Screening for new metabolites and Strain development

 Screening for new metabolites and Strain development:

Azharul Islam Shovon, B.Sc, Biochemistry and Molecular Biology, NSTU

• Screening:

The use of highly selective procedures to allow the detection & isolation of only those microorganisms which are of interest from among a large microbial population. It is very time consuming and expensive process. For example, Eli Lilly & Co. Ltd discovered three species of antibiotic producing organisms in a span of 10 years and after screening 4,00,000 organisms.

 

Although there are many screening techniques, all of them are generally grouped into two broad categories.

They are:

1. Primary screening, and

2. Secondary screening.

 

Primary Screening:

Primary screening allows the detection & isolation of microorganisms that possess potentially interesting industrial application.

Secondary Screening:

Secondary screening allows further sorting out of microorganisms obtained from PS having real value for industrial processes and discarding of those lacking this potential.

 

Primary metabolite:

A primary metabolite is a small chemical compound that is directly involved in the growth, development and reproduction in living organism.

Secondary metabolite:

Secondary metabolites are organic compounds which are not directly involved in the normal growth, development or reproduction of the organism.

 

 

 

What are metabolites?

They are low molecular weight, small compound produced as an intermediate or end product of metabolism.

Metabolites are divided into two groups: Primary and secondary.

Of which primary ones are considered as essential to microorganisms for their proper growth and maintenance of cellular function.

While secondary metabolites do not play a role in growth, development and reproduction and are formed during the end or near the stationary phase of growth and mostly are end products of primary metabolites.

These metabolites can be used in industrial microbiology to obtain amino acids, develop vaccines and antibiotics and isolate chemical necessary for organic synthesis.

The Primary metabolites consist of the vitamins, amino acids, nucleosides and organic acid, which are necessary at the time of logarithmic phase of microbial growth.

But the products like antibiotics, steroids, vitamins, amino acids, gibberellins, toxins are the secondary metabolites compound produces during the stationary phase of the cell growth. The structurally diverse metabolites show a variety of biological activities like antimicrobial agent, inhibitors of enzyme and antitumors, immune suppressive and antiparasitic agents.

Antibiotics: Penicillin-P.notatum, Cephalosporin-c, acremonium and streptomycin -Streptomyces griseus.

Vitamins: Rivoflavin (B2) -Ashbya gossypii, L-Sorbose-Gluconobacter oxidans, vitamin B12-Propionibacterium-shermasnii, Streptomyces.

Amino acids: Tryptophan-E-coli, Threonine-Streptomyces racemosus, phenyl alanine-Corynebacterium glutamicus.  

 

Primary Screening of Microorganisms:

Primary screening may be defined as detection and isolation of the desired microorganism based on its qualitative ability to produce the desired product like antibiotic or amino acid or an enzyme etc. In this process desired microorganism is generally isolated from a natural environment like soil, which contains several different species. Sometimes the desired microorganism has to be isolated from a large population of different species of microorganisms.

 

The following are some of the important primary screening techniques:

The crowded plate technique

Indicator dye technique

Enrichment culture technique

Auxanography technique

Technique of supplementing volatile and organic substrates.

·         Screening for organic acid producing microbes:

The pH including dyes can be used here for detecting microbes able to procedure organic acids. As acid is produced these dyes undergo color change dyes such as neutral red, bromothymol blue are added to poorly buffered nutrient agar media. Culture inoculation is done, incubated and depending on color change colonies are selected. Using CaCO3 can be incorporated in media. On acid production, clear zone of dissolved CaCO3 around can be seen.

·         Screening for Antibiotic production:

The Crowded Plate Technique:

This technique is primarily employed for detecting those microorganisms, which are capable of producing antibiotics. This technique starts with the selection of a natural substratum like soil or other source consisting of microorganisms. Progressive serial dilution of the source is made. Suitable aliquot of the serial dilution is chosen which is able to produce 300 to 400 individual colonies when plated on an agar plate, after incubation. Such a plate is called as crowded plate.

The antibiotic producing activity of a colony is indicated by no growth of any other bacterial colony in its vicinity. This region of no growth is indicated by the formation of a clear and colorless area around the antibiotic producing microorganism’s colony on the agar plate. This region is called as growth inhibitory zone. Such a colony is isolated from the plate and purified either by making repeated sub-culturing or by streaking on a plate containing a suitable medium, before stock culture is made. The purified culture is then tested for its antibiotic spectrum.

However, the crowded plate technique has limited applications, as it will not give indication of antibiotic producing organism against a desired organism. Hence, this technique has been improved later on by employing a test organism to know the specific inhibitory activity of the antibiotic.

