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Non Growth Associated Product Formation Homework

The model equations used for simulation are based on fed-batch biochemical reactions growing baker's yeast. It is assumed that the feed does not contain any product. The differential equations are as follows:

x1 represents the cell mass concentration(g/l),
x2 the substrate concentration(g/l),
x3 the desired product concentration(g/l),
F the feed rate(l/hr),
V the volume of the broth(l),
μ is the specific growth rate (SGR)(hr-1),
σ is the substrate consumption rate (SCR)(hr-1), and
π is the product formation rate (PFR) (hr-1).

Concentration and Rate Profiles in Fed-batch reactor:

Presentation on theme: "Pharmaceutical Biotechnology PHR 403"— Presentation transcript:

1 Pharmaceutical Biotechnology PHR 403
Chapter 2: Fermentation Technology

2 Fermentation Technology
In the context of Microbial biotechnology the term “fermentation” refers to the growth of large quantities of cells under anaerobic or aerobic conditions within a vessel called a fermenter or bioreactorSimilar vessels are used in processes involving cell-free and immobilized enzyme transformations

3 Bioprocess typically involves principle 3 steps(see figure)
Industrial Bioprocess – any large scale operation involving a transformation of a raw material into a product by moss or animal plant cells or enzymes or cell extractsProduct may be useable (eg. Insulin) or have commercial value (waste water treatment)Bioprocess typically involves principle 3 steps(see figure)Each step requires a combination of operating units.Downstream processing may include-extraction, ultra filtration, crystallization, chromatography, drying and packaging

4 Bioprocess stages and the commonly used operations in them
PROCESS STAGES OPERATIONSRaw MaterialSortingSievingComminutingHydrolysisSterilizationPRETREATMENTBiomass productionMetabolite biosynthesisBiotransformationsBIOREACTIONFiltrationCentrifugationSedimentationFlocculationCell DisruptionPrecipitationEvaporationDOWNSTREAM PROCESSINGProduct

5 Microbial Biotechnology
Use of micro-organisms for large scale industrial processesOldest form of microbiology and biotechnology which was used to make wine, beer, sake, bread with use of bacteria and yeasts without knowing scientific basisProduction of ethanol, lactic acid, butanol using microbes and Enzymes like amylase, protease, invertase were used during early 20th centuryPencillin was produced during WWII and other amino acids, nucleotides, enzymes were also produced later

6 MODERNWhite BiotechnologyNow use in bioconversion/biotransformations of chemicals, genetic engineering of MO for non microbial products (insulin, interferons, HGH, vaccines)Microbial Fermentations (organic acids, amino acids, vitamins, antibiotics, enzymes) and fermented foods (dairy, meat, plant, breads, alcoholic beverages)Use of Microbes in increasing crop productivityIcrobes as Biofertilizers and BiopesticidesUseMicrobes as food/feed: single cell proteinBioenergy and BioremediationMining and metallurgy

7 Isolation and culturing Micro-organisms
SourcesSoils, lakes, oceans, river, plant, animal, air, non living objectsIsolation methodsSerial dilution, spread plate, gradient pour plate, streak plateFiltration, centrifugationImportance of eliminating other organisms (antibiotics, heating)

8 Isolation and culturing Micro-organisms
Growth MediaMO require nutrients (C, N, P, Minerals), O2 requirement, temp, pH, salinity etcSynthetic mediaSemi synthetic mediaNatural mediaMedia needs to be economical for large scale productions, consistent quality and available throughout. Raw material can be pre-treated if requiredCheap C and N2 sources can be used

9 Isolation and culturing Micro-organisms
Sources of nutritionCarbon: sugarcane molasses, beet molasses, vegetable oil, starch, cereal grains, whey, glucose, sucrose, lactose, malt, hydrocarbonsNitrogen: corn steep liquor, slaughter house waste, urea, ammonium salts, nitrate, peanut granules, soyabean meal, yeast extract etcGrowth factors: vitamins and amino acids are added when MO cannot synthesize them

10 Antifoams: sunflower oil, olive oil to prevent foaming
Isolation and culturing Micro-organismsSources of nutritionTrace elements: Zn, Mo, Mn, Cu, Co required for metabolism or in metallo-enzymes or in proteins (Hb)Inducers, precursors, repressors: for enzymes to function in metabolic processes inducers are required. Sometimes presence of presursors enhances production of a secondary metabolite or production an enzyme can be repressed due to repressors. Eg streptomycin is nduced by yeast extract, Sec metabolites can be repressed due to some cpds.Antifoams: sunflower oil, olive oil to prevent foamingWater: clean water of consistent composition, dissolved chemicals, pH is measured. Also required for cleaning, washing, rinsing, cooling, heating etc.

