Bioprocess Technology Lecture Notes PDF

BIO62104 Topic 4: Isolation, Bioprocess Preservation and Strain Technology Improvement  Lecture outline Explain the important techniques in isolating industrially important microorganisms Explain the criteria in selection of microorganisms D...

BIO62104 Topic 4: Isolation, Bioprocess Preservation and Strain Technology Improvement  Lecture outline Explain the important techniques in isolating industrially important microorganisms Explain the criteria in selection of microorganisms Describe the preservation of industrially important microorganisms Describe the genetic modification of bacteria strains  Isolation of industrially important microorganisms First stage in fermentation process: – To screen for microbes with potential industrial applications – Objective: to obtain pure or mixed cultures and carry out desired reaction or produce desired product  Isolation of industrially important microorganisms Selection of culture is a compromise between productivity of the microbes and the economic constraints of the process The desired cultures can be obtained from: – Natural resources – Culture collections Question: Is it cheaper to buy than to isolate? Which is better?  Isolation of industrially important microorganisms Major culture collections 1. National collection of type cultures (NCTC) 2. National collection of yeast cultures (NCYC) 3. American type culture collection (ATCC) Criteria in selection of microorganisms 1. Nutritional characteristics 2. Optimum growth temperature 3. Reaction of organism with equipment used and suitability to the type of process to be used 4. Stability of the microorganism and its amenability to genetic manipulation Criteria in selection of microorganisms 5. Productivity of microorganisms 6. Ease of product recovery from culture broth 7. Quality of product produced  Isolation of microorganisms Researchers try to isolate strains from extreme or unusual environments in hope that such strains may be capable of producing new metabolites – For example: high altitudes, cold habitats, sea water, hot spring, desserts, petroleum fields, etc. – Acidophiles, psychrophiles, thermophiles, anaerobes, halophiles  Isolation of microorganisms Isolation methods – Selection of desired characteristics: selective pressure/force – Isolation of organisms that will grow on a particular substrate or in the presence of certain compounds Two different methods: 1. Enrichment liquid culture 2. The use of solidified media  Isolation of microorganisms Enrichment liquid culture – Increase in the number of desired organism compared to other types in the original inoculum – By establishing a medium and a set of incubation conditions that are selective for the desired organisms and screen out undesired organisms – Batch/Continuous enrichment technique  Isolation of microorganisms Enrichment liquid culture: 1. Batch enrichment technique –provide specific conditions (provision of particular substrate or inclusion of inhibitors) to a mixed population and select specific microbes Problems: changes in the selective force may allow growth of other undesired types of bacteria, e.g. excretory product of microbe A may favour growth of microbe B  Isolation of microorganisms Enrichment liquid culture: 1. Batch enrichment technique – selective force can be re-established by inoculating the enriched culture into identical fresh medium for several times Sub-culturing may be repeated several times before the dominant organism is isolated by spreading a small inoculum of the enriched culture on to solid medium  Isolation of microorganisms Enrichment liquid culture: 1. Batch enrichment technique – time of subculture is critical and should correspond to the point at which the desired organism is dominant The prevalence of an organism in an enrichment culture will depend on the maximum specific growth rate (µmax) However: microbe with highest µmax ≠ most useful or desired microbe  Isolation of microorganisms How to overcome this problem? – The problem of time of transfer and selection based on the µmax may be overcome by continuous enrichment technique *continuous enrichment technique: fresh medium is added to the culture broth at a constant rate  Isolation of microorganisms Enrichment liquid culture: 2. Continuous enrichment technique – an open system of constant volume to which fresh medium is added continuously and used culture medium and cells are removed continuously, at a constant rate Chemostat: device used to maintain cell populations in exponential phase for long period of time  Isolation of microorganisms Enrichment liquid culture: 2. Continuous enrichment Fi = Fo = F technique – Dilution rate, D = F/V ❖ F = Flow rate of medium into the vessel ❖ V = Volume of the vessel culture medium  Isolation of microorganisms Enrichment liquid culture: 2. Continuous enrichment technique – Once a chemostat system is in equilibrium, the system is said to be in steady state: Formation of new biomass is balanced by the loss of cells from the vessel Net change in the cell concentration over a period of time: dX dX = growth − output = µX - DX dt dt  Isolation of microorganisms Enrichment liquid culture: 2. Continuous enrichment technique – under steady state conditions, the cell concentration remains constant: dX = 0 and µ = D dt Different microbes have different µ at steady state for different limiting substrate concentration – Dilution rate (D) control the growth rate of the culture  Isolation of microorganisms Example: competition between two microorganisms, A and B capable of growing in a continuous culture Q: How to isolate and separate A and B from a continuous enrichment culture? µ A X B Y Limiting substrate concentration  Isolation of microorganisms The use of solidified media – carried out directly on plate to test for the biological activity of microorganisms: – Only those colonies