In this modified procedure, suitable serially diluted soil suspension is spread on the sterilized agar plate to allow the growth of isolated and individual microbial colonies (approximately 30 to 300 per plate) after incubation. Then the plates are flooded with a suspension of test organism and the plates are incubated further to allow the growth of the test organism. The formation of inhibitory zone of growth around certain colonies indicates the antibiotic activity against the test organism.

A rough estimation of the relative amounts of antibiotic produced by a microbial colony can be estimated by measuring the diameter of the zone of inhibited test organism’s growth. Antibiotic producing colonies are later on isolated from the plate and are purified before putting to further testing to confirm the antibiotic activity of a microorganism.

Fig: The Crowded Plate Technique

 

Indicator Dye Technique:

Microorganisms capable of producing acids or amines from natural sources can be detected using this method by incorporating certain pH indicator dyes such as neutral red or bromothymol blue into nutrient agar medium. The change in the color of a particular dye in the vicinity of a colony will indicate the ability of that colony to produce an organic acid or base.

Production of an organic acid can also be detected by an alternative method. In this method calcium carbonate is incorporated into the agar medium. The production of organic acid is indicated by the formation of a clear zone around those colonies which release organic acid into the medium. The identified colonies are isolated and purified either by repeated sub-culturing or by streaking methods and a stock culture is made which may be used for further qualitative or quantitative screening tests.

·         Screening for extracellular metabolite producing microbes:

Auxanotrophic Technique:

This technique is employed for the detection and isolation of microorganisms capable of producing certain extra cellular substances such as growth stimulating factors like amino acids, vitamins etc. A test organism with a definite growth requirement for the particular metabolite is used in this method.

For this purpose, spread a suitable aliquot on the surface of a sterilized agar plate and allow the growth of isolated colonies, after incubation. A suspension of test organism with growth requirement for the particular metabolite is flooded on the above plate containing isolated colonies, which are subjected to further incubation.

The production of the particular metabolite required by the test organism is indicated by its increased growth adjacent to colonies that have produced the required metabolite. Such colonies are isolated, purified and stock cultures are prepared which are used for further screening process.

·         Enrichment Culture Technique:

This technique is generally employed to isolate those microorganisms that are very less in number in a soil sample and possess specific nutrient requirement and are important industrially. They can be isolated if the nutrients required by them is incorporated into the medium or by adjusting the incubation conditions.

 

 

Technique of Supplementing Volatile Organic Substances:

This technique is employed for the detection and isolation of microorganisms capable of utilizing carbon source from volatile substrates like hydrocarbons, low molecular weight alcohols and similar carbon sources. Suitable dilution of a microbial source like soil suspension are spread on to the surface of sterile agar medium containing all the nutrients except the one mentioned above.

The required volatile substrate is applied on to the lid of the petri plates, which are incubated by placing them in an inverted position. Enough vapors from the volatile substrate spread to the surface of agar within the closed atmosphere to provide the required specific nutrient to the microorganism, which grows and form colonies by absorbing the supplemented nutrient. The colonies are isolated, purified and stock cultures are made which may be utilized for further screening tests.

 

 

 

Secondary Screening of Microorganisms:

Primary screening helps in the detection and isolation of microorganisms from the natural substrates that can be used for industrial fermentations for the production of

compounds of human utility, but it cannot give the details of production potential or yield of the organism. Such details can be ascertained by further experimentation.

This is known as secondary screening, which can provide broad range of information pertaining to the:

Ability or potentiality of the organism to produce metabolite that can be used as an industrial organism.

The quality of the yield product.

The type of fermentation process that is able to perform.

Elimination of the organisms, which are not industrially important.

To evaluate the true potential of the isolated microorganisms both qualitative and quantitative analysis are generally conducted. The sensitivity of the test organism towards a newly discovered antibiotic is generally analysed during qualitative analysis, while the quantum yield of newly discovered antibiotic is estimated by the quantitative analysis.

Evaluation of Potentialities of Microorganisms:

Microorganisms isolated in the primary screening are critically evaluated in the secondary screening so that industrially important and viable potentialities can be assessed.

They include:

To determine the product produced by an organism is a new compound or not.

A determination should be made about the yield potentialities of various isolated microorganisms that are detected in primary screening for that new compound.

It should determine about the various requirements of the microorganism such as pH, aeration, temperature etc.

It should detect whether the isolated organism is genetically stable or not.

It should reveal whether the isolated organism is able to destroy or alter chemically their own fermentative product by producing adaptive enzymes if they accumulate in higher quantities.

