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Chapter 4 Fermentation MonitoringAlargevesselMadeofstainlesssteelEquippedwithtemperature,pHanddissolvedoxygenmeasurementandcontrolsystemsCommon Features of a Fermentorself priming fermentormechanical agitation fermentor Types of FermentorAirlift fermentorTower fermentor Unit 1 Fermentation Basics Four Phases of Bacterial Growth CurveLag phase Log phase Stationary phase Death phase Lag PhasePeriod of adjustment to new conditionsCell growth is minimal Log Phase Cell growth rate and metabolic activity are the highest Number of cells produced Number of cells dyingCells are most susceptible to adverse environmental factors(e.g.radiation,antibiotics)Stationary Phase Population size begins to stabilize Number of cells produced=Number of cells dyingFactors that slow down microbial growthAccumulation of toxic waste materialsAcidic pH of media Limited nutrients Insufficient oxygen supply Death or Decline PhasePopulation size begins to decrease Number of cells dying Number of cells produced Most of the nutrients in the medium have been consumed Methods for measuring microbial growthMeasurementofthechangesinnumberofcellsormassofpopulation.Measurement of Cell NumbersDirectcellcounts-countingchambersViablecellcounts-platingmethods Measurement of Cell MassDry weight-time consuming and not very sensitive Turbidimetric measures-quick,easy and sensitiveoptical density the number of microbes E.coli,1 OD600=8 x 108 cells/ml12Types of Fermentation ProcessBatch,continuousandfedbatchprocessesbatchfedbatchchemostat Batch cultureThesterilegrowthmediumisinoculatedwiththemicroorganismsandnoadditionalgrowthmediumisadded.Allthenutrientsneededforcellgrowthwillonlybeaddedonceatthebeginningoffermentation.Batch Fermentor AdvantagesOptimumlevelsofproductrecoverySimpleoperation DisadvantagesThewastageofunusednutrientsLabourandtimelostbetweenbatches Continuous culture Maintains cells in log phase at a constant biomass concentration for extended periods.It can be controlled in two ways1.Turbidostatic control(internally controlled)2.Chemostatic control(externally controlled)The TurbidostatRegulates the flow rate of media through vessel to maintain a predetermined turbidityMaintain the highest growth rateNo limiting nutrientThe ChemostatRate of incoming medium=rate of removal of medium from vesselMaintain the exponential growth phaseAn essential nutrient is in limiting quantitiesBatch Culture and Continuous Culture Advantages The growth rate is maintained at optimal levels Disadvantages Low biomass and product concentration Relatively prone to be contaminated Strain degeneration Q:Use chemostat as bioreactor,the yield is far more than batch,but why batch culture is far more widely used than continuous culture？1.Secondary products 2.Genetic instability 3.Operability and reliability 4.Market economics Fed-batch culture A production technique between batch and continuous culture.During fermentation,additional nutrients will be added in a batch way to promote the cell growth or product formation and to avoid nutrient deficiency.Fed-Batch FermentorFeedstockvessel(sterile)Pump Advantages Low concentration of specific substrates (e.g.carbon source)High concentration of end-product Providing limiting level of a required nutrient for an auxotrophic strainSome examplesProductMass feedyeastmalt wort、N、P、Mgglutamic acidNH3 or ureacitric acidammonium nitrogen、glucosemalic acidglucoseamylaseglucose Unit 2 Fermentation Control The success of a fermentation process ishighly dependent on environmental factors such as temperature,pH,and dissolved oxygen levels.Fermentation Control Sample AnalysispHDOSugarAmmoniaPhosphateSulphateProductsPrecursorsContaminationPressureprobeLevelprobepHprobeTemp.probeDOprobeAntifoamAcid/BaseCoolingAir/agitationSugar/OilfeedOrganisms exhibit distinct growth temperatures -minimal,maximal,optimalTemperature HotColdC(Minimum)B(Optimum)A(Maximum)Temperature Control Why?Why?Microbes brings about fermentation by secreting certain enzymes which