Industrial Microorganisms: Preservation and Improvement Techniques PDF

Chapter II Isolation, preservation and improvement of industrial microorganisms. Industrial Microbes Originally isolated from nature, but its quality is improved by genetic manipulation via - mutagenesis and selection, or - nuclear fusion or - protoplast fusion (fungi, and streptomyc...

Chapter II Isolation, preservation and improvement of industrial microorganisms. Industrial Microbes Originally isolated from nature, but its quality is improved by genetic manipulation via - mutagenesis and selection, or - nuclear fusion or - protoplast fusion (fungi, and streptomycetes and actinomycetes) or - recombinant DNA technology  Characters of industrial organisms 1-The nutritional characteristics of the organism; is it able to grow in very cheap medium or pre- determined one. 2-The optimum temperature of the organism; for maximum productivity, above 40oC. 3- be available in pure culture 4- be genetically stable, but amenable to genetic manipulation ‫قابل للتعديل الجيني‬ 5- have high productivity of desired metabolites or products 6- the ease of handling, e.g., produce spores or other reproductive structures to allow easy inoculation. Grow in such a way that the cells are easily separated from the product (i,e., the ease of product recovery) 7- grow rapidly and produce product quickly in large-scale culture. 8- Also not be harmful to humans, plants and animals, etc.  Sources of Microbes Microbes are obtained from two sources 1- Culture collection such as American Type Culture Collection (ATCC). These organisms are called standard organisms are mainly used for assays (microbiological, vitamins), training on running the fermentation process, and educational purposes never used for production of required materials. Because they give very low yield of the required product.  Culture collection Culture collection Address National Collection of Type London, UK Cultures (NCTC) American Type Culture Rockville, USA Collection (ATCC) Deutsche Sammlung von Braunschweig, Mikroorganismen und Germany Zelkulturen (DSM) Collection of Microorganisms Saitama, Japan (JCM) 2- Isolation from nature Usually from soil since the soil is very rich in microbial contents by using enrichment liquid culture. Isolation methods are based on selection of M. O of the desired characteristic(s). Enrichment culture will increase the number of a given organism relative to numbers of other types in the original inoculum.  i.e., use selective force for isolation of desired organism by: a- addition of certain substance in the batch enrichment liquid culture to enhance the growth of desired organism or to inhibit or retard growth of undesired organism(s).  Prior to the culture stage, it is often advantageous to subject the environmental source (normally soil) to conditions which favour the survival of the organisms in question. For example, air-drying the soil will favour to inhibit the vegetative cells but not spore forming cells as in case of actinomycetes.  b-Enrichment liquid culture is carried out in shake flasks (for aeration) to increase growth rate of desired organism. c-The selective force may also be re- established (repeated sub culturing) by inoculating the culture into identical fresh medium at time interval relating to max growth rate of desired organism.  The prevalence of an organism in a batch enrichment culture will depend on its maximum specific growth rate compared with the maximum specific growth rates of the other organisms capable of growth in the inoculum.  Thus, provided ‫ بشرط‬that the enrichment broth is sub-cultured at the correct times, the dominant organism will be the fastest growing of those capable of growth.  Such sub-culturing will be repeated several times (to allow greater increase of desired organism in liquid culture) before the dominant organism is isolated by spreading a small inoculum of the enriched culture onto solid medium.  Isolation of bacteria from nature 1- Inoculating 3-Selective prepared soil sample isolation of desired into enrichment liquid organism medium 4-Preservation of Pure organism 2- sub- culturing onto plate Culture isolated from soil  The preservation of industrially important microorganisms The culture used to initiate an industrial fermentation must be viable and free from contamination and keeping its productivity. Thus, industrial cultures must be stored in such way to eliminate genetic change. The preservation (Storing either) at 1-Reduced temperature or 2- Dehydrated form.  1 - Storage at reduced temperature: A) Storage on agar slopes: a-Cultures grown on agar slops may be stored in a refrigerator (5oC ) and sub-cultured at approximately 6- monthly intervals. The time of subculture may be extended to one year if the slops are covered with sterile medicinal grade mineral oil. b- or culture grown in broth may be stored in a freezer at - 20 oC, after