It should reveal the suitability of the medium or its constituent chemicals for the growth of a microorganism and its yield potentialities.

It should determine the chemical stability of the product.

It should reveal the physical properties of the product.

It should determine whether the product produced by a microorganism in a fermentative process is toxic or not.

Secondary screening should reveal that whether the product produced in fermentation process exists in more than one chemical form. If so, the amount of formation of each chemical formation of these additional products is particularly important since their recovery and sale as byproducts can greatly improve the economic status of the fermentation industry.

The new organism should be identified to the species level. This will help in making a comparison of growth pattern, yield potentialities and other requirements of test organism with those already described in the scientific and patent literature, as being able to synthesize products of commercial value.

It should select industrially important microorganisms and discard others, which are not useful for fermentation industry.

It should determine the economic status of a fermentation process undertaken by employing newly isolated microorganism.

 

Methods of Secondary Screening:

Secondary screening gives very useful information pertaining to the newly isolated microorganisms that can be employed in fermentation processes of commercial value. These screening tests are conducted by using petri dish containing solid media or by using flasks or small fermenters containing liquid media. Each method has some advantages and disadvantages. Sometimes both the methods are employed simultaneously.

Liquid media method is more sensitive than agar plate method because it provides more useful information about the nutritional, physical and production responses of an organism to actual fermentation production conditions. Erlenmeyer flasks with baffles containing highly nutritive liquid media are used for this method. Flasks are fully aerated with glass baffles and continuously shaken on a mechanical shaker in order to have optimum product yield.

There are several techniques and procedures that can be employed for secondary screening. However, only a specific example of estimation of antibiotic substance produced by species of Streptomyces, is described in the following paragraph. Similar methods could be used for the detection and isolation of microorganisms capable of producing other industrial products.

(i)                 Giant Colony Technique:

This technique is used for isolation and detection of those antibiotics, which diffuse through solid medium. Species of Streptomyces, is capable of producing antibiotics during primary screening. The isolated Streptomyces culture is inoculated into the central area of a sterilized petri plates containing nutrient agar medium and are selected. The plates are incubated until sufficient microbial growth takes place.

Cultures of test organism, whose antibiotic sensitivity is to be measured are streaked from the edges of plate’s upto but not touching the growth of Streptomyces and are further incubated to allow the growth of the test organisms. Then the distance over which the growth of different test organisms is inhibited by the antibiotic secreted Streptomyces is measured in millimeters.

The relative inhibition of growth of different test organisms by the antibiotic is called inhibition spectrum. Those organisms whose growth is inhibited to a considerable distance are considered more sensitive to the antibiotic than those organisms, which can grow close to the antibiotic. Such species of Streptomyces, which have potentiality of inhibiting microorganisms is preserved for further testing.

(ii) Filtration Method:

This method is employed for testing those antibiotics which are poorly soluble in water or do not diffuse through the solid medium. The Streptomyces is grown in a broth and its mycelium is separated by filtration to get culture filtrate. Various dilutions of antibiotic filtrates are prepared and added to molten agar plating medium and allowed to solidify.

Later on cultures of various test organisms are streaked on parallel lines on the solidified medium and such plates are incubated. The inhibitory effect of antibiotic against the test organisms is measured by their degree of growth in different antibiotic dilutions.

(iii) Liquid Medium Method:

This method is generally employed for further screening to determine the exact amount of antibiotic produced by a microorganism like Streptomyces.

Erlenmeyer conical flasks containing highly nutritive medium are inoculated with Streptomyces and incubated at room temperature. They are also aerated by shaking continuously and vigorously during incubation period to allow Streptomyces to produce the antibiotic in an optimum quantity.

 

Samples of culture fluids are periodically withdrawn aseptically for undertaking the following routine checks:

To check the suitability of different media for maximum antibiotic production.

To determine the value of pH at which there will be maximum growth of the microorganism and antibiotic production.

To check for contamination.

To determine whether the antibiotic produced is new or not.

To check the stability of the antibiotic at various pH levels and temperatures.

To determine the solubility of the antibiotic in various organic solvents.

To check about the toxicity of the antibiotic against the experimental animals.

After carrying out the above mentioned routine tests further studies are also conducted to know the following additional information:

Effect of incubation temperature and antifoaming agents on fermentation.

Rate of resistance developed among the test organisms.

Checking the antibiotic for its bacteriostatic or bactericidal properties. Its ability to precipitate serum proteins to cause hemolysis of blood or to harm phagocytes.

Checking for possibility of inclusion of precursor chemical of the antibiotic production in the medium.

Suitability of the organism for mutation and other genetic studies.

