第四章微生物发酵常用菌种及扩大课件

1OutlineofafermentationprocessRawMaterialsProductionmicroorganismFermentationProductpurificationProductEffluentwaste1OutlineofafermentationproChapter3StrainBreedingandscale-up

Chapter3StrainBreedinganFiveCommonCharacteristicsofMicroorganisms1.Smallvolume,largesurfacearea2.Fastabsorptionandconversion3.Rapidduplicationandgrowth4.Strongadaptability5.WidespreaddistributionanddiversifiedspeciesFiveCommonCharacteristicsofWhattypesofmicroorganismscausefermentationtooccur?BacteriaYeastMoldActinomycetesWhattypesofmicroorganismscBacteria

Bacteria

aretypicallyone-celledorganismsthatmultiplybysimpledivisionandcanbeseenonlywithamicroscope.Bacteria1.LacticacidbacteriaLactobacillus——yoghourtStreptococcus——yoghourt

Pediococcus——sourpicklesBifidobacterium——probiotics1.LacticacidbacteriaLactobacillusLactobacillusStreptococcusStreptococcusPediococcusPediococcusBifidobacteriumBifidobacterium2.Aceticacidbacteria

Acetobacter——vinegar2.Aceticacidbacteria3.GlutamicacidfermentativebacteriumCorynebacterium

glutamicumBrevibacteriumflavum3.Glutamicacidfermentativeb4.BacillusBacillussubtilis--protease,amylaseBacilluslicheniformis--protease,amylase4.BacillusBacillussubtilis--

Yeast

Yeasts

areunicellular(single-celled)fungiandthatordinarilyreproducebybudding.HappyYeastYeastHappyYeastSaccharomycescerevisiaeSaccharomycescerevisiae

1.Saccharomyces

Saccharomycescerevisiae——beer

Saccharomycesellipsoideus——wine

2.Candida

Candidautilis——SCP

Mould

Fungithatgrowintheformofmulticellularfilaments,calledhyphae.Incontrast,microscopicfungithatgrowassinglecellsarecalledyeasts.Mould1.Mucor——chinesecheese2.RhizopusR.oryzoe

——amylase3.Aspergillus

A.niger——citricacid,glucoamylase

A.oryzae——soysauce,

pasta(sweet)sauces

1.Mucor——chinesecheeseFungiPlantoranimalsActinomycetesBacteriaActinomycetesMicrobesrelatedtoantibioticproductingFungiPlantoranimalsActinStreptomyces----streptomycinStreptomyces----streptomycinAboutSCP,whichiscorrect?A.Isakindofproteinwhichextractedfrommicrobialcells.B.Ismicrobialcellwhichproducedbyfermentation.C.Isantibioticssecretedbymicrobialcells.D.Couldnotberegardedasfood.AboutSCP,whichiscorrect?Canantibioticproductingmicrobesbeusedintheaminoacidsproduction?CanantibioticproductingmicrChoosingMicroorganismsforIndustrialMicrobiology–1.Thenutritionalcharacteristicsoftheorganism.–

2.Theproductivityoftheorganism,

–3.Thestabilityoftheorganismanditsamenabilitytogeneticmanipulation.–4.Theeaseofproductrecoveryfromtheculture.ChoosingMicroorganismsStrainSelectionPurchasefromCultureCollectionsScreeningofnaturecircumstancesMutationsGeneticengineeringStrainSelectionPurchasefromInternationalCultureCollectionsAbbreviationNameLocationATCCAmericanTypeCultureCollectionUnitedStatesIAMInstituteofAppliedMicrobiologyUniversityofTokyo,JapanNCTCNationalCollectionofTypeCulturesLondon,UnitedkingdomChinaCultureCollectionsCGMCCChinaGeneralMicrobiologicalCultureCollectionCenterBeijingCultureCollectionsInternationalCultureCollecti原种斜面初筛，复筛，再复筛采样设计实验方案调查研究及查阅充分的资料

增殖培养平板分离单株纯种分离菌种鉴定Stepsofnewspeciesseparationandscreening原种斜面初筛，复筛，再复筛采样设计实验方案调查研究及查阅充分Screeningofnaturecircumstances

