基于代谢调控提高克雷伯菌hq-3利用有机废弃物生产氢气的分析-analysis of improving hydrogen production by klebsiella hq - 3 using organic waste based on metabolic regulation.docxVIP

最适温度为33 °C、最适初始pH为6.5。利用CCD-RSM对氢气产量进一步响应面优化，发酵产氢的最佳工艺条件为：接种量21.26%，温度31.75 °C，初始pH6.67。在此条件下，实验验证产氢量为3.214molH2/mol葡萄糖，与预测值3.236molH2/mol 葡萄糖非常接近，回归方程比较准确地预测了各因素的影响。3. 油脂抽提藻渣（LMBRs）预处理发酵产氢的初步研究。藻渣纤维素“预处理-酶解法”采用超声、稀HCl、稀NaOH、氨水浸泡预处理，中和后，再以纤维素酶处理。经过不同预处理的还原糖得率以热碱结合纤维素酶法效果最佳，并在发酵制氢中氢累积量也最高，达到652mL/L。藻渣处理后的剩余残渣中还含有难降解蛋白质及微量矿物质，该残渣与酵母废弃物（LYBRs）可实现共降解。关键词：厌氧发酵产氢；克雷伯菌；同源表达；响应面优化；藻渣预处理AbstractWiththedepletion offossil energy,deteriorationofenvironmentpollution,and shortageofnaturalresourcesbecomingincreasinglyprominent,hydrogenasan environmentallyfriendlyalternativeenergyisattractingworldwideattention.Dark hydrogenfermentationisapromisingmethodduetoitshighhydrogen-producingrate, mildreactionconditions,low-costequipmentandbioconversionfeasibilityfrom the recyclingorganicwastes.However,lowhydrogenproductionofwildstrainsandhighcost ofthesubstratearethemainbottlenecksofbiohydrogenindustrialization. Therefore, investigatingmechanismandregulatingmetabolicpathwayofhydrogenproductionare becomingthehotfieldforpracticalbiohydrogenproduction.Meanwhile,moreresearches arefocusingontheexplorationofnewsubstrate toreducefermentationcost.Inthispaper,severalgenesinvolvinginthemetabolicpathwayofhydrogen productionwerehomologouslyoverexpressedinwild-typestrainKlebsiellasp.HQ-3and recombinantstrainHQ-3-P(Δppc),whosephosphoenolpyruvatecarboxylasegene(ppc) hadbeendeletedinourpreviousstudies.Eightmutantswereachievedbyhomologously expressingthreeforwardregulatoryfactorsofformatehydrogenlyasepathway(fhlA,fnr, fdhF)andacofactorofNADHpathway(pncB),respectively.Furthermore,hydrogen fermentationparametersofrecombinantstrainHQ-3-P/fhlAwereoptimizedbycentral compositedesign-responsesurfacemethod(CCD-RSM).Finally,differentpretreatment methodswereusedforhydrolysateoflipid-extractedmicroalgalbiomassresidues (LMBRs),andwasusedasacarbonsourcefordarkfermentationofhydrogenproduction. Themainworkandresults werelistedasfollows:Threeforwardregulatoryfactorsandacofactorwerehomologouslyoverexpressed in Klebsiellas