Biology

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Fermentors of Industrail Microbiology

The main function of a fermenter is to provide a suitable environment in which an organism can efficiently produce a target product. Most of them are designed to maintain high biomass concentrations, which are essential for many fermentation processes.

Fermentor design, quality of construction, mode of operation and the level of sophistication largely depend upon the production organism, the optimal operating conditions required for target product formation, product value and the scale of production.

The performance of any fermenter depends on many factors, but the key physical and chemical parameters that must be controlled are agitation rate, oxygen transfer, pH, and temperature and foam production.

Basic Design of a Fermenter

The materials used for construction of fermenter withstand repeated steam sterilization and are nontoxic. The reaction vessel is designed to withstand vacuum or else it may collapse while cooling. The internal surface is smooth and corrosion resistant. Either stainless steel or glass is used for construction.

Conventional bioreactors are cylindrical vessels with dome top and bottom (Figure 6.5).

It is surrounded by a jacket and sparger at the bottom through which air is introduced. The agitator (for mixing of cells and medium) shaft is connected to a motor at the bottom. It has ports for pH, temperature, dissolved Oxygen sensors for regulation. Antifoam agents like animal vegetable oil, lard oil, corn oil and soya bean oil are used to control the foam.

Modern fermentors are usually integrated with computers for efficient process monitoring and data acquisition. Parts of the fermenter and their functions are given in Table 6.2.
Components of fermenter and their uses:

Media Used in the Industrial Productions

Fermentation Medium Most fermentation requires liquid media, often referred to as broth, although some solid-substrate fermentations are also operated. Fermentation media must satisfy all the nutritional requirements of the microorganism and fulfill the technical objectives of the process.

Animal fats and plant oils are also incorporated into some media, often as supplements to the main carbon source. Medium used for large scale production should have the following characteristics.

1. It should be cheap and easily available.
2. It should maximize the growth of the microorganism productivity and the rate of formation of the desired product.
3. It should minimize the formation of undesired products.

It should contain carbon source, nitrogen source, energy source, micro nutrients required for the industrial production. Table 6.3 shows common substances used in the industrial fermentation process.

Waste products from other industrial processes such as molasses, ligno cellulosic waste, and corn steep liquor are generally used as substrates for industrial fermentation.

Apart from carbon and nitrogen sources, some other components like minerals, vitamins, growth factors are also used in Industrial fermentations.

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. For example, corn steep liquor contains a wide range of minerals that will usually satisfy the minor and trace mineral needs.

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, for economic reasons, as less expensive plant and animal extracts.

Precursors

Some fermentation 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: Phenylacetic acid or phenylacetamide added as side chain precursors in penicillin production.

Large Scale Production

Basic Steps of Industrial Fermentation

Successful development of a fermentation process and fermentors requires major contributions from a wide range of other disciplines, particularly biochemistry, genetics, molecular biology, chemistry, chemical engineering and process engineering, mathematics and computer technology. A typical operation involves both upstream processing (USP) and downstream processing (DSP) stages (Figure 6.6).

Upstream Processing

It is the first step in which biomolecules like bacteria or other cells are grown in a fermentor. Upstream processing involves inoculation development, scale up, medium preparation and sterilization of media and fermentation process.

Inoculum development

It is a preparation of a population of micro organisms from a stock dormant culture to a state useful for inoculating a final production fermentor. It is a critical stage in fermentation process. It is a stepwise sequence employing increasing volume of media. Inoculum media is usually balanced for rapid cell growth and not for product formation.

Inoculum scale up

It is the preparations of the seed culture in amounts sufficient to be used in the larger fermenter vessel. It involves growing the microorganisms obtained from the pure stock culture in several consecutive fermenter. By doing this, the time required for the growth of microbes in the fermenter is cut down, so that the rate of productivity is increased. The seed culture obtained is then used for inoculation in fermentation medium. The size of the inoculums is generally 1 – 10% of the total volume of the medium.

In general, fermentation/bioprocess techniques are developed in stages starting from a laboratory and finally leading to an industry. The phenomenon of developing industrial fermentation process in stages is referred to as scale-up. Scale-up is necessary for implementing new fermentation technique developed using mutant organisms.

The very purpose of scale-up is to develop optimal environmental and operating conditions at different levels for a successful fermentation industry where conditions like substrate concentration agitation and mixing, aeration, power consumption and rate of Oxygen transfer are studied. In a conventional scale-up, a fermentation technique is developed in 3-4 stages.

