Biotechnological process Content 1) Basic definition 2) Materials for the fermentation process 3) Sterilisation 4) Bioreactors and fermenters 5) Cultivation of microorganisms 6) Downstream processing 7) Separation and isolation methods Biotechnological process The main products are ➢ Biomass ➢ Extracellular product, i.e. metabolite metabolite- primary - secondary Phase 1 (“upstream processing“) Phase 2 - bioprocess Phase 3 (“downstream processing“) SUBSTRATE PRODUCT Phases of biotechnological process Preparation of inoculum Inoculum fermentation Crude material Medium preparation Medium sterilisation biomassCell separation Purification and the final product processing Product extraction supernatant without cells Liquid culture product Effluent removal Cell disintegration Materials for the fermentation process 1. Water 2. Air 3. Sources of carbon • Saccharides • Complex substrates • Plant oils and animal fats • Petrochemical sources • Synthetic alcohols • Organic acids Water in the fermentation process Normal drinkable, treated water (deionised water) ➢ Preparation of growth media ➢ Washing of biomass Technical water ➢ Cooling of the growth media ➢ Regulation of the cultivation temperature ➢ Washing of the equipment Sources of nitrogen • Ammonia, ammonium salts • Amino acids, urea • Corn-steep liquor, plant flours • peptone, yeast extract Air • Aerobic processes, mixing, aeration Phosphate sources • Inorganic phosphorus (K3PO4, Na3PO4, (NH4)3PO4) • Natural sources (corn-steep liquor, peanuts flour, soya flour, waste from meat and fish processing, bones Macro elements in the fermentation process Growth factors, precursors, protective compounds • Vitamins (B = food yeasts), amino acids (as pure chemicals or natural sources) • Precursors (adding of phenylacetic acid or phenyl acetamide) improve the yield of penicillin G • Buffers (maintenance of pH, e.g. CaCO3) • Antibiotics (if they don't interfere with production and purification processes) Sources of other important elements • Biogenic elements (K, S, Ca, Mg, Na) • Trace elements (Fe, Zn, Mn, Cu, Co…) • Inorganic salts, mostly sulphates and chlorides • industry (Corn-steep liquor, soya and peanuts flours, beet molasses, whey …) Microelements in the fermentation process • Foaming is a typical accompanying feature of most industrial substrates used for fermentation in which high concentration of compounds is present • Foam structure is influenced by several factors (pH, temperature, viscosity, …) • Each substrate influences foaming Antifoam agents • Antifoam agents (natural plant and animal or synthetic) are frequently used as sources of carbon • They usually work at low concentrations and for a long time, must not be toxic to the organism • Natural oils and fats, higher alcohols, derivatives of sorbitol, polyether's and various silicones are used The aim of sterilisation is to remove all microorganisms • Heat • Filtration • Chemically (β-propiolactone, ethylenoxide, propylenoxide and glutaraldehyde) • Radiation (RTG, β-waves, UV light and ultrasound) Sterilisation Methods of sterilisation Sterilizers Sterilisation is an absolute concept = there are no degrees of sterility 13 !!! We cannot guarantee sterility, only express the probability of sterility !!! Almost sterile Partially sterile Partially? The concept of sterilisation 14 It is expressed as 10-6, i.e. 1 surviving microorganism per million A sterility probability of 10-6 means that there is a 1 in a million chance that the item is still contaminated Probability of sterility 15 So, if I make a million vials of medicine, one will be contaminated? Yes, but there's no way of knowing which Probability of sterility Destruction of microorganisms by heating Sterilisation of growth media and tools - I • Temperature denaturation of one or more enzymes, which have essential functions in the organism • The speed of resulting inactivation is influenced by: – Environment (amount of water, growth medium pH, concentration of solutes, etc.) – Physiological state of cells Spores are highly heat resistant Batch sterilisation by heating Sterilisation of growth media and tools - II • The growth medium is heated directly in a reaction vessel (bioreactor) • After the exposure to a high temperature, the content of the reaction vessel is cooled • Heating is direct by hot steam or by heat exchanger • Efficiency of sterilisation depends on the temperature and time of sterilisation 121 °C……………… 15 min 126 °C……………… 10 min 134 °C……………….. 