showing activity will be isolated – Usually involves selective medium (substrate for enzyme) which encourages the growth of enzyme producers  Preservation of industrially important microorganisms The purpose of preservation is to: – Maintain viability – Avoid contamination – Maintain require behaviour/property of microbes Three methods: – Storage at reduced temperature – Storage in a dehydrated form – Storage by subculture Preservation of industrially important microorganisms 1. Storage at reduced temperature a. Agar slopes – can be stored in refrigerator (4°C) or freezer (-20°C, -70°C) and subculture every 6 months interval; slopes can be covered with sterile mineral oil b. Spore suspension – suspend in a sterile distilled water or glycerol (15-30%) at 4°C c. Cell suspension in sterile glycerol – bacteria and yeast culture can be stored in 15-30% glycerol and keep at -80°C; can last for few years Preservation of industrially important microorganisms 1. Storage at reduced temperature d. Liquid nitrogen – storage at very low temperature (-150°C to -196°C); storage of cells in the presence of cryoprotective agent glycerol Best results can be obtained by: - Slowly freezing the cells before storage - Thawing the cells rapidly Preservation of industrially important microorganisms 2. Storage in a dehydrated form (lyophilized) A process in which microorganisms are frozen and water is removed as vapour directly from ice, without passing through the liquid state – sublimation  Preservation of industrially important microorganisms 2. Storage in a dehydrated form (lyophilized) Resuspend cells in a Grow culture to the Transfer a few protective medium maximum drops of suspension (skimmed milk, stationary phase to ampoule sucrose, serum) Freeze dry the Store the ampoules ampoule until Seal ampoule in situ in refrigerator (for sublimation is 10 years or more) complete** ** The freeze drying process involve three steps: prefreezing, primary drying, secondary drying Preservation of industrially important microorganisms 2. Storage in a dehydrated form (lyophilized) a) Prefreezing Products to be freeze dried consist of water (solvent) and materials dissolved or suspended in water (microbes) As cooling proceeds, water separated from solutes as it changes to ice, creating more concentrated areas of solutes Preservation of industrially important microorganisms 2. Storage in a dehydrated form (lyophilized) b) Primary drying Ice can be removed from frozen product (obtained from prefreezing stage) via sublimation, resulting in a dry and structurally intact product These requires very careful control of two parameters: temperature and pressure Preservation of industrially important microorganisms 2. Storage in a dehydrated form (lyophilized) c) Secondary drying After primary drying, though all ice has sublimed, bound moisture is still present in the product with residual moisture content of about 7-8% Continued drying is necessary at warmer temperature to reduce residual moisture content to optimal values Preservation of industrially important microorganisms 3. Subculture Inoculation of a culture onto a suitable medium contained in a tube or bottle The process can be repeated at intervals to ensure the preparation of a fresh culture before the old one dies  Problem: How to overcome fermentation reactions with low concentration of products? Answer: to increase yield and productivity of the selected microbial strains – How to increase yield? Optimizing culture medium and growth conditions However, this approach is limited by the organisms’ maximum ability to synthesize the product  Improvement of industrial microbes The potential productivity is controlled by its genome – the genome must be modified to increase potential yield Genetic modification can be achieved by: 1. Selecting natural variants 2. Selecting induced mutants 3. Selecting recombinants  Improvement of industrial microbes 1. Selection of natural variants – Each time a cell divides, there may be some genetic changes and the culture become more heterogeneous – Heterogeneity causes serious problems of yield: variants are usually inferior producers compared to original culture  Improvement of industrial microbes 1. Selection of natural variants – Example: monospore isolation using solidified media – each colonies isolated from the progeny shall be tested for the ability to produce product and select stable homokaryon with superior production capabilities  Improvement of industrial microbes Mutation technique – is used to modify the genome of microorganisms to obtain the following properties: 1. Resistant to catabolite repression: microbes are able to grow in substrate and produce enzymes 2. Able to produce enzymes without addition of inducers 3. Able to produce enzymes with the presence of repressors  Improvement of industrial microbes 2. Selecting induced mutants – Involves treatment of cells with mutagenic agents, (to the extent of 95-99% mortality) e.g. UV irradiation, ionizing radiation, alkylating agents, and base analogs – The survivor cells are then cultured in a complete media and the growing colonies are isolated for production of desired product  Improvement of industrial microbes 3. Selecting recombinant or genetically engineered microorganisms – Recombination: process which help to generate the combination of new genes which originated from different individuals – Considerations for recombination technique: Prior knowledge on the genetic regulation system in industrial strain  Improvement of industrial microbes Improvement of industrial strains other than the yield of the product: – Selection of stable strains – Selection of strains resistant to infection – Selection of non-foaming strains – Selection of strains tolerant to low oxygen tension – Elimination of undesirable products from production strain – Isolation of mutants producing new fermentation product