The initial stage involves a screening process using Petri dishes or Erlenmeyer flasks followed by a pilot project to determine the optimal operating conditions for a fermentation process with a capacity of 5-200 litres. The final stage involves the transfer of technology developed in the laboratory to industry. (Figure 6.7)

It has to be continuously noted that a fermentation process that works well at the laboratory scale may work poorly or may not work at all on industrial scale. Therefore it is not always possible to blindly apply the laboratory conditions of a fermentation technique developed to industry. At the laboratory scale, one is interested in the maximum yield of the product for unit time.

At the industry level, besides the product yield, minimal operating cost is another important factor for consideration.

Preparation and sterilization of media

According to the specific industrial production basic components needed to carry out fermentation are selected as per the required volume.

Medium components should be free from contamination. So all the medium components employed in the fermentation process are sterilized. Sterilization is mostly carried out by applying heat and to lesser extent other physical methods, chemical methods (disinfectants) and radiation (using UV rays, γ rays). Batch Sterilization is carried out at 121°C (20 to 60 mins) where as continuous sterilization is done at 140°C for (30 to 120 secs).

Much energy is wasted on batch sterilization on compared with continuous sterilization nearly 80 to 90% of energy saved during this process. Air and heat sensitive components are sterilizied by membrane filters.

Fermentation Process

It involves the propagation of the microorganism and the production of the desired product. Fermentation process is divided depending on the feeding strategy of the culture and medium as follows.

1. Batch Fermentation
2. Continuous Fermentation
3. Fed batch Fermentation

1. Batch Fermentation

The medium and culture are initially fed into the vessel and it is then closed. After that, no components are added apart from Oxygen. The pH is adjusted during the course of process by adding either acid or alkali. The fermentation is allowed to run for a predetermined period of time and the product is harvested at the end.

Foaming is controlled by adding antifoam agents such as palm oil or soybeans oil. Heat generated is regulated by providing water circulation system around the vessel for heat exchange.

2. Continuous fermentation

This is an open system. It involves the removal of culture medium continuously and replacement of them with a fresh sterile medium in a bioreactor. In this method, homogenously mixing reactors which include chemo stat and turbid stat bioreactors are used. Examples: production of antibiotics, organic solvents,
beer, ethanol and SCP.

3. Fed batch system

It is a combination of both batch and continuous systems. In this, additional nutrients are added to the fermentors as the fermentation is in progress. This extends the time of operation, but the products are harvested at the end of the production cycle as in batch fermenter. Followed by the fermentation, production, products are harvested or separated by downstream processing.

Downstream Processing

The various processes used for the actual recovery of useful products from fermentation or any other industrial processes are called downstream processing. The cost of downstream processing (DSP) is often more than 50% of the manufacturing cost, and there is product loss at each step of DSP.

Therefore, the DSP should be efficient, involve as few steps as possible and be cost-effective. Methods involved in the downstream processing are outlined in the flowchart (6.2). Table 6.4 shows Difference between upstream and downstream processing.

Table 6.4: Diffrence between upstream (usp) and downstream (dsp) processing

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Preservation of Industrially Important Microorganisms

The selected microorganism of industrial interest must be preserved in its original form for any further use and research. There are different methods for microbial preservation. Suitable methods are selected based on the:

a. Type of micro-organism
b. Effect of the preservation method on the viability of micro-organism
c. Frequency at which the cultures are withdrawn
d. Size of the microbial population to be preserved
e. Availability of resources
f. Cost of the preservation method. Followings are some of the methods of microbial preservation:

a. Desiccation
This involves removal of water from the culture. Desiccation is used to preserve actinomycetes (a form of fungi-like bacteria) for very long period of time. The microorganisms can be preserved by desiccating on sand, silica gel, or paper strips.

b. Agar Slopes
Microorganisms are grown on agar slopes in test tubes and stored at 5 to – 20 °C for six months. If the surface area for growth is covered with mineral oil the microorganisms can be stored for one year.

c. Liquid Nitrogen
This is the most commonly used technique to store micro-organisms for a long period. Storage takes place at temperatures of less than – 196 °C and even less in vapour phase. Microorganisms are made stationary and suspended in a cryoprotective agent before storing in liquid nitrogen.

d. Drying
This method is especially used for sporulating microorganisms (organisms that produce spores). They are sterilized, inoculated, and incubated to allow microbial growth, then dried at room temperature. The resultant dry soil is stored at 4° to 5 °C.

e. Lyophilization
This process is also known as freezedrying. The microbial culture is first filled in ampoules (glass vessels) and frozen, then dried under vacuum. This is a most convenient technique, since it is cheap to store and easy to ship. The disadvantage is that it is difficult to open the freeze dried ampoules; also, several subcultures have to be done to restore the original characteristics of the microorganisms.