11 Culturing methods for Micro-organisms
Sterilization: devoid of MO (aseptic conditions)Contamination free seed cultureMoist heat (121oC/15psi/20min), radiation, ultrasonic treatment, chemicals, mechanical, gases (ozone), filtration for sterilizing airSterilization of equipment, media and airCULTURINGAvoidance of contamination can be achieved byUse pure inoculum to start fermentationSterilize the mediaSterilize fermenter vesselSterilize all materials to be added to the fermentation during the processMaintaining aseptic conditions during the fermentation

12 Control of environmental conditions for
Microbial growthTemperaturepHAgitationO2 concTo be carefully monitored and maintainedAcidic pH: fungi and yeastPsychrophiles, acidophiles etc

13 Sterilization:Elimination of threads and welding of pipes and tubes to reduce contaminationFermenters have pipes which flush steam into the systemMedia along with fermenter is sterilizedAmong the several factors that influence killing are-temperature,pH,osmotic pressure,shear,mass transport, andconcentrations of extraneous substances that react with the killing agent.These factors operate synergistically, and temperature plays roles other than simply affecting the kinetics of a reaction

14 Aeration and MixingShake culture: flasks are kept on a shaker for required rotationsFermenters: Stirrers for O2 mixing and baffles for increasing turbulenceV shaped notchIncrease turbulenceIncrease efficiency ofO2 transferImproves growth of MO

15 FermentationFervere: to boilIn MicrobiologyAny process for the production of useful products through mass culture of MOIn Biochemistry-The numerous Oxidation-Reduction reactions in which organic compounds used as carbon and energy act as acceptors and donors of H2 ion. The organic compound gives rise to various products of fermentation which accumulate in the growth medium-Takes place in absence of O2-Now term industrial fermentation for large scale cultivation of micro-organisms…most of them is aerobic

16 Fermenter and bioreactor
A biorector is a device in which the organisms are cultivated and motivated to form a desired productClosed vessel or containment vessel to give a right environment for optimal growth and metabolic activity of the organismFermenter: for microbes/ Bioreactor : for eukaryotic cellsSize variable ranging from million litres or more.Large scale production (10-100L to1000-million L capacity)Helps to meet requirements of:pHtemperatureaerationagitationdrain or overflowcontrol systemssensorscooling to achieve maximum microbial yield

17 What is fermentation technique?
Techniques for large-scale production of microbial products:It must both provide an optimum environment for the microbial synthesis of the desired product and be economically feasible on a large scale.They can be divided into surface (immersion) and submersion techniques. The latter may be run in batch, fed batch, continuous reactors.In the surface techniques, the microorganisms are cultivated on the surface of a liquid or solid substrate. These techniques are very complicated and rarely used in industry

18 What is fermentation technique?
In the submersion processes, the microorganisms grow in a liquid medium.Except in traditional beer and wine fermentation, the medium is held in fermenters and stirred to obtain a homogeneous distribution of cells and medium.Most processes are aerobic, and for these the medium must be vigorously aerated.All important industrial processes (production of biomass and protein, antibiotics, enzymes and sewage treatment) are carried out by submersion processes.

