isolationofindustrialmicroorganisms-210610090245 (1).pptx

Isolation f industrially important microoranisms

1. BIOPROCESS TECHNOLOGY

2. What are industrial microorganisms? Industrial microbiology includes the use of microorganisms to manufacture food or industrial products in large quantities. Numerous microorganisms are used within industrial microbiology; these include naturally occurring organisms, laboratory selected mutants, or even genetically modified organisms (GMOs)

3. Microorganisms and its uses Production of dairy products: Bacteria are the key players here. Bread Baking: A species of Streptococcus is added to the dough before making bread to bring about the required fermentation . Alcoholic Drinks Organic acids Enzymes Steroid production Help in sewage treatment Used as insecticides

4. WHAT IS A CULTURE? Population of microorganisms grown under well defined conditions. WHAT IS MIXED CULTURE? When a particular species of microbe is present in a very small number in comparison to the total number of microorganisms , such culture is called as mixed culture. WHAT IS PURE CULTURE? A culture containing only one species of microbe is called pure culture. SPECIES- a collection of bacterial cells which share an overall similar pattern of traits in contrast to other bacteria whose pattern differs significantly. STRAIN- A strain is a subset of a bacterial species differing from other bacteria of the same species by some minor but identifiable difference.

5. Strain selection Important characteristics for strain The selection of strains resistant to infection The selection of non- foaming strains The Selection of strains which are resistant to components in the medium The selection of morphologically favourable strains The selection of strains which are tolerant of low oxygen tension The elimination of undesirable Products from a production strain The development of strains producing New fermentation products

6. Choosing microorganisms for Industrial microbiology and Biotechnology The characteristics of microbes that are desirable to the industrial microbiologist are: genetic stability, easy maintenance and growth, and amenability to procedures for extraction and purification of desired product

7. Finding microorganisms in nature major sources of microorganisms for use in industrial processes are soil, water, and spoiled bread and fruits; only a minor portion of microbial species in most environments have been identified

8. COMMON METHODS OF ISOLATION OF PURE CULTURE The process of screening a pure culture by separating one type of microbes from a mixture is called Isolation. Some common isolation methods are; Streak plate method Pour plate method- L oop dilution technique Serial Dilution technique Spread plate method Micromanipulator method Roll tube method

9. Streak plate method Prepare nutrient agar or any required medium and poured into the petri plates. Allow the plates to solidify. Sterilize the inoculation loop using flaming technique. Transfer microbial mixture from a tube to the edge of an agar plate with an inoculation loop as per illustration. Incubate plates at 37 C for 24hours.

10. Streak plate method

11. TYPES Different types of streaking techniques are available. They are T - streak, Quadrant streak, simple streak, radiant streak and continuous streak. These techniques are named based on the types of line of streak drawn on the surface of the medium.

12. Pour plate method To isolate and get pure culture of microorganisms Prepare nutrient agar and PDA potato Dextrose Agar medium (one plate needs approximately 15 - 20 ml) and sterilize at 121 C for 15 minutes. Dilute the sample up to 1: 10000000 (10 -7 ) using diluents. Add 1 ml of samples from 1:1000 (10 - 3 ). Pour the medium into sample added petriplates. Rotate the petriplates clockwise and anticlockwise direction. Allow the plates to solidify. Similarly perform pour plating for other dilutions like 10 -4 , 10 - 5 and 10 - 6 . Incubate all nutrient agar plates at 37 C for 24 hours and PDA plates at 25 – 30 C for 48 hours and record the results.

13. Pour plate method

14. Spread plate method The method is performed for the assay of chemicals like antibiotics, vitamins etc. This method is called spread because L rod or cotton swab is used to spread the sample. This techniques also used for the isolation and enumeration of microorganisms from samples with lower populations of bacteria and other microorganisms.

15. Spread plate method Prepare nutrient agar medium, sterilize at 121 C and pour in to the petri plates. Dilute the sample up to 1:100000 (10 - 5 ). Add 1 ml of sample from 10 -3 on to the centre of an agar medium using sterile pipette. Perform similar procedure for 10 -4 and 10 - 5 dilutions. Dip the glass spreader (L- rod) in to a beaker of ethanol/spirit. Briefly flame ethanol soaked spreader on Bunsen burner and allowed it to cool. Spread the sample evenly over the agar surface. Incubate all the plates at appropriate temperature (for bacteria 37 C and for fungus 25 – 30 C) for 24 to 48 hours.

16. SERIAL DILUTION

17. MICROMANIPULATOR METHOD Micromanipulators • Micromanipulators have been built, which permit one to pick out a single cell from a mixed culture. This instrument is used in conjunction with a microscope to pick a single cell (particularly bacterial cell) from a hanging drop preparation. ADVANTAGES OF MICROMANIPULATOR METHOD • The advantages of this method are that one can be reasonably sure that the cultures come from a single cell and one can obtain strains with in the species. DISADVANTAGES • Disadvantages are that the equipment is expensive, • Its manipulation is very tedious, and it requires a skilled operator

18. ROLL TUBE METHOD Prepare ten-fold dilution and add 0.1 ml of diluted culture to molten agar cooled to 50 C poured in test tube. Now tilt the tube and roll so that medium is formed as a thin film around the wall of tube. Incubate and count the no of colonies on next day .

