生物反應(yīng)器細胞收集細胞破碎提取純化產(chǎn)品制劑BioreactorExtractionRecoveryCelldisruptionCellharvestingPurificationProductformulationPrecipitationandcrystallizationSupernatantChapter2Filtrationandcentrifugation2.1pretreatment2.2filtration2.3centrifugation基本要求：掌握過濾前物料預(yù)處理的基本方法，了解轉(zhuǎn)鼓過濾器的操作原理與過程，以及過濾設(shè)備的基本結(jié)構(gòu)；了解管式離心機、碟片式離心機及螺旋式離心機，掌握差速離心方法和密度梯度離心方法。2.1pretreatmentCharacteristicsoffermentationbrothPretreatmentmethodsHeatingAdjustingpHCoagulationandflocculation凝聚和絮凝Addingfilteraids助濾劑上的吸附Addingreactant反應(yīng)劑Methodsforremovingimpurities

protein,insolublecarbohydrates,multi-valentmetalionsCharacteristicsoffermentationbrothviscous,highlynon-NewtonianslurryverydiluteaqueoussystemssmallparticlesizeminimaldensitydifferencesbetweenthesolidandtheliquidphasehighsolidscompressibilityManyfermentationbrothsareunstable.發(fā)酵viscous,highlynon-Newtonianslurrysmallparticlesize

minimaldensitydifferencesbetweenthesolidandtheliquidphasesmallparticlesize

minimaldensitydifferencesbetweenthesolidandtheliquidphaseVShighsolidscompressibilityFiltrationcentrifugationHeatingThesimplestandtheleastexpensivemethodimprovethefeed’shandlingcharacteristicsmaypasteurizethefeedchiefconstraint

：thethermal

stabilityoftheproduct亮氨酸脫氫酶AdjustingpHReducethesolubilityoftheamphotericmolecules,suchasproteinandaminoacid.Controlmembranefoulingbychangingtheelectrostaticchargeofchargedcomponents.Cells,celldebrisandsomecolloidalparticlesareeasytoflocculateaslargeaggregates.例如，鏈霉素發(fā)酵液，酸化至pH3.0后，直接蒸汽加熱至70－75℃，維持30min的方法去除蛋白質(zhì)，能使過濾速度增大10～100倍，濾液粘度可降低1/6。CoagulationandflocculationElectricaldoublelayer雙電層

Coagulationandflocculationoccurinsuccessivestepsintendedtoovercometheforcesstabilizingthesuspendedparticles,allowingparticlecollisionandgrowthoffloc.agents：simpleelectrolytes

，polyelectrolytesElectricaldoublelayerElectricaldoublelayer

Innerlayer：sternlayer（吸附層或stern層）Outerlayer：diffuselayer（擴散層）Why？Electrostaticinteraction靜電作用Thermalmotion熱運動Howcancolloidalstabilitybeachieved?electricaldoublelayerelectrostaticrepulsion靜電排斥hydrationshell(hydrationlayer)水化層Sternlayer：ionsofoppositechargearestronglybound

Diffuselayer：looselyassociatedwiththeobjectmadeoffreeionswhichmoveinthefluidSchematicofelectricdoublelayerSimpleelectrolytes（簡單電解質(zhì)）reducingtheelectrostaticrepulsion（靜電排斥）betweencolloidalparticles（膠體粒子）.

attractiveLondon-vanderwaalsforces（范德華引力）predominate.Thecolloidscanthencoagulateaslarger,denseparticleswhicharemoreeasilyfiltered.Al3+>Fe3+>H+>Ca2+>Mg2+>K+>Na+>Li+Al2(SO4)3·18H2O、AlCl3·6H2O、FeCl3等豆腐的制作石膏（CaSO4·2H2O）鹵水（MgCl2，CaSO4，CaCl2）過濾定型？？？Polyelectrolytesflocculantschemicalstructureofflocculantsmanyactivefunctionalgroupslongchainhighmolecularweightmechanism

adsorptionprovidebridgesclassification(accordingtofunctionalgroups)offlocculantsinfluencefactorsclassificationofPolyelectrolyteflocculantscationic（陽離子型）

maybeusedaloneneutralizethesurfacecharge

ofthecolloidalparticlesandreduceelectrostaticrepulsionorincombinationwiththealuminumandirontypecoagulants

anionic（陰離子型）oftenusedwithmetalcoagulantsnonionic（非離子型）oftenusedwithmetalcoagulantsinfluencefactorsmolecularweight,typeandamountofflocculantspHofthesolutionstirvelocityandstirtime右圖是α淀粉酶發(fā)酵液的絮凝試驗中，絮凝劑加量對絮凝效果（濾速）的影響結(jié)果。filterAids助濾劑Adsorbcolloidsandreducecakecompressibility