3 min Given temperatures correlate with the pressure of saturated vapour Continuous sterilisation by heating Sterilisation of the growth media and tools - III • Cost effective method, less steam and cooling water is necessary • Shorter sterilisation time = 5-8 min • Higher temperature = 135°C • Heat-labile compounds in medium are less degraded • More correct and automatic regulation of the process Continuous sterilisation by heating Sterilisation of the growth media and tools - III 1) Direct steam is transported to the liquid medium through pipes, then the material travels into the expansion tank, where it is rapidly cooled 2) Heating and cooling of the plate heat exchanger, time is shortened to approximately 20 s – 5 min, sterilisation temperature 135 °C is sustained for about 2 – 3 min Continuous sterilisation is suitable for complex media, which don't contain solid phase but may contain heat-labile growth factors Medium sterilisation by filtration - I Sterilisation of the growth media and tools - IV • Only in media, which contain heat-labile compounds and sterilisation by heating is therefore not possible • Only soluble compounds may be present • Filters have pores 0.2 μm in diameter Medium sterilisation by filtration - II Sterilisation of the growth media and tools - V • It is necessary to sterilise the equipment before the medium sterilisation • Sterilisation is performed by heating, vaporing, chemically or by UV lighting • The equipment is usually distributed sterile directly from producers Bioreactor sterilisation Sterilisation of the growth media and tools - VI • Bioreactor must be sterilised when empty if continuous sterilisation of media or filtration are used • Hot steam (121 °C) • Hot air (150 – 180 °C) • Chemically ➢ heating, ➢ UV lighting ➢ Electromagnetic waves ➢ Filtration – mostly in industry due to financial reasons Air sterilisation Rough pre-filtration of air – porous materials, e.g. Powder coal and lignite coke, glass fibres Filtration ➢ On membranes (nitrocellulose) ➢ In-depth = air flows through a thick layer (several dozens of cm) of filter (glass fibres, nitrate cellulose, teflon, nylon or polyacryl) Bioreactors and fermentors Basic parts of the bioreactor ➢ Inlet and outlet of the growth media ➢ Inoculum supply ➢ Motor-driven impeller ➢ Air supply valve ➢ Sample collector ➢ Heating system ➢ Thermometer, manometer, pH, O2 and CO2 levels controlling devices, etc. Bioreactor (fermentor) = heart of any production line in the biotechnological process Material flow through the bioreactor/fermentor Bioreactor/fermentor scheme Types of bioreactors For cultivation of freely growing cells, immobilised cells, enzymes According to the process type: batch, semicontinuous (fed) and continuous According to the size: laboratory, pilot plant and operational According to the shape: cylindrical, with spherical bottom, circulatory, tower-like According to the manner of stirring: fitted with mechanical, pneumatic or hydrodynamic stirring Non-sterile, sterile (special) Liquid medium, solid medium etc. ➢ Stirred-tank ➢ Airlift bioreactor ➢ Fixed-bed bioreactor ➢ Membrane bioreactors (e.g. hollow fibre perfusion bioreactor) 30 Division of bioreactors according to cultivation method 31 Stirred-tank nutrients cooling water base acid sterile air controller controller controller harvest sparger air out cells baffle temperature probe cooling water 32 Airlift bioreactor Air Draft tube 33 Fixed-bed bioreactor Air Gas exchanger Pump Microcarrier beads 34 Membrane bioreactor productnutrients productInoculation port waterjacket Cells in annular space Lumen Inner membrane Outer membrane nutrients 35 Batch Fed-batch Continues Classification of bioreactors according to cell growth kinetics and product formation 36 What do the growth curves look like then Examples of bioreactors Cultivation of microorganisms • Continuous- log phase is prolonged → steady state (large volume of cultivation media, sewage purification) – chemostat – turbidostat • Dis-continuous (one-shot, “batch“) → after nutrients exhausting the growth is slowed • Semi-continuous (“fed batch“) → periodical adding of nutrients, gradual growth slowing (production of yeast) Cultivation with continuous exchange Types of cultivation • Submersion • Surface cultivation on liquid medium – especially for filamentous microorganisms, e.g. Aspergilus niger – producing citric acid Requirements for materials used in the construction of fermenters ➢ Corrosion resistant – no metals must be released into the media ➢ Non-toxic to the cell population ➢ Can be sterilised by highly pressurized steam ➢ Resistant to deformation – stirrers, inlet valves ➢ Transparent materials (glass) Inoculum preparation ➢ Inactivated cell cultures are transfered, which is in into the growth medium, where the cells start to proliferate and reproduce ➢ From several thousands of cells → to several hundreds liters of culture ➢ Transfer into bioreactor is performed at the end of the log growth phase ➢ Inoculum is used in an amount of approximately 5% of the volume of the growth media Growth monitoring ➢ Changes of pH ➢ Oxygen depletion ➢ Change of cell weight after centrifugation Process of inoculation ➢ Transfer of inactivated cells into the fresh media ➢ Prevention of contamination of the media ➢ Monitoring of quantity and physiological stage of cells (growth