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Strain Improvement of Industrail Microbiology

Improvement of the production strain(s) offers the great opportunities for cost reduction without significant capital outlay in industries. Moreover, success in making and keeping a fermentation industry competitive depends greatly on continuous improvement of the production strain(s). Improvement usually resides in increased yields of the desired metabolite.

The science and technology of manipulating and improving microbial strains, in order to enhance their metabolic capacities for biotechnological applications, are referred to as strain improvement. Need for strain improvement Microbes exist in the nature produce certain compounds of biological interest.

However the industrial application of producing those compounds by natural strains is not an economical one so, wild strains are changed by the changing their gene pattern or by regulating their enzymes production. As a result, the specific product is produced in excess.

Knowledge of the function of enzymes, rate limiting steps in pathways, and environmental factors controlling synthesis further helps in designing screening strategies.

Attributes of Improved strains

1. Assimilate inexpensive and complex raw materials efficiently.
2. Alter product ratios and eliminate impurities or by products in downstream processing.
3. Reduce demand on utilities during fermentation (air, cooling water, or power).
4. Provide cellular morphology in a form suitable for product separation.
5. Create tolerance to high product concentration.
6. Shorten fermentation times.
7. Overproduce natural products or bioactive molecules not synthesized naturally for example insulin.
8. Excrete the product to facilitate product recovery.

Generally wild strains of microorganisms produce low quantities of commercially important metabolites. So, genetic improvements have to be made and new strains need to be developed for any substantial increase in the product formation in a cost effective manner.

The following techniques at practical genomic level help to improve the microbial strain. They are:

1. Selection of mutants
2. Recombination
3. Regulation
4. Genetic engineering
5. Protoplast fusion

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Screening of Industrially Important Microorganism

Isolation of industrially important microorganisms

Success of fermentation depends upon the isolation of microorganism. The microorganisms are isolated from their natural habitats like soil, lakes, river mud or even in unusual habitats or environments such as extreme cold, high altitude, deserts, and deep sea and petroleum fields and are tested directly for the product formation and isolated or it can be genetically modified.

Different types of microorganisms are isolated by different methods. Different microbes with desired activity are isolated using various culture techniques. The next step after isolation of microorganisms is the selection or screening. For the successful fermentation process, selection of microorganisms is the prime important step. Screening includes primary screening and secondary screening.

Primary screening:
The elementary steps that are performed to select the desired organisms and eliminate the undesirable organisms are termed as primary screening. Methods such as crowded plate technique, auxanography and enrichment culture technique are some of the techniques used in primary screening. For screening of antibiotic producing organisms crowded plate technique is described here,

Crowded plate technique

1. Soil is serially diluted
2. The serially diluted sample is spread on the nutrient agar plates
3. The plates are incubated and the agar plate having 300 to 400 colonies are observed for antibiotic producing activity
4. The ability of a colony to exhibit antibiotic activity is indicated by the presence of a zone of inhibition surrounding the colony
5. The technique is improved by using test organism
6. The antibiotic produced by the organisms in the soil may inhibit the growth of test organism
7. The formation of inhibitory zones around certain colonies indicates their antibiotic sensitivity
8. The diameter of the zones of inhibition is measured in millimeters. Crowded plate technique is depicted in the diagram (Figure 6.3).

Enrichment isolation

The process of enrichment provides a suitable condition to support the growth of microorganisms. It allows the growth of the specific microbe while inhibiting the other non-target microbe. The growth of target microorganisms is enriched by providing sole carbon source.

For screening microorganisms degrading the compound, different inhibitors are employed which have the ability to block a specific metabolic pathway of the non-target microbe.

pH and temperature are also adjusted favoring the growth of desired microorganisms. Soil Calcium carbonate enrichment technique is used for isolation of secondary metabolite producing microorganisms (actinomycetes).

Secondary screening

It is very useful in sorting out microorganisms that have real commercial value from many isolates obtained during primary screening.

1. As primary screening allows the detection and isolation of microorganisms which posses, potentially interesting industrial applications. It is further followed by secondary screening, to check the capabilities and gain information about these organisms.