 

 

Ex. For Riboflavin:

Lactic acid bacteria are known for riboflavin production. We get isolate in primary screening by growing them on riboflavin devoid medium and check for its production. In secondary screening we do riboflavin assay, do media optimization and characterize the culture.

Ex. For Gibberellic Acid:

Rhizobacteria are known for gibberellic acid production. We get isolate and do primary screening for GA production by growing culture media and doing liq-liq extraction and measure by UV spectrometer. For secondary screening we setup different expt. For screening highest yield, differing different parameters then we optimize the media for fermentation.

Ex. For Amylase:

Industrially very important enzyme. Primary screening is done by using starch agar plate, for isolation. In secondary screening enzyme activity assay is done. Experiments are set up for getting highest yield by media and parameter optimization.

Characterization of isolate is also important (pathogen/ nonpathogen).

 

 

 

 

Strain Development

Strain: A strain is a group of species with one or more characteristics that distinguish it from other sub group of the same species of the strain. Each strain is identified by a name, number or letter. Example: E.coli strain K12.

 

What is the strain improvement/development?

Strain improvement is defined as the science and technology of genetically modifying microbial strains to improve their potentials for numerous biotechnological applications and it majorly involves in iteration the genetic alterations, fermentation techniques and assay.

 

What is industrial strain development?

Industrial strain development: Mutants which synthesize one component as the main product are preferable, since they make possible a simplified process for product recovery.

 

Industrial strains and strain improvement

Irrespective of the origins of an industrial microorganism, it should ideally exhibit:

1. Genetic stability;

2. Efficient production of the target product, whose route of biosynthesis should preferably be well characterized;

3. Limited or no need for vitamins and additional growth factors;

4. Utilization of a wide range of low-cost and readily available carbon sources;

5. Amenability to genetic manipulation;

6. Safety, non-pathogenicity and should not produce toxic agents, unless this is the target product;

7. Ready harvesting from the fermentation;

8. Ready breakage, if the target product is intracellular; and

9. Production of limited byproducts to ease subsequent purification problems.

Other features that may be exploited are thermophilic or halophilic properties, which may be useful in a fermentation environment. Also, particularly for cells grown in suspension, they should grow well in conventional bioreactors to avoid the necessity to develop alternative systems. Consequently, they should not be shear sensitive, or generate excessive foam, nor be prone to attachment to surfaces.

 

 

Strain improvement:

Further strain improvement is a vital part of process development in most fermentation industries. It provides a means by which production costs can be reduced through increases in productivity or reduction of manufacturing costs. Examples of some targets for strain improvement are given in the following Table:

 

Examples of targets for strain improvement:

Rapid growth Genetic stability Non-toxicity to humans Large cell size, for easy removal from the culture fluid Ability to use cheaper substrates Modification of submerged morphology Elimination of the production of compounds that may interfere with downstream processing Catabolite derepression Phosphate deregulation Permeability alterations to improve product export rates Metabolite resistance Production of   additional enzymes   compounds to inhibit contaminant microorganisms   heterologous proteins that may also be engineered   with downstream processing ‘aids’, e.g. polyarginine tails

 

In many cases strain improvement has been accomplished using natural methods of genetic recombination, which bring together genetic elements from two different genomes into one unit to form new genotypes. An alternative strategy is via mutagenesis. Those recombinants and mutants are then subjected to screening and selection to obtain strains whose characteristics are more specifically suited to the industrial fermentation process. However, such strains are unlikely to survive well in nature, as they often have altered regulatory controls that create metabolic imbalances. Also, they must then be maintained on specific media that select for, and help retain, the special characteristic(s).

 

 

 

NATURAL RECOMBINATION

Bacterial DNA is usually in the form of a single chromosome and plasmids; the latter are autonomous self replicating accessory pieces of DNA.

Fig.  Plasmid pBR322, which contains 4361 base pairs and is a typical example of a cloning vector (*restriction sites).