curve) Growing curve Log phase ➢ The most suitable from the technological perspective ➢ Time-limited • Lag-phase • Phase of accelerated growth • Log or exponential phase • Phase of decelerated growth • Stationary phase • Death phase Physical ➢ Temperature, steam, water and air pressure, power input, foam formation and its amount, gases and liquids flows Measurement and regulation of the biotechnology process Physico-chemical ➢ pH, redox potential, amount of soluble oxygen, chemical agents (measurement of concentration of growth stimulators and inhibitors or products formation - C, N, P, S, Mg, K, Na, Fe, growth regulators, precursors, etc. Measurement of NH4+, Mg2+, Na+, Ca2+ and PO4 3- concentration by specific electrodes) Downstream processing Saccharomyces cerevisiae Products isolation and purification Crystallisation, vaporization, drying, lyophilisation Liquid culture Growth medium processing Adjustment of pH, heating, adding compounds for coagulation of proteins and cells Cell separation Centrifugation or filtration Product in liquid (extracellular) Product in cells (intracellular) Product isolation precipitation, extraction, chromatograph y Cell disintegration Enzymatic, chemical, physical Cell walls separation product Final product processing Centrifugation or filtration Products isolation and purification Crystallisation, vaporization, drying, lyophilisation Liquid culture Growth medium processing Adjustment of pH, heating, adding compounds for coagulation of proteins and cells Cell separation Centrifugation or filtration Product in liquid (extracellular) Product in cells (intracellular) Product isolation precipitation, extraction, chromatography Cell disintegration Enzymatic, chemical, physical Cell walls separation product Final product processing Centrifugation or filtration ➢ Centrifugation (batch or continuous) ➢ Filtration (rotary vacuum drum filters) Cell separation ➢ enzymatic: lysozyme ➢ chemical: alkali, detergents ➢ physical: osmotic shock, cell disintegration by abrasives, ultrasound Cell disintegration ➢ centrifugation ➢ filtration Separation of cell walls ➢ Extraction ➢ System of two miscible solvents ➢ Protein isolation = PEG and dextran or PEG and specific salts, such as K3PO4 or NH4SO4 ➢ Precipitation ➢ Protein salting by NH4SO4 ➢ Precipitation by organic solvents (ethanol, acetone, …) ➢ Chromatography (gel, ionex, bioaffinity, adsorption) ➢ Electro migration (electrophoresis, isoelectric focusing, isotachophoresis) Isolation of product from liquid phase Evaporisation Final processing of the product Drying ➢ Vacuum vaporizer ➢ Mind heat-labile compounds! ➢ Desktop vaporizers are better for heat-labile enzymes ➢ Removing water and volatile compounds from the product ➢ Dryers – belt, tray, drum, spray ➢ Fluid-air heat exchanger (passage of warm air through the material) – frequent in pharmaceutical industry Chromatography - I ➢ Biologically active substances constitute a big group of compounds with special functions ➢ Changes of pH, ions forces, metal ions concentration, cofactors, etc. can strongly influence isolated biologically active molecules ➢ To avoid the loss of biological activity during the isolation process, it is necessary to use as mild separation methods as possible Chromatography - II ➢ Development of effective isolation methods (gel, ionex, bio-affinity chromatography, etc.) enabled establishment of new branches of chemical industry ➢ No development of chemistry of proteins and nucleic acids – molecular biology, gene engineering, immunochemistry, etc. would be possible Purification strategy - I ➢ Low concentration of biologically active compounds ➢ Mixture of many similar substances The first stage of isolation = adsorption ➢ Bio-specific affinity chromatography ➢ Most proteins have negative charge at physiological levels of pH → sorption to annex Next stage of isolation ➢ gel chromatography ➢ electrophoresis Purification strategy - II Combination of several methods of separation is mostly successful for isolation of pure and biologically active substances Don't repeat methods based on the same purification principle - when you prepare purification schedule Gel chromatography Separation of bio macromolecules based on different size of individual substances; the substances are separated on porous stationary phase (gel filtration) ➢ agarose ➢ cross-linked dextran (Sephadex) ➢ polyacrylamide (BioGel P) ➢ cellulose (Cellufin) ➢ material based on Silica Gel or porous glass Stationary phase – inert porous material saturated by liquid Principle of gel chromatography Small molecules diffuse into pores of matrix during the flow of mixture of compounds through the porous stationary phase. It means that the movement of small molecules is slowed down. Large molecules are not captured by pores and move quicker. The bigger the molecule, the quicker the movement the through chromatography column is. ➢ Repeated