Industrial Microorganisms

Basic Knowledge about industrial microorganism. why industry choose microorganism rather than chemical. isolation technique of microorganism. source of microorganisms. Process of using microorganism. Disadvantages of using microorganisms in industry. Process of genetic modification of microorganisms. Storage process of microorganism. preservation methods of microorganism. Reculture methods of microorganism.

1. INDUSTRIAL MICROORGANISMS

2. Presented by Md. Rakibul Islam M.Sc. Student Dept. of Biotechnology and genetic Engineering Islamic university, Kushtia .

3. What is Industrial Microorganism ? Industrial Microorganism is the branch of biotechnology that applies in industry for creating industrial products in mass quantities. Industrial biotechnology or white biotechnology uses enzymes or microorganisms to make biobased products such as chemicals, food and feed, detergents, paper and pulp, textile and bioenergy with less waste generation & reduce energy consumption . E.g - Saccharomyces cerevisiae , Aspergillus Niger, Acetobacter acete etc

4. Interaction between industrial biotechnology & industrial microorganism Low cost Ethical issue Genetically modifiable Others Increase yield Make stable Purity Microorganisms purity Product purity More nutrient

5. Importance of Industrial Microorganisms Safety Environment friendly Purity Cost effective Easily available

6. Feature of Industrial Microorganism Genetically stable Efficiency Culture efficiency Product efficiency Simple nutritional value Cheap carbon and energy source Genetically manupulatable Addition Deletion Insertion Safety Easy product recovery Extracellular Intracellular Profitable byproduct

7. Sources of Industrially important Microorganisms Way to collect of desired microorganisms The place where we can get M.Os readily & Get M.Os. from culturing sample How to select the sources ? Study Select the area Cost Culture cost Maintenance cost Production cost Environment Health

8. Sources of Industrially important Microorganisms Sources of industrially important microorganism: a) Institution: Commercial source e.g - IFO(Osaka, japan), ATCC(USA), CMI(England) etc. Educational source Lab University Personal source

9. Sources of Industrially important Microorganisms b)Natural sources: Soil Industrial area soil Municipal waste soil Farmland soil Water Lake & river water Industrial area sewage Municipal area sewage Food & vegetables Animal and plants

10. Screening of Microorganism The procedure of isolation, detection, and separation of microorganism or metabolize of our interest from a mixed population by using highly selective methods is called screening. Which deals with low cost, rapid growth and easy to handle.

11. Screening 1. Primary screening: Sample collection Culture Media formulation Inoculums preparation Incubation Activity test Characterization Identification

12. Screening 2. Secondary screening Commercial value Environment friendly New product Toxicity Pathogenicity Economical stability Scale up Genetic stability Culture condition and media composition Fermentation condition

13. Strain improvement 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.

14. Why strain improvement is necessary ? Strain improvement is necessary to Reduce production cost Media and product cost To ensure safety Toxicity Pathogenicity Increase efficiency Yield Purity Longevity Increase stability Genetic & continuous culture

15. Methods of strain improvement Recombinant DNA technology Mutation Recombination Hybridization

16. 1. Recombinant DNA technology Genetic engineering, also known as recombinant DNA technology, molecular cloning or gene cloning. Recombinant DNA Technology enables isolation of genes from an organism, this gene can be amplified, studied, altered & put into another organism.

17. Recombinant DNA procedure i . Cutting of donor DNA ii. Cloning of a gene iii. Transformation

18. 2. Mutation Mutation: A mutation is a sudden and heritable change in the traits of an organisms. Application of mutagens to induce mutation is called mutagenesis. Types of mutation: 1. Natural mutation 2. Induced mutation

19. 1. Natural mutation Natural mutation: Mutation occurring without any specific treatment are called natural mutation. The causes of spontaneous mutation which are thus for understood include integration and excision of transposons along with errors in the functioning of enzyme such as DNA polymerase recombination enzymes and DNA repair enzyme.

20. 2. Induced mutation Induced mutation: Mutation are resulting due to a certain treatment are known as induced mutation. Induced mutation also classified into two type : Physical mutation Chemical mutation

21. Induced mutation Physical mutation caused by physical agents include X ray, gamma ray and ultraviolet radiations. X rays and gamma ray break the covalent bonds in DNA molecules thereby producing fragment. Double stand break result in major structural changes such as translocation, inversion or similar chromosomal mutation. Chemical mutation is the interaction of certain chemical compounds and cell metabolism may result in genetic changes in DNA structure, affecting one or more genes. Some chemical mutagenic agents are HNO 2 (nitrous acids), Alkylating agents, formaldehyde, benzene, DDT.