Each plasmid carries up to a few hundred additional genes and there may be as many as 1000 copies of a plasmid per cell. They contain supplemental genetic information coding for traits not found in the bacterium’s chromosomal DNA. Unlike most eukaryotic organisms, bacteria have no form of sexual reproduction. However, they are able to exchange some genetic material via the processes of conjugation, transduction and transformation. Conjugation involves cell-to-cell contact, where the donor contacts the recipient with a filamentous protein structure called a sex pilus, which draws the two cells close together. The donor copies all or a part of its plasmid or chromosomal DNA and passes it through the pilus to the recipient. In transduction, a bacterial virus (bacteriophage) acts as a vector in transferring genes between bacteria. The bacteriophage attaches to a bacterial cell and injects its DNA into the host to become incorporated into the host chromosome. During bacteriophage replication the phage may acquire pieces of the adjacent host DNA. If the phages go on to enter new hosts, they are able to integrate their original DNA, and the genes picked up from their previous host, into the new host’s chromosome. Bacteriophages, like plasmids, may also acquire transposons, which are pieces of DNA that can ‘jump’ from one piece of DNA to another, e.g. from a plasmid to a chromosome and vice versa. The bacteriophages can carry transposons on to new host bacterial cells, where they are able to ‘jump’ onto a plasmid or the host chromosomal DNA. The third process, transformation, involves cellular uptake of a naked piece of DNA from the surrounding medium, which then becomes incorporated into the cell. In natural environments this is a totally random process, the DNA fragments available for uptake being derived from cells that have lysed. The DNA fragments can be relatively large and may contain several genes. However, they are capable of entering and thus transforming only so-called ‘competent’ cells, which are in a specific physiological state rendering them permeable to DNA. In eukaryotes, genetic recombination naturally occurs during sexual reproduction. New genotypes result from the combination of parental chromosomes and as a consequence of crossing-over events during meiosis. The latter involves breakage of sections of chromosomal DNA and the exchange of these segments between homologous chromosomes to form new combinations. Some industrially important fungi, including Penicillium and Aspergillus, do not have a true sexual phase. However, a parasexual cycle has provided a route by which new strains can be produced. This is promoted when two genetically different haploid strains are grown together, allowing fusion of their hyphae. These events result in the formation of a heterokaryon, composed of mycelium containing nuclei derived from each strain. Direct formation of heterokaryons can now be performed in vitro by fusing protoplasts, which are cells that have had their walls removed. Also, certain eukaryotes, including some yeasts and filamentous fungi, possess autonomous plasmids, such as the 2µm plasmid of Saccharomyces cerevisiae, which have proved useful as vectors in genetic engineering.

 

MUTAGENESIS: A CONVENTIONAL TOOL FOR STRAIN IMPROVEMENT Mutations result from a physical change to the DNA of a cell, such as deletion, insertion, duplication, inversion and translocation of a piece of DNA, or a change in the number of copies of an entire gene or chromosome. Subjection of microorganisms to repeated rounds of mutagenesis, followed by suitable selection and screening of the survivors, has been a very effective tool in improving many industrial microorganisms. As mutants can arise naturally or be induced, they are considered to be the product of natural events. Consequently, there are fewer problems in gaining approval from the regulatory authorities than when recombinant DNA technology is used to develop an industrial microorganism. Spontaneous mutation rates are low; in most bacterial genes for example, the rate is approximately 10–10 per generation per gene. The mutation rate can be greatly increased by using mutagens, which are of two types. Physical mutagens include ultraviolet, g and X radiation; and chemical mutagens are compounds such as ethane methane sulphonate (EMS), nitroso methyl guanidine (NTG), nitrous acid and acridine mustards. Mutants are formed when the mutagens induce modifi- cations of the base sequences of DNA that result in basepair substitutions, frame-shift mutations or large deletions that go unrepaired. Mutagenesis can also be induced using transposons delivered by a suitable vector. They produce insertion mutants whose normal nucleotide sequence is interrupted by the transposon sequence. Mutagenesis methods generally have rather limited use as they primarily achieve either loss of an undesirable characteristic or increasing production of a product, due to impairment of a control mechanism. These traditional methods have been successfully employed in removing the yellow colour of early penicillin prepara tions caused by chrysogenin, a yellow pigment produced by Penicillium chrysogenum. Mutagenesis programmes have also been highly effective in increasing the yield of penicillin in industrial strains of the same organism. Other notable examples of impairment of control processes, resulting in greater product yields, are seen in several microorganisms used for amino acid production. More recently, methods have been developed to enhance both the overall mutability and mutation rate of specific genes, in order to obtain the maximum frequency of desired mutant types. This directed mutagenesis obviously requires a knowledge of the genes that control the target product and often a genetic map of the organism. In addition, in vitro mutagenesis is now used in combination with genetic engineering (see below) to modify isolated genes or parts of genes.