吸附膠體，降低濾餅的可壓縮性Diatomaceousearth（硅藻土）orperlite（珍珠巖）

commonlyusedmethodsusedasaprecoataddedtothebrothbeforefiltrationselection選擇和使用助濾劑的要點（1）根據(jù)過濾介質(zhì)和過濾情況選擇助濾劑品種粗目濾網(wǎng)宜使用石棉粉、纖維素、淀粉；細目濾布宜使用細硅藻土；燒結(jié)或粘結(jié)材料宜使用纖維素。（2）粒度選擇當(dāng)粒度一定時，過濾速率與澄清度成反比。助濾劑的粒度必須與懸浮液中固體顆粒的尺寸相適應(yīng)。（3）使用量的選擇當(dāng)預(yù)涂時，間歇操作助濾劑的最小厚度為2mm；連續(xù)操作則要根據(jù)所需過濾率來確定。當(dāng)直接加入時，一般情況下，若用量與懸浮液中固形物含量相等，過濾速度最快。2.2Filtration過濾definitionFiltrationseparatessolidfromaliquidbyforcingtheliquidthroughasolidsupportorfiltermedium.classificationoperationbatchoperationcontinuousoperationclassificationmechanismclarificationfiltrationcakefiltrationMotivepowerforfluid

gravityfiltrationpressurefiltrationvacuumfiltrationcentrifugefiltrationBatchoperation（分批操作）UsefulforsmallfermentationbatchesFiltrationequipment

plateandframefilter（板框過濾機）Graduallyaccumulatesbiomass（生物量）,thenisopenedandclearedofthefiltercake廣泛應(yīng)用于培養(yǎng)基制備的過濾及霉菌、放線菌、酵母菌和細菌等多種發(fā)酵液的固液分離。推動力來自泵產(chǎn)生的液壓或進料貯槽中的氣壓比較適合于固體含量1％一10％的懸浮液的分離。板框過濾機優(yōu)點結(jié)構(gòu)簡單、裝配緊湊、過濾面積大；推動力能大幅度調(diào)整，能耐受較高的壓力差（可超過0.1MPa,這是真空過濾器無法達到的）；固相含水分低，并能適應(yīng)不同過濾性質(zhì)的發(fā)酵液的過濾；輔助設(shè)備少、維修方便、價格低和動力消耗少等。缺點設(shè)備笨重、間歇操作、勞動強度大、衛(wèi)生條件差、輔助時間多和生產(chǎn)效率低。Continuousoperation（連續(xù)操作）Usedinlargerprocessestoreducedowntime（停工時間）filtersrotarydrumvacuumfilters真空轉(zhuǎn)鼓過濾器OperatingprincipleCharacteristicsapplicationcrossflowfiltration錯流過濾diatomitefilter硅藻土過濾機OperatingprincipleDirectionofRotationKnifeDischargecakeFeedWashersWashingDryingLoadingRotaryDrumFilterCharacteristics自動化程度高操作連續(xù)和處理量大特別適合于固體含量較大(＞10％)的懸浮液的分離受推動力(真空度)的制約，一般不適合于菌體較小和粘度較大的細菌發(fā)酵液的過濾。過濾所得固相的干度不如加壓過濾。Crossflowfiltrationdead-endfiltrationthefeed(slurry)flowinthesamedirectionasthepermeateflow.Crossflowfiltration(tangentialflowfiltration)Fluidflowsparallel,orcrossflowtothefiltermediasurface.

Mainadvantage:theshearingactionoftheflowreducesthethicknessofthefiltercake.Membraneseparation:microfiltration,ultrafiltrationCharacteristics

CrossflowfiltrationCharacteristics不能得到干濾餅?zāi)芎谋纫话氵^濾高需要大的膜面積收率高（97-98%）濾液質(zhì)量好減少處理步驟對染菌罐批易于處理diatomitefilter右圖為垂直葉片式硅藻土過濾機適合過濾固形物含量<0.1%的懸浮液。2.3centrifugationdefinitionF,RCF,rpmTypesofcentrifugesLaboratorycentrifuges實驗室離心機Preparativecentrifuges制備型離心機tubularbowlcentrifuge（管式離心機）disk/disccentrifuge（碟片式離心機）scroll/decantercentrifuge（螺旋式離心機）UltracentrifugationDifferentialcentrifugation差速離心Densitygradientcentrifugation密度梯度離心Ratezonalcentrifugation速率區(qū)帶離心Isopycniccentrifugation等密度離心Whenasuspensionisallowedtostand,thedensersolidsslowlysettletothebottomofthecontainerundertheinfluenceofgravity,aprocesscalledsedimentation（沉降）.Whenthissettlingisacceleratedwithacentrifugalfield（離心場）,theprocessiscalledcentrifugation.Centrifugationutilizesthedensitydifferencebetweenthesolidsandthesurroundingfluid.