MostmajorsourcesofmicrobesforuseinindustrialmicrobiologyarenaturalmaterialsScreeningofnaturecircumstanMicrobialstraincollection

1.soilslurry

2.Enrichment

Specialmedia

Controlcondition

InhibitunwantedstrainMicrobialstraincollection3.Isolationcultivation

Streakplatemethod

PourplatemethodSpreadplatemethod3.IsolationcultivationStreakplate

method

Bacteriaare“streaked”overthesurfaceofanagarplatesoastoobtainsinglecolonies.Itcanalsohighlightthepresenceofcontaminatingmicro-organisms.StreakplatemethodBacteriaThisisanexampleofagoodstreakforisolationusingthe"fourcorners"method.ThisisanexampleofagoodsPerformingaPlateStreakIPerformingaPlateStreakIPerformingaPlateStreakIIPerformingaPlateStreakII第四章微生物发酵常用菌种及扩大课件Pourplate

method

Bacteriaaremixedwithmeltedagar.Pouredintoanemptyplateandallowedtosolidify.Pourplatemethod第四章微生物发酵常用菌种及扩大课件Spreadplatemethod

Smallsamplesofthedilutedbacteriaarepipettedontothesurfaceofagarplates.Asterile,bent-glassrodisthenusedtospreadthebacteriaevenlyovertheentireagarsurface.稀释的Spreadplatemethod稀释的第四章微生物发酵常用菌种及扩大课件第四章微生物发酵常用菌种及扩大课件4.PurecultivationGrowonlyonekindofmicrobeMustuseaseptictechniquetoavoidcontaminatingthecultureTransferasinglecolonyfromagarplatetoliquidmedium4.Purecultivation5.Productivity

measurement

Primaryscreening：quantity

Secondaryscreening：quality5.ProductivitymeasurementIfIcouldn’tfindanidealstrainfromnature,howwouldIdo?IfIcouldn’tfindanidealstSpontaneous(natural)mutationInducedmutationmutationSpontaneous(natural)mutationTechniqueforinducingmutationPhysicalmutagens

e.g.X-rays,g-rays,UVChemicalmutagense.g.baseanalogs,nitrousacid,alkylatingagents碱基类似物亚硝酸烷化剂TechniqueforinducingmutatioSteps:分离和筛选出发菌株的选择处理菌悬液的制备诱变处理中间培养Steps:分离和筛选出发菌株的选择处理菌悬液的制备诱变处理第四章微生物发酵常用菌种及扩大课件

Howaremutantsdetectedbyscientists?VisibleNutritional(auxotrophic)Whatisreplicaplating,andhowisitusedtodetectauxotrophicmutants?Resistancemutations(plateonmediacontainingthechemical)营养缺陷型影印培养法Howaremutantsdetectedb降解纤维素产生透明圈摇床复筛降解纤维素摇床复筛Replica-platingtechniquetoscreenformutantstrainsofacolony-formingmicroorganism.Replica-platingtechniquetosQ:Howwouldyouselectforprotease(amylase,celluaseorlase)-producingmutantstrain?Q:HowwouldyouselectforprCellBiologyTechniquesProtoplastRemovingthecellwallwithlyticenzymesinthepresenceofosmoticstabilizers.Inthepresenceoffusogenicagentsuchaspolyethyleneglycol(PEG),protoplastsareinducedtofuseandformtransienthybridsordiploids.Regenerationofviablecellsfromthefusedprotoplasts.融合剂二倍体CellBiologyTechniquesProtoplGeneticEngineeringVarioushighvalueaddedproductshavebeenproducedfromgeneticengineeringmethods.GeneticEngineeringVarioushigCulturepreservation