19 Some important submerged fermentation products
OrganismUseEthanolSaccharomyces cerevisiaeIndustrial solvents, beveragesGlycerolProduction of explosivesLactic acidLactobacillus bulgaricusFood and pharmaceuticalAcetone and butanolClostridium acetobutylicumSolvents-amylaseBacillus subtilisStarch hydrolysis

20 General Aspects of Fermentation Processes

21 Fermenter The heart of the fermentation process is the fermenter.
In general:Stirred vessel, H/D  3Volume m3 (80 % filled)Biomass up to 100 kg dry weight/m3Product 10 mg/l –200 g/l

22 Component parts of a fermenter
Formulation of media to be used in culturing the organism during development of inoculum and in the production fermenterSterilization of the medium, fermenter and ancillary equipmentProduction of an active, pure culture in sufficient quantity to inoculate the production vesselThe growth of the organism in the production fermenter under optimum conditions for product formationThe extraction of the product and its purificationDisposal of effluents produced by the process

23 Production fermenter Biomass Culture Cell fluid separation Cell free
DOWNSTREAMPROCESSINGCulturefluidCellseparationStockcultureShakeflaskSeedfermenterCell freesupernatantMedium STERILIZATIONMedium FORMULATIONProductextractionMedium raw materialProductpurificationEffluent treatmentProduct packaging

24 Cross section of a fermenter for Penicillin production ( Copyright:  

25 Cross section of a fermenter for Penicillin production ( Copyright:

26 Flow sheet of a multipurpose fermenter and its auxiliary equipment

27 Batch fermentation refers to
Basic modes of operations of a fermenterBatch cultureBatch fermentation refers toa partially closed system in which most of the materials required are loaded onto the fermentor, decontaminated before the process starts and then, removed at the end.The only material added and removed during the course of a batch fermentation is the gas exchange and pH control solutions.In this mode of operation, conditions are continuously changing with time, and the fermentor is an unsteady-state system, although in a well-mixed reactor, conditions are supposed to be uniform throughout the reactor at any instant time.The principal disadvantage of batch processing is the high proportion of unproductive time (down-time) between batches, comprising the charge and discharge of the fermenter vessel, the cleaning, sterilization and re-start process

28 Basic modes of operations of a fermenter
Continuous cultureContinuous culture is a technique involving feeding the microorganism used for the fermentation with fresh nutrients and, at the same time, removing spent medium plus cells from the systemA unique feature of the continuous culture is that a time-independent steady-state can be attained which enables one to determine the relations between microbial behavior (genetic and phenotypic expression) and the environmental conditions.

29 Basic modes of operations of a fermenter
Fed-batch processesThe fed-batch technique was originally devised by yeast producers in the early 1900s to regulate the growth in batch culture of Saccharomyces.Yeast producers observed that in the presence of high concentrations of malt, a by-product - ethanol - was produced, while in low concentrations of malt, the yeast growth was restricted. The problem was then solved by a controlled feeding regime, so that yeast growth remained substrate limited.The concept was then extended to the production of other products, such as some enzymes, antibiotics, growth hormones, microbial cells, vitamins, amino acids and other organic acids.An alternative description of the method is that of a culture in which "a base medium supports initial cell culture and a feed medium is added to prevent nutrient depletion"

30 By products may lead to cell death
Fed-batch processesBasically, cells are grown under a batch regime for some time, usually until close to the end of the exponential growth phase.At this point, the reactor is fed with a solution of substrates, without the removal of culture fluid.This feed should be balanced enough to keep the growth of the microorganisms at a desired specific growth rate and reducing simultaneously the production of by-products (that can be growth or product production inhibitory and make the system not as effective).By products may lead to cell death

31 A fed-batch is useful in achieving high concentration of products as a result of high concentration of cells for a relative large span of time.Two cases can be considered:the production of a growth associated product and the production of a non-growth associated product. In the first case, it is desirable to extend the growth phase as much as possible, minimizing the changes in the fermenter as far as specific growth rate, production of the product of interest and avoiding the production of by-products.Growth associated products- These are those products formed simultaneously with microbial growth. The specific rate of product formation is proportional to the specific rate of growth. Example: production of constitutive enzyme.Non-growth associated products- Products are formed during the stationary phase when the net growth rate is zero. The specific rate of product formation is constant. Example: Secondary metabolites like antibiotics (penicillin).Mixed growth associated products- Here products are formed during the slow growth and stationary phases. Example: Production of lactic acid fermentation.