19. Enrichment methods for isolation of microorganisms Enrichment methods are useful for quick isolation of specific types of organisms. Types of organisms Enrichment method Thermophiles High temp (42- 100 C) Psychrotrophs low temp (5- 15 C) Acidophiles Low pH (2- 4) Halophiles High NaCl concentration Anaerobes N2 atmosphere Actinoplanes Pollen grains Myxobacteria Wood bark

20. Strains of microorganisms from unusual environments The unusual environments such as cold habitats, high altitudes, deserts, deep sea and petroleum fields are constantly being tried for this purpose. Such strains may be capable of producing new products of industrial importance

21. Screening of metabolites for isolation of microorganisms The microorganisms can be tested directly for the product formation, and isolated. For example – if the product is an antibiotic, then the test system consists of the strains of organisms which inhibit zones, on the agar plates. Another example is the isolation of microorganisms producing amylases. When grown on agar plates containing starch, and then stained with iodine, amylase producing organisms can be identified and isolated.

22. Screening of microbes 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.

23. 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. The following are some of the important primary screening techniques : Indicator dye technique The crowded plate technique Enrichment culture technique Auxanographic technique

24. PRIMARY SCREENING OF ORGANIC ACID PRODUCING MICROORGANISMS The ph indicating dyes may be used for detecting microorganism that are capable of producing organic acids. These dyes undergo colour changes according to its ph. Dyes such as Neutral red, Bromothymol blue are added to the poorly buffered nutrient agar media . Colonies are sub cultured to make stock culture. Further testing is needed since inorganic acids, bases are also metabolic products of microbial growth

25. Incorporation of CaCO3 in medium is also used to screen organic acid producing microbes on basis of formation of clear zone of dissolved CaCO3 around the colony.

26. PRIMARY SCREENING OF ANTIBIOTIC PRODUCING MICROORGANISMS Crowded plate technique is used for screening of antibiotic producing microorganisms. Does not give information about the sensitivity of antibiotics towards other microorganisms. Dilutions are made and then pouring and spreading of soil samples that give 300 to 400 or more colonies per plate. Colonies showing antibiotic activity are indicated by zone of inhibition around the colony . Such colonies are sub cultured and purified by streak before making stock cultures.

27. Antibiotic producing microbe Amylase producing microbes

28. Antibiotic activity against microbe

29. AUXANOGRAPHY TECHNIQUE This technique is largely employed for detecting microorganisms are able to produce growth factors (e.g. Amino acid and vitamins) extracellular. ENRICHMENT CULTURE TECHNIQUE This technique was used to isolate the desired microorganisms form a heterogeneous microbial population present in the sample. Either medium or incubation conditions are adjusted so as to favor the growth of the desired microorganism.

30. SECONDARY SCREENING It’s a systematic screening programme intended to isolate industrially important or useful microorganisms . SOME IMPORTANT POINTS ASSOCIATED WITH SECONDARY SCREENING ARE It is useful in sorting of microorganisms that have real commercial value. The microorganisms having poor applicability in fermentation process are discarded. Provides the information whether the product formed by microorganisms is new or not. This may be accomplished by paper , thin layer, chromatographic technique.

31. Screening for new metabolites, and isolation of microorganisms Research work is particularly directed for identifying chemotherapeutically important products for the treatment of tumors, bacterial diseases (newer antibiotics against resistant strains) and viral diseases, besides several other substances (e.g. hormones, enzyme inhibitors). In addition, isolation of microorganisms for improvement of food industry, and for efficient degradation of the environmental pollutants and hazardous chemicals also assumes significance.

32. An isolated producer strain should possess the following characters: 1. It should be able to grow on relatively cheaper substrates. 2. It should grow well in an ambient temperature preferably at 30-40°C. This reduces the cooling costs. 3. It should yield high quantity of the end product. 4. It should possess minimum reaction time with the equipment used in a fermentation process. 5. It should possess stable biochemical characteristics. 6. It should yield only the desired substance without producing undesirable substances. 7. It should possess optimum growth rate so that it can be easily cultivated on a large scale.

33. Microbes in Industrial Products Beverages. Yeasts are the widely used microorganism for the production of beverages like beer, brandy, rum, wine, whiskey, etc. Organic acids. Microbes are also used for the industrial production of certain organic acids. Enzymes. Antibiotic. Vitamins.