InternationalCultureCollectionsAbbreviationNameLocationATCCAmericanTypeCultureCollectionUnitedStatesIAMInstituteofAppliedMicrobiologyUniversityofTokyo,JapanNCTCNationalCollectionofTypeCulturesLondon,UnitedkingdomChinaCultureCollectionsCGMCCChinaGeneralMicrobiologicalCultureCollectionCenterBeijingCulturepreservationInternatidryoxygendeficit

lowtemperaureCulturepreservationOligotrophedrylowCultureOligotrophe菌种保藏的方法很多，一般有下面几种：A：斜面冰箱保藏法B：沙土管保藏法C：石蜡油封存法D：真空冷冻干燥保藏法E：液氮超低温保藏法菌种保藏的方法很多，一般有下面几种：Iffermentationinalargetankreceiveonlyoneloopofinoculum,aprolongedperiodwouldresult……..energyconsumingcontamination

NecessityofinoculumdevelopmentIffermentationinalargetanInoculumDevelopment

Thepreparationofapopulationofmicroorganismsfromadormantstockculturetoanactivestateofgrowththatissuitableforinoculationinthefinalproductionstage.休眠的InoculumDevelopment休眠的

SotheinoculumvolumehastobequitelargeAseedfermenterisusuallyrequiredtoproducetheinoculumvolumeTheseedfermenter’spurposeisnottoproduceproductbuttoprepareinoculumSotheinoculumvolumehastAeration:通风,通气Aeration:通风,通气IninoculumdevelopmentInoculumlevel:approximately0.5to5percentinoculumbyvolumefromtheprecedingstep.Incubationperiod:short,inloggrowthphase,littlefermentationproductInoculummedia:balancedforrapidcellgrowthandnotforproductformation.IninoculumdevelopmentTheprocessofinoculumdevelopmentTheprocessofinoculumdevelo第四章微生物发酵常用菌种及扩大课件实验室阶段1、培养物选择的原则目的：种子扩培到一定的量和质，根据菌种的特点最终的培养物可分为两类：

对于不产孢子和芽胞的微生物——获得一定数量和质量的菌体对于不产芽孢和孢子的微生物，实验室阶段的种子扩培最终是获得一定数量和质量的菌体，如谷氨酸的种子培养。实验室阶段1、培养物选择的原则目的：种子扩培到一定的量和质，第四章微生物发酵常用菌种及扩大课件获得一定数量和质量的孢子菌丝体培养步骤少，因而更容易获得量和质稳定的种子，但操作繁琐。对于产孢子的微生物获得一定数量和质量的菌丝体便于操作，但需要更仔细的控制。获得一定数量和质量的孢子菌丝体培养步骤少，因而更容易获得第四章微生物发酵常用菌种及扩大课件2、培养基选择的原则培养基的选择应该是有利于菌体的生长，对孢子培养基应该是有利于孢子的生长。在原料方面，实验室种子培养阶段，规模一般比较小，因此为了保证培养基的质量，培养基的原料一般都比较精细。2、培养基选择的原则培养基的选择应该是有利于菌体的生长，对孢3、起始接种物的传代问题

细菌保藏斜面→活化斜面

产孢子保藏→母斜面→子斜面目的：使菌种的传代次数尽可能的少。3、起始接种物的传代问题细菌保藏斜面→活化斜面母瓶：活化、纯化，使保藏菌种生长，并去除变异株。所以接种时要稀一点、便于纯化生长到单菌落。子瓶:大量繁殖，得到大量孢子。接种：①从母斜面上点接种，选取生长好的单菌落②接种时密一点，得到大量的孢子。孢子培养时注意湿度，子斜面使用一般不超过1个月孢子培养母瓶：活化、纯化，使保藏菌种生长，并去除变异株。子生产车间阶段1、培养物的选择原则在生产车间阶段，最终一般都是获得一定数量的菌丝体。缩短发酵时间有利于获得好的发酵结果菌丝体比孢子要有利：生产车间阶段1、培养物的选择原则在生产车间阶段，最终一般都是InoculationofaFermenter

fromanotherSeedTankSeedTankFermenterABTrap TrapSteamCJDIHEFGInoculationofaFermenterfro第四章微生物发酵常用菌种及扩大课件2、培养基选择的原则目的：获得一定数量和质量的菌体，因此培养基的选择应首先考虑的是有利于孢子的发育和菌体的生长.在原料方面：不如实验室阶段那么精细，而是基本接近于发酵培养基，这有两个方面的原因:

一是成本二是驯化2、培养基选择的原则目的：获得一定数量和质量的菌体，因此培养

薯干粉成分斜面种子罐发酵麦芽汁麦汁薯干粉10%22-28%(NH4)2SO40.5%琼脂2%薯干粉成分斜面第四章微生物发酵常用菌种及扩大课件3、发酵级数的确定谷氨酸：三级发酵一级种子（摇瓶）→二级种子（小罐）→发酵青霉素：三级发酵一级种子（小罐）→二级种子（中罐）→发酵

一般由菌丝体培养开始计算发酵级数，但有时，工厂从第一级种子罐开始计算发酵级数3、发酵级数的确定谷氨酸：三级发酵一级种子（摇瓶）→二级种子发酵级数确定的依据级数受发酵规模、菌体生长特性、接种量的影响

级数大，难控制、易染菌、易变异，管理困难，一般2-4级。

在发酵产品的放大中，反应级数的确定是非常重要的一个方面发酵级数确定的依据级数受发酵规模、菌体生长特性、接种量Aeration:通风,通气Aeration:通风,通气第四章微生物发酵常用菌种及扩大课件第四章微生物发酵常用菌种及扩大课件IndustrialFermentationSettingIndustrialFermentationSettinReviewQuestions1.Citesomemicro-organismsusedwidelyinfoodindustryandtheircorrespondingproducts.2.Howtogetanidealstrainforfermentationindustry?3.What’sthepurposeofinoculumdevelopment?Citetheprocessofit.ReviewQuestions3.Whycannotthemicro-organismsproductexcessivemetabolitesspontaneously?Bywhichmethodscanwegettheexcessivemetabolites?4.Howtoreducetheconcentrationofendproductsinfermentation?5.Howtoimprovethepermeabilityofcellmembrane?3.Whycannotthemicro-organ84OutlineofafermentationprocessRawMaterialsProductionmicroorganismFermentationProductpurificationProductEffluentwaste1OutlineofafermentationproChapter3StrainBreedingandscale-up

Chapter3StrainBreedinganFiveCommonCharacteristicsofMicroorganisms1.Smallvolume,largesurfacearea2.Fastabsorptionandconversion3.Rapidduplicationandgrowth4.Strongadaptability5.WidespreaddistributionanddiversifiedspeciesFiveCommonCharacteristicsofWhattypesofmicroorganismscausefermentationtooccur?BacteriaYeastMoldActinomycetesWhattypesofmicroorganismscBacteria

Bacteria

aretypicallyone-celledorganismsthatmultiplybysimpledivisionandcanbeseenonlywithamicroscope.Bacteria1.LacticacidbacteriaLactobacillus——yoghourtStreptococcus——yoghourt

Pediococcus——sourpicklesBifidobacterium——probiotics1.LacticacidbacteriaLactobacillusLactobacillusStreptococcusStreptococcusPediococcusPediococcusBifidobacteriumBifidobacterium2.Aceticacidbacteria

Acetobacter——vinegar2.Aceticacidbacteria3.GlutamicacidfermentativebacteriumCorynebacterium

glutamicumBrevibacteriumflavum3.Glutamicacidfermentativeb4.BacillusBacillussubtilis--protease,amylaseBacilluslicheniformis--protease,amylase4.BacillusBacillussubtilis--

Yeast

Yeasts

areunicellular(single-celled)fungiandthatordinarilyreproducebybudding.HappyYeastYeastHappyYeastSaccharomycescerevisiaeSaccharomycescerevisiae

1.Saccharomyces

Saccharomycescerevisiae——beer

Saccharomycesellipsoideus——wine

2.Candida

Candidautilis——SCP

Mould

Fungithatgrowintheformofmulticellularfilaments,calledhyphae.Incontrast,microscopicfungithatgrowassinglecellsarecalledyeasts.Mould1.Mucor——chinesecheese2.RhizopusR.oryzoe