 

GENETIC ENGINEERING OF MICROORGANISMS Over the last 20 years the development of recombinant DNA technology and methods of cell fusion, such as hybridoma formation for monoclonal antibody production , have had a major impact on industrial microbiology. In contrast to natural recombination processes, modern recombinant DNA technology provides almost unlimited opportunities for the production of novel combinations of genes. These methods are also highly specific and well controlled, and a vast range of genetic information is available from almost any living and even extinct organisms. Recombinant DNA technology has allowed specific gene sequences to be transferred from one organism to another and allows additional methods to be introduced into strain improvement schemes. This can be used to increase the product yield by removing metabolic bottlenecks in pathways and by amplifying or modifying specific metabolic steps. Overall, genetic engineering procedures allow totally new properties to be added to the capabilities of industrial microorganisms. Microorganisms may be manipulated to synthesize and often excrete enhanced ranges of enzymes, which may facilitate the production of novel compounds or allow the utilization of cheaper complex substrates. As there is no restriction to the origins of the genes that microorganisms express, the production of plant and animal proteins is made possible. Valuable products already produced include human growth hormone, insulin and interferons. Nevertheless, these methods have not totally replaced traditional mutatagenesis methods and the two approaches should be viewed as complementary strategies for strain improvement.

 

Strategies for the genetic engineering of bacteria

Genetic engineering involves manipulation of DNA outside the cell. It necessitates the initial isolation and recovery of the gene(s) of interest from the donor organism’s genome. Isolated DNA sequences may then be modified and the regulation of their expression altered, before insertion into host organisms via a suitable easily manipulated vector system. The first step requires total DNA extraction from the donor organism, which is then cut into smaller sequences using a specific restriction endonuclease. Many of these restriction enzymes, found in various species of bacteria, make a staggered cut through a double-stranded DNA molecule at a specific sequence or palindrome.

As a result, the ends of cut molecules have complementary single-stranded sequences. The small sections of DNA (restriction fragments) can then be joined or spliced into vector DNA molecules that have been cut with the same restriction enzyme. Splicing is performed by an enzyme, DNA ligase, and creates a synthetic DNA molecule. Plasmids and bacteriophages have been the most useful cloning vectors. They play an important role as delivery systems to introduce the recombinant molecules into host cells via transformation or transduction. Once inside they are capable of autonomous replication, which maintains the recombinant DNA within the host cell. Introduction of recombinant plasmids into bacterial cells can be achieved following calcium chloride treatment, which renders the cell membranes more permeable to DNA. After introduction the plasmids replicate autonomously. In some cases, numerous copies are produced within the host cell to increase the amount of the recombinant DNA per cell. Plasmids can be designed to contain selectable genetic markers, such as antibiotic resistance, vitamin requirement, etc. These markers may be used to select only those host cells that have incorporated the plasmid during transformation, e.g. the 4.3kb plasmid pBR322, carrying ampicillin and tetracycline resistance markers. Bacteriophages are particularly useful cloning vectors as up to half of their genome can be removed and replaced with foreign DNA. This is achieved in vitro using restriction enzymes in a similar manner to plasmid manipulation. Suitable DNA fragments are then packaged into phage particles, which are able to infect a selected host. The mixture of restriction fragments, originating from a whole DNA extract, once packaged within phages or plasmids, is used to transform or transduce host cells. This generates a DNA library consisting of individual clones that contain different recombinant DNA molecules, representing all DNA sequences/genes of the donor genome. Once the library has been established, the clones are allowed to form colonies on solid selective media. At this stage, the specific clone containing the recombinant DNA molecule of interest can be identified. If the foreign gene is successfully expressed in the host bacterium and a heterologous protein is made, detection can be achieved by use of a specific antibody reaction with the protein. Alternatively, if the recombinant protein is an enzyme that is not normally produced by the host, the enzyme activity can be detected.

 

Strain stability

A key factor in the development of new strains is their stability. An important aspect of this is the means of preservation and storage of stock cultures so that their carefully selected attributes are not lost. This may involve storage in liquid nitrogen or lyophilization. Strains transformed by plasmids must be maintained under continual selection to ensure that plasmid stability is retained. Instability may result from deletion and rearrangements of recombinant plasmids, which is referred to as structural instability, or complete loss of a plasmid, termed segregational stability. Some of these problems can be overcome by careful construction of the plasmid and the placement of essential genes within it. Segregational instability can also be overcome by constructing so-called suicidal strains that require specific markers on the plasmid for survival. Consequently, plasmid-free cells die and do not accumulate in the culture. These strains are constructed with a lethal marker in the chromosome and a repressor of this marker is located on the plasmid. Cells express the repressor as long as they possess the plasmid, but if it is lost the cells express the lethal gene. However, integration of a gene(s) into the chromosome is normally the best solution, as it overcomes many of these instability problems.

 

 

 

 

 

Fermentation Equipment

Fermentation

Fermentation can be defined as a metabolic process in which raw materials such as sugar or carbohydrates are converted into acids, gases and alcohols. Micro-organisms such as yeast and bacteria play a central role in the fermentation process.