22. Results of mutation a)Genome mutation: : Mutation occur in chromosome level is called genome mutation. b) Chromosome mutation: when mutation occur in gene level it is called chromosome mutation. ( eg : deletion ,inversion, duplication or translation) c )Gene or point mutation: May result from changes in the base sequence in a gene. - Transition: purine - purine (A-G) and Pyrimidine-Pyrimidine (C-T) - Transversion : Pyrimidine–purine or purine-pyrimidine

23. Results of mutation Frameshift : When one nucleotide or more is inserted ,deleted thus altering the reading frame in the following transcription and translation processes and lead to a changed amino acid sequence in the resulting protein.

24. 3.Recombination Recombination: when sequence change with any process that is called recombination . Recombination is used for both genetic analysis as well as strain improvement. Recombination is two types Sexual Parasexual

25. Recombination Sexual: Some fungi used industrially have a complete sexual cycle. In these organisms, nuclear fusion results after the fusion of hypae has led to a mix together of nuclei in the heterokaryotic mycelium. Parasexual : Some of the most economically useful fungi such as penicillium chrysogenum and cephalosporium acremonium do not have sexual recombination . In parasexuality , the fusion of two hypae of equal or different polarity results in a mycelium with nuclei of both parent strain.

26. Others types Homologous: recombination occur in same locus is called homologous recombination. Heterologous : recombination occur in foreign locus is called heterologous recombination.

27. 4. Hybridization Hybridization: the process of an animal or plant breeding where protoplast fused with an individual of another species or variety is called hybridization. Types of hybridization - Interspecies Intraspecies

28. Hybridization Interspecies: This includes crosses between different species of the same genus or of different genera. When two species of the same genus are crossed, it is known as inter-species hybridization. E.g - E.coli with bacillus. Intraspecies : The parents involved in hybridization belong to the same species; they may be two strains, varieties or races of the same species. It is also known as intraspecies hybridization. e.g - hybridization between two E.coli

29. Preservation Preservation usually involves preventing the growth of bacteria, fungi (such as yeasts), and other microorganisms, as well as retarding the oxidation of fats which cause rancidity. Methods of preservation Sub-Culturing cryopreservation With Mineral Oil Storage under Liquid Nitrogen Freeze drying

30. 1. Repeated Sub-Culturing This is the most common, simplest and routine method of preservation of microorganisms. Selected microorganisms are initially grown on agar slants. After sufficient growth has taken place, they are transferred to fresh medium before they lose their viability. The appropriate time period for such transfer ranges from a week to few months (generally four to eight months).

31. Repeated Sub-Culturing Advantages: 1. This method is cheap, 2. Needs no special equipment, 3. Recommended for small collection centers and 4. Retrieval easy Disadvantages: 1. Change in physiological and genetical characters and 2. Time consuming.

32. Preservation with Mineral Oil In this method tubes with sterile agar slants are inoculated with a given culture. The tubes are incubated till sufficient growth of the given microbe takes place. The grown up culture is covered with a suitable mineral oil to a depth of about 1 cm above the top of the slanted surface using sterile technique. Thus, over laid cultures can be stored at room temperature or preferably at low temperature by about 15°C.

33. With Mineral Oil Advantages: This method of maintenance has the unique advantage that you can remove some of the growth under the oil with a transfer needle, inoculate a fresh medium and still original culture can be preserved. It is easy to control mites problem. Disadvantages: 1. Chances of air-borne contamination during sub-culturing are more, 2. Chances of mutations are more and 3. Retarded growth or inability to sporulate on retrieval.

34. Storage under Liquid Nitrogen This method is also called as cryogenic storage method, because a cryoprotective agent in the form of 10% glycerol is used. Industrially useful microorganisms are stored under very low temperature ranging from -150°C – 196°C. In this method ranging, low temperatures are created by employing liquid nitrogen. Metabolic activities of microorganisms are reduced considerably at this low temperature. This method is generally employed for the preservation of fungi, bacteriophages, viruses, algae, yeasts, animal and plant cells, and tissue cultures.