2. Through primary screening only few or many microorganism that produce a industrially important product, are isolated. The information about the product formed is very less. So, through secondary screening, further sorting out is performed. In this method, only microorganisms with real commercial
value are selected and those that lack the potential are discarded.

3. Secondary screening should yield the types of information which are needed in order to evaluate the true potential of a microorganisms industrially usage.

4. Secondary screening may be qualitative and quantitative in its approach.

5. It is done by using paper, thin layer or other chromatographic techniques.

6. The product’s physical, clinical, and biological properties are determined.

7. It detects gross genetic instability in microbial cultures.

8. It gives information about the number of products produced in a single fermentation.

9. It determines the optimum conditions for growth or accumulation of a product associated with particular culture.

10. It gives information about the different components of the fermentation medium.

11. It helps in providing information regarding the product yield potential of different isolates.

12. It reveals whether microorganisms are capable of a chemical change or it destroys their fermentation product.

There are various methods employed for secondary screening which includes test conducting on petridish containing solid media or by using flasks or small fermentors containing liquid media, giant colony technique, and filtration method liquid medium method (using Erlenmeyer flask). Here giant colony technique is explained in detail.

Giant Colony Technique

The Streptomyces culture is inoculated onto the central areas of petriplates containing a nutritious agar medium or they are streaked in a narrow band across the centre of plates. The plates are then incubated until growth and possibly, sporulation have occurred. Strains of micro organisms to be tested for possible
sensitivity to the antibiotics (the test organisms) are then streaked from the edges of the plates up to but not touching the Streptomycete growth.

The plates are further incubated to allow the growth of the test organism. The growth of the test organism inhibited by antibiotic in the vicinity of the Streptomycete is then measured in millimeters. These Streptomycetes that have produced antibiotics with observable microbial inhibition spectrum are retained for further testing (Figure 6.4).

The microbes used in the industrial microbiology should have following characters.

1. The strain should be a high-yielding strain.
2. The strain should have stable biochemical and genetical characteristics.
3. It should not produce undesirable substances.
4. It should be easily cultivated on large scale.

The strain should be in pure culture, free from other microorganisms including Bacteriophages. These characters are screened for the production of desirable products from microorganisms.

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Industrially Important Microorganisms and their Products of Microbiology

Microorganisms have the powerful capacity to produce numerous products, during their life cycle. Flowchart 6.1 shows the production of valuable metabolic products during the growth of microorganisms on a suitable medium under controlled environmental conditions. Microbial products are often classified as primary and secondary metabolites.

Primary metabolites consist of compounds related to the synthesis by microbial cells in the growth phase. Primary metabolites such as amino acids, vitamins, enzymes, organic acids and nitrogenous bases are produced by wide variety of microorganisms. These primary metabolites are essential for the growth of microorganisms and they are produced during Logarithmic phase.

Secondary metabolites do not play a role in development, growth and reproduction of microorganisms. They are produced at the end of growth phase near stationary phase. They usually accumulate during the period of nutrient limitation or waste product accumulation that follows the exponential phase. These compounds have no direct relationship to the synthesis of cell materials and normal growth.

They are the end products of the primary metabolism. Products such as steroids, alkaloids, antibiotics are secondary metabolites. Excessive production of the primary and secondary metabolites produced by the microorganisms are useful in the large scale in industrial production. Unlike primary metabolites, secondary metabolites are produced in small quantities and their extraction is difficult (Figure 6.2).

Some industrially important products are,

• microbial cells (living or dead), microbial biomass and components of microbial cells
• microbial metabolites
• intracellular or extracellular enzymes
• modified compounds that has been microbiologically transformed, and recombinant products through the DNA recombinant technology. (Table 6.1 shows some industrially important microorganisms)

Industrially important microorganisms

The industrial production of commercial products is carried out by fermentation process. The term fermentation is defined scientifically in a strict sense as a biological process that occurs in the absence of oxygen (anaerobic).

In industrial sense any process mediated by or involving microorganisms in which a product of economic value is obtained is called fermentation. The term Industrial fermentation also means large scale cultivation of microorganisms even though most of them are aerobic.

There are many microbiological processes that occur in the presence of air (aerobically) yielding incomplete oxidation products.
Examples:

1. The formation of acetic acid (vinegar) from alcohol by vinegar bacteria
2. Citric acid from sugar by certain molds such as Aspergillus niger. These microbial processes are often referred to as fermentations, although they do not decompose in the absence of air.