34. A List of important microorganisms and their products Microorganism Product Algae Chlorella sorokiniana Single cell protein Spirulina maxima Single cell protein Bacteria Acetobacter aceti Acetic acid Bacillus subtilis Bacitracin Actinomycetes Streptomyces aureofaciens Tetracycline Fungi Aspergillus niger Citric acid

35. Strategies for improvement of industrially important strains -

36. What are strains? A strain is a genetic variant or subtype of a microorganism (e.g., a virus, bacterium or fungus). Microbial strains can also be differentiated by their genetic makeup using metagenomic methods to maximize resolution within species. What are industrial strains? Strains which synthesize one component as the main product are preferable, since they make possible a simplified process for product recovery. Why is strain development important in industrial microbes? Prerequisite for efficient biotechnological processes at industrial scale is the use of microbial strains which produce high titre of the desired product. The process of enhancing the biosynthetic capabilities of microbes to produce desired product in higher quantities is defined as microbial strain improvement.

37. From where we can find industrial strains? The first step in developing producer strains is the isolation of concerned microorganisms from the natural habitats. What we are looking? From where we can get? • The procedure of isolation, detection, and separation of microorganisms of our interest from a mixed population by using procedures is highly selective called Screening.

38. Proper strain used in industry genetically regarded as safe (GRAS)

39. Methods of strain improvement Mutation and mutant selection Recombination Transduction Transformation Conjugation Protoplast fusion Parasexual recombination Recombinant DNA technology

40. Mutation and mutant selection A mutation is a sudden and heritable change in the traits of an organism. Mutations occurring without any specific treatment are called “spontaneous mutation”. Mutation are resulting due to a treatment with certain agents are known as “induced mutation”. Application of mutagens to induce mutation is called mutagenesis. Agents capable to induce mutations are called mutagens.

41. Mutagens Physical Radiation Heat Chemical Base analogs Intercalating agents Deaminating agents Metals Biological Transposon Virus Bacteria

42. The practical isolation of mutants

43. Transduction

44. Transformation Bacterial transformation is a process of horizontal gene transfer by which some bacteria take up foreign genetic material (naked DNA) from the environment. Cells in which transformation can occur are ‘competent’ cells.

45. Conjugation Bacterial conjugation : is the transfer of genetic material between bacterial cells by direct cell-to- cell contact or by a bridge- like connection between two cells. Conjugation types: F + x F - Conjugation Hfr x F - Conjugation F’ x F - conjugation

46. Protoplast fusion

47. Parasexual recombination

48. Genetic Engineering Genetic engineering (GE) is the process of using recombinant DNA (rDNA) technology to alter the genetic makeup of an organism. Genetic engineering is accomplished in three basic steps: The isolation of DNA fragments from a donor organism The insertion of an isolated donor DNA fragment into a vector genome The growth of a recombinant vector in an appropriate host.

49. Improvement of microbial processes by GE Primary metabolites New processes for the production of amino acids and vitamins have been developed by recombinant DNA technology. Escherichia coli strains were constructed with plasmids bearing amino acid biosynthetic operons. Cloning extra copies of threonine export genes into E. coli led to increased threonine production. An engineered strain of Corynebacterium glutamicum producing L - tryptophan was further modified by cloning in additional copies of its own transketolase gene . Biotin has been made traditionally by chemical synthesis but recombinant microbes have approached a competitive economic position. The cloning of a biotin operon ( bioABFCD) on a multicopy plasmid allowed E. coli to produce 10000 times more biotin than did the wild- type strain. Riboflavin production in Corynebacterium moniagenes - was developed by cloning and overexpressing the organism's own riboflavin biosynthesis genes and its own promoter sequences . A novel process for vitamin C synthesis involved the use of a genetically engineered Erwinia herbicola strain containing a gene from Corynebacterium sp.

50. Improvement of microbial processes by GE Secondary metabolites Studies revealed that many antibiotic biosynthesis genes were arranged in clusters. The entire cephamycin C pathway was cloned and expressed from a cephamycin- producing strain of Streptomyces cattleya into another cephamycin producer, Streptomyces lactamgens , a two- to three-fold improvement was obtained. Microbial enzymes Genes encoding many microbial enzymes have been cloned and the enzymes expressed at levels hundreds of times higher than those naturally produced. Scientists at Novo Nordisk isolated a very desirable lipase for use in detergents from a species of Humicola . For production purposes, the gene was cloned into Aspergillus oryzae , where it produced 1000- fold more enzyme and is now a commercial product. The α-amylase gene from Bacillus amyloliquefaciens was cloned using multicopy plasmid pUB110 in B. subtilis

51. Improvement of microbial processes by GE Polymers, fuels, foods and beverages Recombinant DNA manipulation of Xanthomonas campestris increased titers of xanthan by two- fold. Alcohol dehydrogenase II and pyruvate decarboxylase genes from Zymomonas mobilis were inserted in E. coli. Beer wort contains barley β- glucans which reduce the filtrability of beer and lead to precipitates and haze in the final product. The gene coding for endoglucanase was transferred from Trichoderma reesei to brewer's yeast and the engineered yeast strain efficiently hydrolyzed the β- glucans. Bioconversions Recombinant Candida pasteurianum can carry out the conversion of glycerol to 1,3-propanediol.