——amylase3.Aspergillus

A.niger——citricacid,glucoamylase

A.oryzae——soysauce,

pasta(sweet)sauces

1.Mucor——chinesecheeseFungiPlantoranimalsActinomycetesBacteriaActinomycetesMicrobesrelatedtoantibioticproductingFungiPlantoranimalsActinStreptomyces----streptomycinStreptomyces----streptomycinAboutSCP,whichiscorrect?A.Isakindofproteinwhichextractedfrommicrobialcells.B.Ismicrobialcellwhichproducedbyfermentation.C.Isantibioticssecretedbymicrobialcells.D.Couldnotberegardedasfood.AboutSCP,whichiscorrect?Canantibioticproductingmicrobesbeusedintheaminoacidsproduction?CanantibioticproductingmicrChoosingMicroorganismsforIndustrialMicrobiology–1.Thenutritionalcharacteristicsoftheorganism.–

2.Theproductivityoftheorganism,

–3.Thestabilityoftheorganismanditsamenabilitytogeneticmanipulation.–4.Theeaseofproductrecoveryfromtheculture.ChoosingMicroorganismsStrainSelectionPurchasefromCultureCollectionsScreeningofnaturecircumstancesMutationsGeneticengineeringStrainSelectionPurchasefromInternationalCultureCollectionsAbbreviationNameLocationATCCAmericanTypeCultureCollectionUnitedStatesIAMInstituteofAppliedMicrobiologyUniversityofTokyo,JapanNCTCNationalCollectionofTypeCulturesLondon,UnitedkingdomChinaCultureCollectionsCGMCCChinaGeneralMicrobiologicalCultureCollectionCenterBeijingCultureCollectionsInternationalCultureCollecti原种斜面初筛，复筛，再复筛采样设计实验方案调查研究及查阅充分的资料

增殖培养平板分离单株纯种分离菌种鉴定Stepsofnewspeciesseparationandscreening原种斜面初筛，复筛，再复筛采样设计实验方案调查研究及查阅充分Screeningofnaturecircumstances

MostmajorsourcesofmicrobesforuseinindustrialmicrobiologyarenaturalmaterialsScreeningofnaturecircumstanMicrobialstraincollection

1.soilslurry

2.Enrichment

Specialmedia

Controlcondition

InhibitunwantedstrainMicrobialstraincollection3.Isolationcultivation

Streakplatemethod

PourplatemethodSpreadplatemethod3.IsolationcultivationStreakplate

method

Bacteriaare“streaked”overthesurfaceofanagarplatesoastoobtainsinglecolonies.Itcanalsohighlightthepresenceofcontaminatingmicro-organisms.StreakplatemethodBacteriaThisisanexampleofagoodstreakforisolationusingthe"fourcorners"method.ThisisanexampleofagoodsPerformingaPlateStreakIPerformingaPlateStreakIPerformingaPlateStreakIIPerformingaPlateStreakII第四章微生物发酵常用菌种及扩大课件Pourplate

method

Bacteriaaremixedwithmeltedagar.Pouredintoanemptyplateandallowedtosolidify.Pourplatemethod第四章微生物发酵常用菌种及扩大课件Spreadplatemethod

Smallsamplesofthedilutedbacteriaarepipettedontothesurfaceofagarplates.Asterile,bent-glassrodisthenusedtospreadthebacteriaevenlyovertheentireagarsurface.稀释的Spreadplatemethod稀释的第四章微生物发酵常用菌种及扩大课件第四章微生物发酵常用菌种及扩大课件4.PurecultivationGrowonlyonekindofmicrobeMustuseaseptictechniquetoavoidcontaminatingthecultureTransferasinglecolonyfromagarplatetoliquidmedium4.Purecultivation5.Productivity

measurement

Primaryscreening：quantity

Secondaryscreening：quality5.ProductivitymeasurementIfIcouldn’tfindanidealstrainfromnature,howwouldIdo?IfIcouldn’tfindanidealstSpontaneous(natural)mutationInducedmutationmutationSpontaneous(natural)mutationTechniqueforinducingmutationPhysicalmutagens

e.g.X-rays,g-rays,UVChemicalmutagense.g.baseanalogs,nitrousacid,alkylatingagents碱基类似物亚硝酸烷化剂TechniqueforinducingmutatioSteps:分离和筛选出发菌株的选择处理菌悬液的制备诱变处理中间培养Steps:分离和筛选出发菌株的选择处理菌悬液的制备诱变处理第四章微生物发酵常用菌种及扩大课件