have an optimum temperature or a temperature range.An energy balance on fermentation yields is the following equation:Qacc=Qf+Qag Qevap Qsurr Qacc net heat accumulation Qf heat of fermentation Qagheat generated by mechanical agitationQevap heat of evaporation Qsurr heat dissipated to the surroundingsQ:Usually,optimum growth temperature is difference with optimum production temperature,how would you do?AnswerTemperature-shifting fermentation Temp.control methodsCooling water in cooling jacket or coilSterilization-steamRefrigerator or freezer pH control Most microorganisms have narrow pH growth ranges(pH 5-7)The buffering in culture media is generally lowMetabolites which released into the medium can change the pHHow to maintain optimum pH?Addition of base(NaOH)or acid(HCl)Addition of physiological acid substance(NH(NH4 4)2 2SOSO4 4)or physiological alkali substance(ammonium hydroxide)Q:In many fermentation factories,urea or ammonia water is often used as a fed-batch substance,whats the function of it?Adjusting pH of mediaUsing as a nitrogen source Dissolved oxygen(DO）ControlMostindustrialfermentationsareaerobicprocessesSupplyingoxygentoaerobiccellshasaproblem：oxygenispoorlysolubleinwater(8mg/Lat4oC,sucroseissolubleto600g/L)OxygensupplyingistheratelimitingstepinanaerobicfermentationTemperatureElevationSalinityTurbulenceFactors affecting dissolved oxygen concentrationatmospherewaterUndersaturatedSaturatedSupersaturatedo2o2o2o2o2o2Q:How to maintain a constant dissolved oxygen concentration during the fermentation process?A:Increase aeration and agitation rate(oxygensupply)Aeration:by bubbling air through the liquidAgitation:using impellers(agitator)Other influence factors(oxygendemand)The most important is cell concentration substrate concentration fed-batch operation High cell density fermentationMedium optimizationFed-batch cultureIncreasing DORemoval of toxic componentsOxygen is the most important gaseous substrate for microbial metabolism.Carbon dioxide is the most important gaseous metabolic product.Foaming controlFoamsconsistofliquidlamellasfilledwithgas How is foam formed?Surface-activemedia(peptides)andfoamingcomponents(proteins)Highaerationandagitationrates Excess foaming will.displace the fermentation broth and cause it to leak from the vessel.lead to losses in productivity and culture contamination.Foam control methods1.Mechanical defoaming Foam-breaker（Defoamers）DefoamingbladesinfermentationtankS Separaterotorintheupperareaoftheculturetank 2.2.Chemical defoaming Addition of chemical antifoam agents in the beginning（1）Animal and vegetable oils,0.1%0.2%（2）Polyether surfactant（Defoamer GPE-1）,0.02%-0.03%FreshMediaFeed(substrates)DissolveO2SensorThermocoupleInletAirFlowExitGasFlowAcid/baseAntifoampHSensorLevelSensorAgitatorSpargerExitLiquidFlow(cells&products)Aerobic Fermentation Contamination control Detection methods 1.Observation -OD(optical density)-DO(dissolved oxygen)、pH、CO2 abnormal -foam 2.Microscopy3.Culture techniques plate streaking phenol-red broth base Sources of contaminationMicrobiologicalcontaminationoninitialinoculumAirFiltrationsystemfailureEquipmentleakageMediumsterilizationisunqualified Analysis of contamination Single fermenter Whole fermenter In the earlier stage of fermentation In the middle or later stage of fermentationPhage contamination&control lWhats phage?lDefinition-Obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinerylComposition-Nucleic acid -ProteinStructure-Head-TailComposition and Structure Phage control 1.phage detection Agar plate Cross-streaking plates Liquid culture 2.phage control methodUsing phage-insensitive strainsMinimizing the concentration of phage in processing plants ReviewQuestions1.Statethedefinitionsofbatch,continuousandfedbatchculture.Showtheadvantagesoffed-batchculturebygivingsomeexamples.2.Pleaseillustratehowtocontroltemperature、pH、DOandfoaminfermentation.3.Statethereasonsandsolutionsforthebacteriacontaminationandphageinfectionduringfermentation.
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