• Fermentation process requires a fermenter, substrate, inoculum  and optimum conditions.
• The equipment used to carry out fermentation is called a fermenter.

 

 

What is a fermenter?

A fermenter can be defined as an equipment or a vessel in which sterile nutrient media and pure culture of microorganism are mixed and fermentation process is carried out under aseptic and optimum condition.

Fermenter provides a sterile environment and optimum condition that are important for growth of microorganisms and synthesis of desired product.

 

Design of a fermenter

1. A fermenter should be large enough to allow creation of fermenter bacteria.
2. It should have an inlet for oxygen and outlet for carbon dioxide in case aerobic microbes are used for fermentation.
3. It should be completely sealed from outside environment to avoid contamination.
4. It should provide a facility to add anti-foaming agents as well as a temperature controlling system.
5. A pH detecting system is also important. An outlet for the withdrawal of media for determination of pH as well as inlet for addition of acids and alkalis should be provided. It is important to maintain pH at optimum levels for growth of culture.
6. The most important thing is that a fermenter should provide aseptic means of withdrawal of fermented product and introduction of culture samples.
7. The fermenter facilitate the stirring of media so that inoculum and fermentation media mix properly. It also ensures that the gases required for growth of microbial culture are available to all microbes.

 

A fermenter should be constructed in such a way that it can make provisions for the below activities:

1. Sterilization

2. Temperature control

3. pH control

4. Foam control

5. Aeration and Agitation.

6. Sampling point

7. Inoculation points for microorganisms, media and supplements.

8. Drainage point for drainage of fermented media

9. Harvesting of product

 

Diagrammatic representation of Fermenter:

Major Parts of fermenter:

1. Material used for fermenter
2. Impellers
3. Baffles
4. Inoculation port
5. Sparger
6. Sampling point
7. pH Control device
8. Temperature control system
9. Foam control device
10. Bottom drainage system.

1. Material used for fermenter:

The material used for designing of a fermenter should have some important functions.

It should not be corrosive. It should not add any toxic substances to the fermentation media. It should tolerate steam sterilization process. It should be able to tolerate high pressure and pH changes.

The fermenter material used is also decided on type of fermentation process. For example, in case of bear, wine, lactic acid fermentation, the fermenter tanks are made up of wooden material. Whereas material such as iron, copper, glass and stainless steel can be use in some cases. Most of the time, 304 and 316 stainless steel is used for designing of a fermenter and these fermenters are mostly coated with epoxy or glass lining. A fermenter should provide the facility to control and monitor various parameters for a successful fermentation process.

 

2. Impellers:

Impellers are an agitation device. They are mounted on the shaft and introduced in the fermenter through its lid. They are made up of impeller blades and position may vary according to its need. These impellers or blades are attach to a motor on lid.The important function of an impeller is to mix microorganisms, media and oxygen uniformly. Impellers blades reduce the size of air bubbles and distributes these air bubbles and distributes these air bubbles uniformly into the fermentation media. Impellers also helps in breaking foam bubbles in the head space of fermenter. This foam formed during fermentation process can cause contamination problem and this problem is avoided by the use of impellers.

 

3. Baffles:

Baffles are mounted on the walls of a fermenter. The important function of baffles is to break the vortex formed during agitation process by the impellers. If the vortex is not broken, the fermentation media may spill out of fermenter and this may result in contamination as well as can lead to different problems. So it is important to break the vortex formed by using a barrier.

Baffles acts as a barrier which break the vortex.

4. Inoculation port:

Inoculation port is a device from which fermentation media, inoculum and substrate are added in the fermentation tank. Care should be taken that the port provides aseptic transfer.The inoculation port should be easy to sterilize.

 

5. Spargers:

A Spargers is an aeration system through which sterile air is introduced in the fermentation tank. Spargers are located at the bottom of the fermentation tank. Glass wool filters are used in sparger for sterilization of air and other gases. The sparger pipes contain small holes of about 5-10 mm. Through these small holes pressurized air is released in the aqueous fermentation media. The air is released is in the form of tiny air bubbles. These air bubbles helps in mixing of media.

 

6. Sampling point:

Sampling point is used for time to time withdrawal of samples to monitor fermentation process and quality control. The sampling point should provide aseptic withdrawal of sample.

 

7. pH control device:

The pH controlling device checks the pH of media at specific intervals of time and adjusts the pH of media at specific intervals of time and adjusts the pH to its optimum level by addition of acids or alkalis.

 

8. Temperature control:

Temperature control device generally contains a thermometer and cooling coils or jackets around fermenter.