Thesolidconcentrateproducedbycentrifugationdiffersfromthatproducedbyfiltration。Abestcentrifugationproducesapaste,oftenityieldsonlyamoreconcentratedsuspension.Filtrationproducesrelativelydrycake,whichisamajoradvantage.However,manybiologicalfeeds,whichcanbecentrifuged，cannotbeeffectivelyfiltered,sothatcentrifugationisoftenanattractivealternative.F,RCF,rpmCentrifugalforce

離心力Relativecentrifugalforce(RCF)

相對離心力Maximumrelativecentrifugalforce最大相對離心力CentrifugalforceF=mω2rF：thecentrifugalforceactingontheparticletomaintainitinthecircularpathm：themassoftheparticleω:theangularvelocityoftheparticler:theradiusofthepathRelativecentrifugalforce(RCF)RCF=G=F/W=mω2r/mg=(2πn)2r/gn：therotationalspeed,measuredinrevolutionsperminute（rpm）r：theradiusofrotationg：earth'sgravitationalaccelerationRCF=1.119×10-5×r×RPM2RPM,RCF,rMaximumrelativecentrifugalforce最大相對離心力實際工作中知道離心轉(zhuǎn)頭達到的最高速度以及該轉(zhuǎn)頭的最大直徑即可計算該轉(zhuǎn)頭工作產(chǎn)生的最大相對離心力。最大分離因素工業(yè)又把離心機能夠產(chǎn)生的額定最高RCF稱為最大分離因素。離心機能夠承受的RCF是離心機性能的重要參數(shù)。LaboratorycentrifugesRotorsFixedangledrotorSwing-outrotor（平拋式）Rmax-RminRmax-RminCentrifugetubePushbuttonDisplayrotorFrequentlyUsedLaboratoryCentrifugesBeckman-Coulter

FrequentlyUsedLaboratoryCentrifugesTubularbowlcentrifugesSuspensionisusuallyfedthroughbottom,andclarifiedliquidisremovedfromthetop.Solidsdepositonthebowl’swallasathickpaste.Thesuspensioncanbefeduntilsolidlossintheeffluent.Mostefficienthighcentrifugalforce（15000g－65000g）；thinsettlingzoneCanbecool,arealadvantageinproteinwork.

TubularbowlcentrifugesTubularbowlcentrifugesDisadvantageSmallsludgeholdingspace------onlyfordilutesuspensions(concentrationlowerthan1%byvolume)Solids

mustbemanuallyremoved.intermittentoperation（間歇操作）Thebowlmustbedismantled（拆卸）andcleaned.Applicationsseparationsofmicrobialcells，celldebris，cellorganelles，viruses，proteinsandnucleicacidsdiscDisktypecentrifugesDisktypecentrifugesFeedusuallyentersatthetop，

clarifiedliquidflowsoutanannularslitnearthefeed.types(accordingtothetypeofdischarge卸渣)Solidsejectingdisccentrifuge:intermittentoperationNozzledisccentrifuge:continuousoperationBasicdisccentrifuge:batchoperationMaximumRCF：1000g－20000gApplicationsseparationsofcelldebrisandmicrobialcells，suchasbacteria