addition of 10-20 % glycerol. B) Storage under liquid nitrogen: The metabolic activities of micro- organisms may be reduced considerably by storage at the very low temperatures (-150 oC to – 196oC ) which may be achieved using a liquid nitrogen refrigerator. This approach is the most universally applicable of all preservation methods of all biological systems, i.e.,  i.e., Fungi, bacteriophage, viruses, algae, yeasts, animal and plant cells and tissue cultures have all been successfully preserved by this technique. The technique involves growing a culture to the maximum stationary phase, re-suspending the cells in a cryoprotective agent (such as 10% glycerol) and freezing the suspension in sealed ampoules before storage under liquid nitrogen. BN1; Some loss of viability is suffered during the freezing and thawing stages ‫ مراحل التجميد والذوبان‬but there is virtually ‫عاده فعليا‬no loss during the storage period. again, It is the best method for long term (for years) preservation of cells that do NOT survive freeze- drying (Lyophilized) ,such as animal and plant cells and tissue cultures.  BN2; Although the equipment is expensive the process is economical on labor. However, the method has the major disadvantage that liquid nitrogen evaporates and must be replenished regularly ‫تتجدد بانتظام‬. If this is not done, or the apparatus fails, then the consequences ‫ النتائج‬are the loss of the collection.  Liquid nitrogen Tank Culture under liquid nitrogen Liquid Nitrogen Tank  2-Storage in a dehydrated form A-Dried cultures: Dried soil cultures have been used widely for culture preservation. Used only for fungi, actinomycetes and other spore forming bacteria.  Moist, sterile soil may be inoculated with culture medium and incubated for several days for some growth to occur and then allowed to dry at room temperature and kept in a dry atmosphere or, preferably, in a refrigeration. B- Lyophilization: Lyophilization, or freeze-drying, involves the freezing of a culture by its drying under vacuum which results in the sublimation of the cell water. The technique involves growing the culture to the maximum stationary phase and resuspending the cells in a protective medium such as milk, serum or sodium glutamate.  A few drops of the suspension are transferred into an ampoule, which is then frozen and subjected to a high vacuum until sublimation is complete, after which the ampoule is sealed. The ampoules may be stored in a refrigerator and the cells may remain viable for 10 years or more.  Lyophilization is very convenient for service culture collections because, once dried, the cultures do not need further attention (i.e., remains stable) and the storage equipment is only refrigerator which is cheap and reliable.  However, freeze-dried cultures are tedious to open and revitalize ‫ تنشيط‬/‫ منحه حياة جديدة‬and several sub-cultures may be needed before the cells regain their typical characteristics. Lyophilization are NOT suitable for preservation of animal and plant cells and tissue cultures. Overall, the technique appears to be second only to liquid nitrogen storage and even when liquid nitrogen is used makes an excellent insurance against the possibility of the breakdown of the nitrogen freezer.  Quality control of preserved stock cultures: Each batch of newly preserved cultures should be routinely checked to ensure their quality: At least 3% of the ampoules are reconstituted and the cultures assessed for the 3 tests; purity, viability and productivity. If the samples fail any one of these tests the entire batch should be destroyed.  How could you increase the yield product To increase the yield 1- Improvement the quality of industrial micro-organisms 2- Improve conditions of production; a- physical condition b- nutritional condition  1- Improvement the quality of industrial micro-organisms A- Mutations : The mutagenic agents could be UV radiation, ionization radiation, chemicals like nitrous acid, nitrosoguanidine,…. etc. The exposure to these agents leads to the death of most of the cells in a culture, however, some survivors may contain some of the mutants exhibiting changed characteristics; desired one.  i.e., A small population of these cells (mutants) could be the one which produces large number of bioactive components of interest. These components are either primary or secondary metabolites. What are the characteristics of primary or secondary metabolites?  A. Primary metabolites. 1- essential for cell growth Or its production are associated with cell activities or energy production 2- its production are not associated to media components 3- its production correlates cell growth and produced at concentrations correlate growth rate, as single compound. and in trophophase 4- examples are amino acids, fatty acids, some vitamins and alcohol. 