During the fermentation process various reactions take place in the fermenter. Heat is generated and released in the fermentation media. This increase in temperature is detrimental to the growth of microorganisms which may slow down the fermentation process.

 

9. Foam controlling device:

A foam controlling device is placed on the top of fermenter with a inlet into fermenter. This device contains a small tank containing anti-foaming agent.

 

10. Bottom drainage system:

It is an aseptic outlet present at the bottom of fermenter for removal of fermented media and products formed.

 

Sterilization of the fermenter:

Fermenter can be sterilized separately for aseptic operation of pilot-scale and industrial fermentation process. Sterile medium which is sterilized in a separate cooker or continuous medium cooker for many fermentation is taken to the sterile empty fermenter.

On the other hand media required for the fermentation process and fermenter both are sterilized together in a single process of sterilization. All the fermentation process are not aseptic but contamination by microbes need to be maintained minimum by carried out media boiling and pasteurization. Or else fermentation medium will be covered by fast growing contaminant which do not allow proper fermentation process due to use of nutrient by contaminant. For small scale fermenter, medium and fermenter both are sterilized by steam under pressure in autoclave.

Sterilized of media and vessel is done using steam at 121 degree C for 15 minutes. Negative pressure should not to be developed inside the fermenter which may lead to contamination.

If the medium is sterilized in a separate batch cooker, or is sterilized continuously, then the fermenter has to be sterilized separately before the sterile medium is added to it. This is normally achieved by heating the jacket or coils of the fermenter with steam and sparging steam into the vessel through all entries, apart from the air outlet from which steam is allowed to exit slowly.

 

Agitation, aeration, pH, temperature controls during fermentation process

Agitation control:

Monitor the rate of rotation (rpm) of the stirrer shaft – Tachometer.

Detection mechanisms – electromagnetic induction voltage generation

                                        Light sensing

                                        Magnetic force

Laboratory fermenters - a.c. slip motor coupled to a thyristor control

Large fermenters – gear boxes, size of wheels and drive belts, changing drive motor.

 

Control of oxygen and aeration:

Oxygen supplied as air

Laboratory scale cultures – shake flask technique

Pilot and industrial scale – stirred vessels, airlift ferments, bubble column fermenters etc.

Maximum biomass production – dissolved oxygen concentration grater than critical level.

 

pH measurement and control:

pH of an actively growing culture – never constant.

Rapid changes in pH reduced by design of media, incorporating buffers.

pH measurement combined glass reference electrode.

 

 On/off controller

Signal received

Pinch valve opened or pump started

Acid or alkali pumped into the fermenter for a short period of time

Addition cycle followed by a mixing cycle

After mixing cycle another pH reading.

 

Temperature:

In a fermenter – Heat is generated by microbial activity & through mechanical agitation. If the heat not ideal – have to add or remove heat from the system.

Laboratory scale – extra heat provided by

• Thermostatically controlled bath
• Internal heating coils
• Heating jacket through which water is circulated

In large vessels – internal coils and cold water circulation.

 

Use of antifoam agents, process and controlling system

Use of antifoam agents:

All aerobic fermentations need and use antifoams. Without them, fermentation processors could encounter massive amounts of foam as organic matter – such as sugars, starch, cellulose and proteins – ferments and transforms into more desirable products. And without Invelychem’s solutions, those processors would likely be using greater quantities of antifoam materials, experiencing lower product yields and encountering higher costs.

Excessive foam in fermentation processes can limit equipment capacity, increase production time and cost, diminish product quality and even impact safety. Therefore, antifoams are commonly used to control foam in fermentation applications during two stages – fermentation and product separation.

During the fermentation stage, antifoams provide excellent foam control for a variety of aerobic fermentation applications, including bacterial and fungal fermentations.

Antifoams can also control foam during the downstream separation of fermentation products from fermentation broth. Because these antifoam products are highly effective at low concentrations, typically between 1 and 10 ppm of fermentation broth, they generate little to no interference in product separation. Invely’s silicone-based antifoam agents can help take foam that helps to minimize antifoam reapplications and save processing time and costs.

 

Control systems:

Process parameters controlled using control loops

Four basic components:

1. A measuring element
2. A controller
3. A final control element
4. The process to be controlled

Automatic control systems – 4 types

1. Two position controllers (on/off)
2. Proportional controllers
3. Integral controllers
4. Derivative controllers
• Measuring element senses a process parameter
• Generates a corresponding output signal
• Controllers compares signal with a predetermined set point value
• Produces an output control signal
• Final control element receives the control signal
• Adjusts the process by changing a valve opening or pump speed
• Process property controlled.