35. Storage under Liquid Nitrogen Advantages: 1. Viable cultures may be preserved for many years by this method, 2. Though the equipment is costly, the process is economical. 3. The cultures remain viable under these conditions for 10-30 years without undergoing any change in their characteristics. Disadvantages: 1. Evaporation of liquid nitrogen and replacement of lost liquid nitrogen regularly and periodically. 2. The method is relatively expensive.

36. Freeze drying F reeze drying involves freezing of a culture followed by its drying under vacuum which results in the temporary inhibition of metabolic activities of microorganisms. 1. The organism is allowed to grow to the maximum stationary phase on a suitable sterilized medium. 2. The cells are suspended in a protective medium like milk, serum or sodium glutamate. 3. A few drops of suspension are transferred to a glass ampoule. 4. The ampoules are then frozen by immersing into a freezing mixture of dry ice and alcohol at -78°C and are subjected to high vacuum until evaporation takes place completely. 5. The ampoules are then sealed and stored in a refrigerator

37. Freeze drying Advantages: 1. Culture once dried needs no further attention 2. It needs very cheap storage equipment like refrigerator and 3. It is easy to transport freeze-dried ampoules to far off places in large numbers in relatively small boxes. Disadvantages: 1. This is expensive Need expertise

38. Isolation of industrially important microorganism The term isolation refers to the separation of a strain from a natural, mixed population of living microbes, as present in the environment, for example in water or soil flora, or from living beings with skin flora, oral flora or gut flora, in order to identify the microbe(s) of interest. Microorganism isolation has 4 different techniques Crowded plate technique Auxanography Enrichment culture Indicator system

39. Crowded plate technique This technique is primarily employed for detecting those microorganisms, which are capable of producing antibiotics. The steps of crowed plate technique are given bellow: This technique starts with the selection of a natural substrate like soil or other source consisting of microorganisms. Then serial dilution of the soil or other source material for antibiotic producing microorganism is made. Then pouring and spreading of dilution soil samples 0.1ml on nutrient agar plate for 24-48 hours that gives 300 to 400 or more colonies per plate.

40. Crowded plate technique Colonies showing antibiotic activity are indicated by zone of inhibition around the colony. Such colonies are sub culture and purified by streak before making stock culture. The purified cultures are than tested to find the microbial inhibition spectrum.

41. Auxanography technique Auxanography technique: This technique is employed for the detection and isolation of microorganisms capable of producing certain extracellular substances such as growth stimulating factors like amino acids, vitamins etc. This technique has two major steps are:- A) Preparation of first plate: A filter paper is put across the bottom of petri dish. The nutrient agar is prepared and poured on the paper disc. Allowed to solidify Soil sample is diluted and proper dilutions are inoculated

42. Auxanography technique B)Preparation of second plate: A minimal media lacking the growth factors is prepared and seeded with the test organism. The seeded medium is poured onto fresh petri plate and the plate allowed to set. The agar in first plate is then lifted and placed on the second plate without inverting. The growth factor produced on agar can diffuse into the lower layer containing test organism The zones of stimulated growth of test organism around colonies is an indication that organism produce growth factor extracellularly .

43. Enrichment culture Enrichment culture is the use of certain growth media to favor the growth of a particular microorganism over others by enriching a component for the microorganism of interest. This is generally done by introducing nutrients or environmental conditions that only allow the growth of an organism of interest. Such as, Skim milk agar is used to selectively isolate protease producing species. Alkaline Peptone Water is used for the cultivation of vibrio .

44. Enrichment culture It consists of following steps: a.) Nutrient broth is inoculated with microbial source material and incubated. b.) A small portion of all inoculums is plated onto the solid medium and well isolated colonies are obtained. c.) Suspected colonies from the plate are sub cultured on fresh media and subjected for further testing.

45. Indicator system Indicator system: 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. The steps of indicator system are given bellow: The pH indicating dye may be used for detecting microorganisms that are capable of producing organic acid

46. Indicator system These dyes undergo color changes according to its pH Dye 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, base are also metabolic products of microbial growth. Incorporation of CaCO 3 in medium is also used to screen organic acid producing microbes on basis of formation of clear zone of dissolved CaCO 3 around the colony.