5- usually produced and kept inside the cell  B. Secondary metabolites 1- are not essential for cell growth 2- its production are not associated with cell growth or energy production 3- its rate of production doesn't correlate with rate of cell growth. 4- its production depends on media components 5- metabolite formed after growth has occurred, during idiophase and usually produced at high concentrations in idiophase and as single or mixture of closely related organic compounds. 5- examples are antibiotics and some vitamins 6- usually produced and secreted outside the cell  Selection of mutant producing improved (higher) level of primary metabolites. The most important commercially available primary metabolites are the amino acids. Two of the most common and commercially available amino acids are - glutamic acid and - lysine  A-Glutamic acid Glutamic acid is also called glutamate, mostly metabolized in brain and is an essential Neuro- transmitter. i.e., is an excitatory ‫ مثيرة‬neurotransmitter. The brain can use glutamic acid as fuel that increases the firing of neurons in the central nervous system. ‫يمكن للدماغ استخدام حمض الجلوتاميك كوقود يزيد من إطالق نشاط الخاليا العصبية‬ ‫في الجهاز العصبي المركزي‬ This is essential for normal, healthy brain function.  It is converted into either glutamine or Gamma-Aminobutyric Acid (GABA),which are the two other amino acids that help pass messages to the brain. If you do not produce the proper quantities of glutamic acid, some serious brain disorders can arise.  - Glutamic acid may prove beneficial in the treatment of muscular dystrophy, Parkinson's Disease and schizophrenia. - Glutamic acid helps to correct personality disorders and is useful in treating childhood behavioral disorders.  - It is used in the treatment of epilepsy, mental retardation, muscular dystrophy, ulcers, and hypoglycemic coma (a complication of insulin treatment for diabetes).  Production of Glutamic acid The organism for the production is Corynebacterium glutamicum. Two basic aspects must be known in addition to the fact that primary metabolites produced in concentration correlating only the cell need.  These two basic aspects are: a- Increasing synthesis of the glutamic acid concentration in the cells above their requirement resulting in induction of Negative feedback mechanism and thus stop the conversion of - ketogutamic acid (the intermediate of production of glutamic acid).  b- Cells naturally requires the vitamin biotin (25- 35 g/mg of dry cells) for synthesis of fatty acid of cell membrane phospholipids providing the high (normal) cell membrane integrity. Weakness of the cell membrane Integrity increases the permeability and thus, release glutamic acid outside  Corynebacterium glutamicum produces glutamic acid during tri-carboxylic acid cycle (Krebs cycle). For commercially production of glutamic acid two essential characters must be present in C. glutamicum. - Lack enzyme which converts - ketogutaric acid to of succinic acid. - biotin auxotroph - Lack enzyme which converts - ketogutaric acid to succinic acid, so direct the cycle for production of glutamic acid with higher rate. ,,,,,,, - biotin auxotroph has cell membrane deficient in phospholipids thus corrupts their selective permeability, resulting in continuous release glutamic acid outside and productivity…….i.e., Inhibits negative feedback mechanism  The explanation of excretion of glutamic in the biotin limiting condition is based upon the fact that it results into the cell membrane being deficient in phospholipids thus corrupts their selective permeability. This will the release out of the amino acid and thus no –ve feedback ,consequently, there is continuous production of amino acid, i.e., increase the productivity  B- Lysine. It plays a role in the production of carnitine (which helps to loss of lipid by oxidation and thus aiding in weight reduction, and transport the amino acids into mitochondria where it used for energy production) Promotes normal growth and development by increasing collagen formation Supports the production of other proteins; like enzymes, antibodies and hormones Promotes bone health by increasing calcium absorption; prevents osteoporosis or weak bones by reducing bone loss  Helps convert fatty acids to energy, aiding in weight reduction Helps lower bad cholesterol levels, thus reducing the risk for heart disease Promotes skin health through increased collagen formation May be used to treat viral infections like herpes simplex, cold sores, shingles, human papilloma virus (HPV) infection such as genital warts, and genital herpes Can relieve migraines and other types of pain and inflammation When taken with other nutrients like vitamin C, it can reduce chest pains (angina) related to heart disease Helps in muscle building, when taken with other amino acids like arginine  production of Lysine: The organism used for production is Corynebacterium glutamicum a- Homoserine auxotroph ….. To direct the conversion of aspartylsemialdehyde to pathway of synthesis of lysine instead of pathway of synthesis of homoserine. b- Increase of cell permeability, i.e., corruption of cell selective permeability to overcome –ve feed back mechanism. This is achieved by 1- surfactants; fatty acid derivatives. 