32 For non-growth associated products, the fed-batch would be having two phases: a growth phase in which the cells are grown to the required concentration and then a production phase in which carbon source and other requirements for production are fed to the fermenter.This case is also of particular interest for recombinant inducible systems: the cells are grown to high concentrations and then induced to express the recombinant product.

33 Simple fermenters (batch and continuous) Fed batch fermenter
Types of Fermenter/BioreactorsSimple fermenters (batch and continuous)Fed batch fermenterAir-lift or bubble fermenterCyclone column fermenterTower fermenterFluidized bed bioreactorsPacked bed bioreactorphotobioreactorOther more advanced systems, etcThe size is few liters (laboratory use) - >500 m3 (industrial applications)

34 Outline of a fermentation process
Upstream processingRaw MaterialsProduction microorganismFermentationDownstream processingProduct purificationProductEffluent waste

35 Fermentation SystemsWe examine conventional fermenters used for microbial, plant and animal cell cultureMost fermentations use liquid mediaSome are non stirred, non aerated and non aseptically operated (beer, wine) while others are stirred, aerated and asepticAlso classified as to organization of biological phase – suspended growth or supported growth

36 Fermentations are performed in fermenters
Often with capacities of several thousand litresRange from simple tanks (stirred or unstirred)To complex integrated systems involving varying levels of computer controlsFermenter and assoc pipe work must be made of stainless steel (for repeated sterilizationn and must not react with mos or metabolites

37 The essential components of a fermenter

38 The Fermentation Process

39 Operating SystemsFermentations in liquid media can be carried out under batch, fed-batch or continuous culture conditionsStirred batch fermentor : a closed system where all nutrients are present at start of fermentation within a fixed volumeFed-batch fermentor: fresh medium is fed in throughout the fermentation and volume of batch increases with timeContinuous culture: fresh medium is fed into the vessel and spent medium and cells are removed (fixed volume)In all systems, pH, temperature, aeration etc is monitored and adjusted

40 Batch Fermentor Medium added Fermentor sterilised Inoculum added
Fermentation followed to completionCulture harvestedGlucoseProductCell biomassTime

41 Fed-Batch FermentorsPumpFeedstockvessel(sterile)

42 Characteristics of Fed Batch Fermentors
Initial medium concentration is relatively low (no inhibition of culture growth)Medium constituents (concentrated C and/or N feeds) are added continuously or in incrementsControlled feed results in higher biomass and product yieldsFermentation is still limited by accumulation of toxic end productsBATCHFED-BATCHGlucoseGlucoseProductProductBiomassBiomass

43 Continuous Fermentor Flow rate1 = Flow rate2 Pump 1 Pump 2 Feedstock
vessel(sterile)Collection vessel

44 Fermentation MediaMedia must satisfy all nutritional requirements of the organism and fulfil the objectives of the processGenerally must providea carbon source (for energy and C units for biosynthesis)Sources of nitrogen, phosphorous ans sulfurMinor and trace elementsSome require added vitamins e.g. biotinand riboflavinMedia generally contain buffers or pH controlled by adding acids / alkalisPotential problemsCompounds that are rapidly metabolized may repress product formationCertain compounds affect morphology

45 Factors affecting final choice of raw materials
Costs and availabilityEase of handling, transporting and storingSterilization requirements and denaturation problemsFormulation, mixing and viscosity characteristicsConcentration of product produced / rate of formation/ yield per gram of substrateLevels and ranges of impurities which may produce undesirable by productsHealth and safety considerations

46 Desirable Characteristics of Media
Should be inexpensive and available year round . Media may account for 60-80% of cost of processShould ideally be stored at room T otherwise costs are highMust be easily sterilized with minimal thermal damageMay influence aeration and cause foaming during process

47 Carbon sources Molasses Malt extract
Byproduct of cane sugar productiona dark viscous syrup containing 50% CHO (sucrose) with 2% nitrogen, vitamins and mineralsMalt extractUse aqueous extracts of malted barley to produce C sources for cultivation of fungi and yeastsContain 90% CHO, 5% nitrogen and proteins, peptides and amino acidsMolasses : cant use pure glucose or sucrose (cost)