andyeastscrolldischargecentrifugesThebowlandthescrewconveyorrotateinthesamedirection,butthelatterrotateswithavelocityslightlylowerorhigherthantheformer.FeedinClarifiedliquidSoliddischargescrolldischargecentrifugesthecontinuousfeeding,separatinganddischargingcanberealizedasitrotatesatitsfullspeed.Operationtemperature:upto300℃Canhandlesuspensionswithconcentrationsinthe1–50%range.applicationdewateringofbiologicalsludgesyeastandbacteriarecoverySummaryParametersTubularBowlDiskTypeScrollDischargeParticlesize0.01-100mm0.5-500mm0.5～2000mmParticleConcentration<1%<1-2%①<10%②<25%③1-50%RCF15000-650001000-200006000Processingcapacity0.1-0.4m3/h300m3/h0.4-60m3/hApplicationBacteria;celldebris,organelles;virus;proteinBacteria;yeast;Actinomycetes;CelldebrisInsulin;cytochrome;mycelia①人工卸料，②自動間歇排料，③噴嘴連續(xù)排料DifferentialcentrifugationDifferentialcentrifugationDifferentialcentrifugation(differentialsedimentation)isaprocessofsuccessivecentrifugation(singleorrepeatedsteps)withincreasingcentrifugeforce.Separationisdependentonparticlemassandsize,whereheavierparticlesorcellssettlefirstatlowergvalues.Application:subcellularfractionation.Protein（蛋白質(zhì)）microsome（微粒體）Golgi（高爾基體）endoplasmicreticulumER（內(nèi)質(zhì)網(wǎng)）roughER,smoothERlysosome（溶酶體）peroxisome（過氧化物體）mitochondria（線粒體）nuclei，nucleus（細胞核）DifferentialcentrifugationAdvantagesEasytohandleSupernatant/Pelletiseasytoseparate.BigVolumefixedanglerotorDisadvantagesPoorPerformanceWalleffectDestroycellcomponentspellet（顆粒）nuclei，nucleus（細胞核）mitochondria（線粒體）lysosome（溶酶體）peroxisome（過氧化物體）microsome（微粒體）Golgi（高爾基體）endoplasmicreticulum，ER（內(nèi)質(zhì)網(wǎng)）roughER,smoothERDensitygradientcentrifugationDensitygradientcentrifugationutilizesaspecificmediumthatgraduallyincreasesindensityfromtoptobottomofacentrifugetube。Thismeansthatundercentrifugalforce，particleswillmovethroughthemediumanddensitygradientandstopatapointinwhichthedensityoftheparticleequalsthedensityofthesurroundingmedium.DensitygradientcentrifugationAdvantagesHighPerformanceWide

UsedKeepthePropertiesDisadvantagesTimeGradientBufferOperationExperienceRequiredDensitygradientcentrifugationCommonprocedures(1)preparegradient制備梯度(2)applysample加樣(3)Centrifuge離心(4)collectandanalyzefractions收集和分析組分DensitygradientcentrifugationIsopycniccentrifugationRatezonalcentrifugationRatezonalcentrifugation差速區(qū)帶離心法：當(dāng)不同的顆粒間存在沉降速度差時，在一定離心力作用下，顆粒各自以一定速度沉降，在密度梯度的不同區(qū)域上形成區(qū)帶的分離方法。Ratezonalseparationtakesadvantageofparticlesizeandmassinsteadofparticledensityforsedimentation.Gradientmaterial:sucrose，F(xiàn)icoll，PercollRatezonalcentrifugationCriteriaforsuccessfulratezonalcentrifugationDensityofthesamplemustbelessthanthatofthelowestdensityportionofthegradient.Densityofthesampleparticlemustbegreaterthanthatofthehighestdensityportionofthegradient.Thepathlengthofthegradientmustbesufficientfortheseparationtooccur.Time

isimportant.Ifyouperformtoolongruns,particlesmayallpelletatthebottomofthetube.Ratezonalcentrifugation100%separationispossiblewithasmallsample.Usedtopurifyenvelopedvirusesandtoseparatevarioustypesofmacromolecules

mixtureofproteins;differenttypesofRNA;separateDNAfromRNA,orproteins;ribosomalsubunits;polyribosomes;cellorganellesfromcrudecellularextracts.BioreactorfluidwithcellsmembraneVirusinactivationZonalcentrifugationConcentratedvirusUFmembrane

hydrolysisβ-propiolactoneβ-丙內(nèi)酯cellsproduct4℃37℃permeateNonantigenicmaterialFigure1.3downstreamprocessesforproductionofrabiesvaccine(狂犬疫苗)0.3μmTakes24hoursSucrosegradient10%-60%,50000g,15hIsopycniccentrifugationDuringthecentrifugation,theCsClgeneratesagradient(“self-generatinggradient”),andthemoleculesmovetothepositioninthegradientwheretheirdensityisthesameasthegradientmaterial.Separatesmoleculesbasedontheirdensity–mayhavethesamesize.因此用這種方法分離顆粒，主要是根據(jù)被分離顆粒的密度差異。只要被分離顆粒間的密度差異大于1%就可用此法分離。IsopycnicpositionIsopycniccentrifugationCriteriaforsuccessfulisopycnicseparation:Densityofthesampleparticlemustfallwithinthelimitsofthegradientdensities.Anygradientlengthisacceptable.Theruntimemustbes