washing at mobile phase also washes out small molecules ➢ No interaction between the solute and matrix; no denaturation of separated substances ➢ The method is based on a reversible exchange of ions between mobile liquid and stationary phases ➢ Stationary phase - ionexes (anion or cation exchangers)             annex exchanges anions             Cation exchanger exchanges cations Ion exchange chromatography Ion exchange chromatography process Activation of column Loading of sample Adsorption of particles ElutionWash of column product Materials for ionexes ➢ Modified cellulose ➢ Sephadex ➢ Ionexes derived from agarose (Trisacryl, Fractogel…) ➢ Ionexes based on silica gels and synthetic polymers Ion exchange chromatography is one of the most widespread methods, which were and still are used for the isolation of different biologically active substances (enzymes, NAs, AAs, antibiotics, vitamins, nucleosides and nucleotides, lipids, etc.) Bio affinity chromatography The principle of isolation method is interaction of isolated protein with ligand connected to solid surface Based on an outstanding feature of biologically active substances to form tightly connected specific reversible complexes with other compounds called affinity ligands enzyme – substrate, cofactor – effector, antibody – antigen, hormone – receptor, etc. Ligand = compound which forms bio specific reversible complex with the isolated product Ligands in affinity chromatography Any compound, which is able to form biospecific reversible complex with the given substance, can be used as a ligand ➢ Immobilised pyridine or adenine nucleotides ➢ Dyes with antraquione structure ➢ Immobilised haemoglobin or casein for proteolytic enzymes ➢ Ligand must contain a function group by which it binds to the solid carrier ➢ It must have sufficient affinity to the isolated substance solid carrier ligand solid carrier binding of particular protein washing solid carrier Elution by soluble anti- ligand solid carriersolid carrier Elution by “deforming” buffer binding of particular protein washing Elution by soluble anti- ligand Elution by “deforming” buffer solid carrier ligand solid carrier solid carriersolid carrier solid carrier High performance bio affinity chromatography (HPLAC) ➢ New method used only in laboratories so far ➢ Fully automated system working on high pressure ➢ Better resolution than in classical methods Electro migration methods Zonal electrophoresis – separation based on the differences in total charge, volume and shape • Mostly in gels – agar or polyacrylamide • Gradient gels (PGE) – electric-field-induced movement of macromolecules in a medium with gradient • SDS-electrophoresis (SDS-PAGE) • Isoelectric focusing (IF) – separation based on the isoelectric point The use of electro migration methods ➢ Biochemistry, molecular biology, genetic engineering, bio analytical chemistry ➢ Medicine (diagnostics, immunochemistry, …) ➢ Accurate and effective analysis of ➢ Simple organic compounds (amino acids, steroids, peptides, alkaloids, vitamins, dyes, antibiotics,…) ➢ Complex macromolecular complexes (membrane receptors, enzymes, immunoglobulins, protein hormones, plasmatic proteins, allergens, etc.) Immobilised bio catalysers Bio catalysers = biological material, which is able to transform any reactant into a product without changes of the bio catalyser itself Immobilisation = process in which enzyme or cell (or its part) is transformed into the form of heterogenic catalyser • Enzymes • Living cells • Dead cells Advantages of immobilisation ➢ Better economy of the catalysis ➢ Continuous process ➢ Better control of reactions ➢ Possibility to use non-compatible enzymes at the same time ➢ Longer enzyme activity ➢ Quicker separation of product and substrate Microencapsulation – closing of a bio catalyser by a membrane into micro-capsules → forming of emulsion 1. Building into polymers - Polymerisation into gel matrix - Dispersion in biopolymers - Polymeric membranes Methods of immobilisation - I membrane catalyser 2. Binding to solid carrier adsorption ➢ Non-covalent binding by hydrogen bonds to inert carrier ➢ By electrostatic interactions to ion exchangers ➢ Non-specific interaction of hydrophobic groups, pseudoaffinity interaction, … H O H Methods of immobilisation - II 2. Binding to solid carrier Covalent bond ➢ Modified natural polymers (cellulose, dextran, agarose, …), or also synthetic polymers (polyacrylates, …) Methods of immobilisation - III 3. Forming of aggregates without any carrier ➢ Cross-linking of enzyme molecules by bi-functional agents or their binding to molecules of other inert proteins (inter-molecular bound) Methods of immobilisation - IV agent enzyme enzyme agent Summary 1) Basic definition 2) Materials for the fermentation process 3) Sterilisation 4) Bioreactors and fermenters 5) Cultivation of microorganisms 6) Downstream processing 7) Separation and isolation methods