黑曲霉发酵过程中菌体形态的分析方法建立及应用-唐文俊

/ChineseJournalofBiotechnology(2):291−299DOI:10.13345/j.cjb.140240©2015ChinJBiotech,AllrightsreservedReceived:April22,2014;Accepted:July4,2014Supportedby:SpecializedResearchFundfortheDoctoralProgramofHigherEducation(No.20110074110015).Correspondingauthor:JuChu.Tel:[phone];E-mail:[e-mail](No.20110074110015)2014-08-20(2):291–299.TangWJ,XiaJY,ChuJ,etal.DevelopmentandapplicationofmorphologicalanalysismethodinAspergillusnigerfermentation.ChinJBiotech,2015,31(2):291–299.:(Filamentousfungi):DevelopmentandapplicationofmorphologicalanalysismethodinAspergillusnigerfermentationWenjunTang,JianyeXia,JuChu,YingpingZhuang,andSiliangZhangStateKeyLaboratoryofBioreactorEngineering,EastChinaUniversityofScienceandTechnology,Shanghai200237,ChinaAbstract:Filamentousfungiarewidelyusedinindustrialfermentation.Particularfungalmorphologyactsasacriticalindexforasuccessfulfermentation.Tobreakthebottleneckofmorphologicalanalysis,wehavedevelopedareliablemethodforfungalmorphologicalanalysis.Bythismethod,wecanpreparehundredsofpelletsamplessimultaneouslyandobtainquantitativemorphologicalinformationatlargescalequickly.Thismethodcanlargelyincreasetheaccuracyandreliabilityofmorphologicalanalysisresult.Basedonthat,thestudiesofAspergillusnigermorphologyunderdifferentISSN1000-3061CN11-1998/QChinJBiotechFebruary25,2015Vol.31No.2:Aspergillusniger,fungalmorphology,imageanalysis,processcontrol(Filamentousfungi)Aspergillusniger[1][2](Dispersedmycelia)(Clumps)(Densepellets)[3](Glucoamylase)[4-5]pH[3,6-11][12]11.1AspergillusnigerCBS513.881.2(CM0139OxoidUK)45.1g/L10g/L0.1g/LCaCl2·2H2O1.0g/LMgSO4·7H2O2g/L3g/L(NH4)2SO43g/LKH2PO41.5g/LNaH2PO4·H2O0.04g/LMnSO4·H2O0.02g/LZnCl20.015g/LCuSO4·5H2O0.015g/LCoCl2·6H2O0.3g/LFeSO4·7H2O163.9g/L1.35L(/[e-mail]293)AE2000(Motic)5DMarkII(Canon)Image-ProPlusv6.0(MediaCybernetics)1.4105/mL400r/min20%40%60%300r/min450r/min500r/min600r/min144h1.51.61.6.140%(V/V)60%(V/V)1mL114[13]1.6.210mL5min4−55mL1.6.320mL0.5%(W/V)1.6.4ImageProPlusv6.0(MediaCybernetics,Singapore)“Contrastenhancement”“Segmentation”“HIS”“Count/Size”(Marco-D)(N)“Bestfitequalization”“Segmentation”HSI“Count/Size”(Micro-D)(Core-D)(A)(R)ImagePro-PlusBasicISSN1000-3061CN11-1998/QChinJBiotechFebruary25,2015Vol.31No.2=IpDocGet(GETNUMDOC,0,numDocs)Fori=0TonumDocs-1......Nexti1.6.512ImageProPlusFilamentousLength(Fl)FilamentousRatio(Fr)GrowthRate(G)1Table1SomecommonpelletparametersobtainedbyimageanalysisClassificationNameDefinitionSizeProjectedarea(A)TheprojectedareaofanobjectPerimeter(P)ThenumberofpixelaroundanobjectMaximumdiameter(dmax)ThelengthbetweentwofarthestpixelsintheobjectMinimumdiameter(dmin)ThelengthbetweentwoclosestpixelsintheobjectMeandiameter(dmean)Taking5°asastep-length,calculatetheFeretDiameteroftheobject.Taketheaveragevalueofall72FeretDiametersofthisobjectasameandiameterCountNumber(N)TheamountofpelletinacertainvolumeofbrothShapeFilamentouslength(Fl)Thelengthofhairypartofapellet.Calculatedas:Fl=(dmax–dmin)/2Filamentousratio(Fr)Thevolumeratioofthewholepelletandthecorepart.Calculatedas:Fr=(dmean/dmin)22Table2SomecommonhyphaeparametersobtainedbyimageanalysisClassificationNameDefinitionSizeProjectedarea(A)TheprojectedareaofanobjectHyphaewidth(W)Thewidthofprojectedhyphae,whichisthediameterofarealhyphaeHyphaelength(L)ThetotalpixelnumberofaskeletonizedobjectCountBranchnumber(Bs)ThebranchnumberofasinglemyceliumShapeGrowthunit(G)Hyphaegrowthrate,definedas:G=L/Bs22.11)2)3)4)1)/[e-mail]2952)3)()4)1)4)2)3)122510()50502001Fig.1Comparisonofpelletstructureusingdifferentpre-treatments.2()Fig.2Influenceonquantifiedmorphologydatawithdifferentpre-treatmentsandmeasurementnumber.Solid:roasted;hollow:agargelimmobilized.2.215L[14-16][17-19]100ISSN1000-3061CN11-1998/QChinJBiotechFebruary25,2015Vol.31No.2μm125μm50−75μm[20]ATP3Fig.3Thedistributionofpelletdiameterandfilamentouslengthunderdifferentoxygensupplycondition.2.324()300μm150−250μm600r/min75μm100μm[21](5)(6)/[e-mail]297[22-23]450r/min300r/min65.66%37.53%96h4Fig.4Thedistributionofpelletdiameterandfilamentouslengthatdifferentshearratelevel.5450r/minFig.5Thedistributionofpelletdiameterandfilamentouslengthat450r/minduringdifferentphases.6Fig.6Comparisonofdifferenthyphalmorphologyatdifferentfermentationphases.3[6-7,24-25]CFDISSN1000-3061CN11-1998/QChinJBiotechFebruary25,2015Vol.31No.2[1]ArcherDB,PeberdyJF.Themolecularbiologyofsecretedenzymeproductionbyfungi.CritRevBiotechnol,1997,17(4):273–306.[2]YuL,ChaoY,WenselP,etal.HydrodynamicandkineticstudyofcellulaseproductionbyTrichoderma