Howaremutantsdetectedbyscientists?VisibleNutritional(auxotrophic)Whatisreplicaplating,andhowisitusedtodetectauxotrophicmutants?Resistancemutations(plateonmediacontainingthechemical)营养缺陷型影印培养法Howaremutantsdetectedb降解纤维素产生透明圈摇床复筛降解纤维素摇床复筛Replica-platingtechniquetoscreenformutantstrainsofacolony-formingmicroorganism.Replica-platingtechniquetosQ:Howwouldyouselectforprotease(amylase,celluaseorlase)-producingmutantstrain?Q:HowwouldyouselectforprCellBiologyTechniquesProtoplastRemovingthecellwallwithlyticenzymesinthepresenceofosmoticstabilizers.Inthepresenceoffusogenicagentsuchaspolyethyleneglycol(PEG),protoplastsareinducedtofuseandformtransienthybridsordiploids.Regenerationofviablecellsfromthefusedprotoplasts.融合剂二倍体CellBiologyTechniquesProtoplGeneticEngineeringVarioushighvalueaddedproductshavebeenproducedfromgeneticengineeringmethods.GeneticEngineeringVarioushigCulturepreservation

InternationalCultureCollectionsAbbreviationNameLocationATCCAmericanTypeCultureCollectionUnitedStatesIAMInstituteofAppliedMicrobiologyUniversityofTokyo,JapanNCTCNationalCollectionofTypeCulturesLondon,UnitedkingdomChinaCultureCollectionsCGMCCChinaGeneralMicrobiologicalCultureCollectionCenterBeijingCulturepreservationInternatidryoxygendeficit

lowtemperaureCulturepreservationOligotrophedrylowCultureOligotrophe菌种保藏的方法很多，一般有下面几种：A：斜面冰箱保藏法B：沙土管保藏法C：石蜡油封存法D：真空冷冻干燥保藏法E：液氮超低温保藏法菌种保藏的方法很多，一般有下面几种：Iffermentationinalargetankreceiveonlyoneloopofinoculum,aprolongedperiodwouldresult……..energyconsumingcontamination

NecessityofinoculumdevelopmentIffermentationinalargetanInoculumDevelopment

Thepreparationofapopulationofmicroorganismsfromadormantstockculturetoanactivestateofgrowththatissuitableforinoculationinthefinalproductionstage.休眠的InoculumDevelopment休眠的

SotheinoculumvolumehastobequitelargeAseedfermenterisusuallyrequiredtoproducetheinoculumvolumeTheseedfermenter’spurposeisnottoproduceproductbuttoprepareinoculumSotheinoculumvolumehastAeration:通风,通气Aeration:通风,通气IninoculumdevelopmentInoculumlevel:approximately0.5to5percentinoculumbyvolumefromtheprecedingstep.Incubationperiod:short,inloggrowthphase,littlefermentationproductInoculummedia:balancedforrapidcellgrowthandnotforproductformation.IninoculumdevelopmentTheprocessofinoculumdevelopmentTheprocessofinoculumdevelo第四章微生物发酵常用菌种及扩大课件实验室阶段1、培养物选择的原则目的：种子扩培到一定的量和质，根据菌种的特点最终的培养物可分为两类：

对于不产孢子和芽胞的微生物——获得一定数量和质量的菌体对于不产芽孢和孢子的微生物，实验室阶段的种子扩培最终是获得一定数量和质量的菌体，如谷氨酸的种子培养。实验室阶段1、培养物选择的原则目的：种子扩培到一定的量和质，第四章微生物发酵常用菌种及扩大课件获得一定数量和质量的孢子菌丝体培养步骤少，因而更容易获得量和质稳定的种子，但操作繁琐。对于产孢子的微生物获得一定数量和质量的菌丝体便于操作，但需要更仔细的控制。获得一定数量和质量的孢子菌丝体培养步骤少，因而更容易获得第四章微生物发酵常用菌种及扩大课件2、培养基选择的原则培养基的选择应该是有利于菌体的生长，对孢子培养基应该是有利于孢子的生