellular microorganisms such as bacteria and yeast cells that would sediment rather than float on top of the culture broth.

Solid state Culture

This is a method by which microorganisms are cultivated on solid substrates in the absence of free water. The microorganisms are inoculated on the substrate where they grow in the solid/gas interface.

On a small/laboratory scale, agar medium containing necessary nutrients for cell growth and product formation can be used as the substrate for solid state fermentation. Polymers, starch, cellulose, pectins, and lignin can also be used but should be mixed with other substances such as rice or wheat bran to improve the nutrient contents and texture of the substrate. In large scale, composite and heterogenous products from agriculture or by-product of agro-industries are used. These include whole or coarsely broken grains, mashed tubers, lignocellusolic materials, and other agricultural products. The substrate must contain carbon, nitrogen, minerals and other nutrients required by the process. Where necessary, the substrates are supplemented with nutrients that are not sufficient in the substrate. The substrate can be buffered to reduce pH changes during the cultivation. Rice bran is known to be rich in nutrients and is usually added to grains as nutrient supplements and also to improve the texture of the substrate. Other desired characteristics of substrates for solid state cultures are i) ability to absorb and retains nutrient supplements, ii) ability to absorb and retain sufficient water to give relatively high water activity at the solid/gas interface for the growth of microorganism, and iii) the particle sizes should be small enough to provide large surface area for the growth of the cells and for good retention of moisture but large enough to allow for good air circulation.

  The substrate is sterilized by steaming which also helps to cook(soften) the substrate for easy metabolism by the microorganism. The steaming process supplies enough water so usually no deliberate effort is made to add more water before inoculation. However, depending on the substrate, they can be soaked in water to absorb sufficient moisture before steaming. Starch, pectins and other flour substrates are wetted with desired water before they are steamed. After cooling, the steamed substrate is then inoculated with the microorganism, heaped in shallow trays or flat surfaces. Air can be circulated around it for aeration and also for temperature control. As the fermentation progresses, metabolic heat is generated and the temperature must be controlled. In a simple system, this is achieved by gradually spreading the substrate for increased surface area and increased heat loss. Modern systems for solid state culture are fully automated in terms of aeration, mixing, temperature, and humidity control. These parameters are not kept constant but varied depending on the stage of the culture. Some of the systems employ static trays that are mixed at specified time intervals while other employ rotating drums with inbuilt static mixers (baffles) for continuous mixing. However, the rotation speed of the drums is controlled. In other processes such as mushroom production, the culture is not mixed after inoculation but temperature and humidity are controlled.

Advantages

1. Solid state cultures are very simple and relatively high productivity can be obtained without rigid control of the culture conditions.
2. The power requirement for solid state culture is relatively low because only limited mixing is done and in some systems the mixing is done manually.
3. Depending on the product, the productivity can be much higher than what is obtained in submerged cultures. In the case of some enzymes, the higher productivity reported for solid state cultures is attributed to higher aeration and or lower water activity.
4. Since there is no free water in solid state cultures, there is little or no effluent from solid state cultures. The whole culture can be formulated into products, thus the cost of effluent treatment is very minimal.
5.  Water consumption is generally much lower than that of submerged cultures.
6. There is generally lower investment cost (cost of equipment) for solid state cultures than submerged cultures.
7. Although strict sterile condition is not maintained, contamination is usually not a very serious problem because of the low water activity which limits the growth of contaminants.

Disadvantages

1. It is very difficult to control culture conditions such as temperature and pH because of the heterogeneity of solid state cultures.
2. Measurement of cell concentration is difficult because of solid particles.

Submerged culture

This is the conventional method of cultivation by which the cells (microorganisms, plants or animal cells) are suspended in liquid medium and remain submerged during the cultivation. Submerged cultures can be classified into static or agitated culture depending on whether the culture is mixed or not. In the case of static culture, there is no mixing so that the cells sediment or remain suspended depending on the relative density of the cell or flocs. Static cultures are usually used for anaerobic microorganisms or when the oxygen demand of the culture is very low. In the agitated culture, mixing is done by bubbling of gasses (as in pneumatically agitated bioreactors), using shakers for flask and test tube cultures (reciprocal or rotary shakers) or using impellers (as in the mechanically agitated bioreactors). All the factors that affect cell growth and product formation are controlled.

Advantages

1. The culture is homogenous, thus it is very easy to control culture conditions such as temperature, pH, dissolved gasses and other parameters.
2. All the nutrients are dissolved in the culture medium and the culture can be operated as batch, fedbatch or continuous system. Submerged culture is the most widely used method of cultivation of cells.

Disadvantages

1. Energy consumption for mixing is very high for agitated submerged cultures.
2. High aeration rate is required because of low solubility of oxygen and other gasses in liquid cultures.
3. High volume of water is used and this results in high volume of effluent with consequent high cost of waste treatment.
4. The investment cost is higher than that of solid state culture.