2- bata- lactams..... also 3- biotin auxotroph  aspartate aspartyl phosphate - aspartylsemialdehyde homoserine - - lysine pathways to - other amino acids  NB: Increasing cell permeability and decreasing the primer metabolites accumulation inside the cell, thus inhibit negative feedback mechanism is achieved by using of a- biotin auxotroph b-surfactants; fatty acid derivatives. c- low concentration ( sub-MIC) of penicillins  Isolation of an auxotroph (e.g., homoserine auxotroph). This is done by using penicillin enrichment technique. Penicillin selectively kills life growing bacterial cells and therefore if the survivors of mutation treatment (by using a mutagenic agent., e.g., UV) were cultured in a medium containing penicillin and lacking the growth requirement (homoserine) of the desired mutant, only those cells unable to grow Would survive(remain life), these are, the desired auxotrophs.  So, if the cells were removed from the penicillin broth lacking homoserine after UV treatment, then washed and re-suspended in a medium containing requirements of desired auxotroph (homoserine) and lacking penicillin, thus the resulting culture should be rich in the desired strain. NB; UV treatment results in - some cells are directly killed and– other cells are life normal and mutants that include homoserine auxotroph. The subculturing in medium containing penicillin and lacking homoserine, penicillin will kill all cells (as they grow) except homoserine auxotroph.The subculturing in medium containing homoserine and lacking penicillin, homoserine auxotroph will grow.  Again; UV treatment results in - some cells are directly killed and - other cells are life normal and mutants that include homoserine auxotroph. The subculturing in medium containing penicillin and lacking homoserine, penicillin will kill the normal and undesired mutants (these are life growling cells) The homoserine auxotroph mutants (desired cells are life and non- growing. Upon their subculturing in medium containing homoserine and lacking penicillin, they will grow and are the desired auxotroph b- Selection of Secondary metabolite producing strains. - Screening for isolation strain from nature and then improve its quality, e.g.; Penicillium notatum produces 1g /ml of penicillin and replaced by Penicillium chrysogenum (10 g/ml) and improve its quality after mutation (U.V.) and increased to yield to 50 mg/ml (500 time more). - Streptomyces kanamyceticus strain are either autotoxic resistant (i.e., resist to its toxic product) by mutation or genetically engineered strain provides high yield of kanamycin.  Autotoxic resistant: a strain which resists high concentration of antibiotic in trophophase and thus able to grow in presence of accumulated relatively higher concentration inside the cell during increased /greater productivity in idiophase. In case of kanamycin, this strain by mutation (mutation of already present gene) produces relatively higher 6/- N. kanamycin acetyltransferase or acquires it by genetic engineering (i.e., introducing cloned gene) and thus is resistant to relatively high concentration of accumulated kanamycin during production phase (the idiophase). Why Autotoxic resistant strain is required for production of kanamycin but not penicillin G?) - in kanamycin the streptomyces strain is bacterium which is affected by kanamycin. This strain acquires more 6/- N. Kanamycin- acetyl- transferase production and thus is resistant to relatively higher concentration of residual kanamycin accumulated inside the cell. consequently, is able to grow at high conc. produced in idiophase. Penicillin G is produced by fungus; penicillium sp. which is not affected by antibacterial penicillin  Very important to know; The antibiotics like kanamycin (as secondary metabolites) released out the cell and 6/- N. Kanamycin-acetyl transferase produced by wild- type (isolated from nature) strain is enough to inactivate normal residually accumulated synthesized antibiotic in the cell. Autotoxic resistant strain produces more and more inactivating enzyme to inactivate more residually accumulated antibiotic in the cell.  i.e., Techniques used either by a- mutation of already defence enzyme encoding gene or by b- genetically engineering; introducing a cloned gene ( which mediates the production of the 6/- N. Kanamycin-acetyl transferase) into the cell. c- Both techniques enabled the cell to produce high conc. of the inactivating enzyme  Again, in general, Improvement the quality of industrial microorganisms A) Mutations discussed before B) Genetic Recombination 1- Nuclear fusion (parasexual recombination) 2- Protoplast fusion 3- Genetic engineering