Molecular Characterization Of Anaerobic Methanogenic Degradation Of Petroleum Hydrocarbon At Low Temperature

In this thesis, Hungate anaerobic techniques was used to construct methanogenic enrichmentsdegrading petroleum hydrocarbon or hexadecane under hypothermia conditions; methods of GC, HPLCand GC-MS were applied to detect the methane production and hydrocarbon degradation, in order toanalysis community construct of hydrocarbon degradation enrichment; T-RFLP、16S rRNA gene clonelibraries、 qPCR and454titanium pyrosequencing were conducted to unveil the key microbialphylotypes during incubation of methanogenic consortia of hydrocarbon degradation. And finally,proteomics technology was used to identify key protein involved in methanongeic degradation ofhydrocarbon.The results were as follows:(1)A methanogenic enrichments not only can degrade hexadecane(200ul), but also can degrade complex compound crude oil(1g) under hypothermia condition, whichproduced2mmol and3.7mmol, respectively. The log phase of anaerobic hexadecane degradation islonger than crude oil degradation; chromatogram was obviously changed by microbial degradation,which shift from single to double peak, with short chain alkans(n<23) degrading is faster than longchain alkans(n>23);(2)Both aceticlastic and hydrogenotrophic methanogens involved in methaneproduction from petroleum hydrocarbon degradation under hypothermia condition. All of the archaelsequences were assigned to four OTUs, One OTU belonged to hydrogenotrophic Methanoculleus andaccount for27%, the other OTU（T-RF284bp）was closed related to Methanosaeta concilii GP6, whichis a aceticlastic methanogen, the abundance of T-RF284bp accounts for67.5%increasing markedly inthe hexadecane-degrading culture with methane accumulating. Therefore, aceticlastic methanogenrepresenting T-RF284bp play an important role in the anaerobic hexadecane degradation;(3)Thesequenced bacterial clones from the hexadecane-degrading microcosms comprised sequences related toDeltaproteobacteria (65.93%), Mollicutes (9.29%), Bacterodia (2.1%), Clostridiales (6.1%),Anaerolineales (6.19%), Betaproteobateria (1.1%) and Alphaproteobacteria (2.1%). T-RFLP andstatistical analysis revealed that T-RFs207bp possibly played a key-role in methanogenic degradation ofhexadecane, showing98.7%similarity to Syntrophus sp.AJ133795, combined with qPCR quantitativeanalysis that the copy of Syntrophaceae is increased with the methane accumulated. So, Syntrophaceaemay play a vital role in anaerobic hexadecane degradation process.;(4)Due to protein concentration islow, there is not identify any key proteins of anaerobic hydrocarbon degradation, but identified enzymesrelated to metabolism of fatty acid oxidation: trans-2-enoyl COA reductase, carbon monoxidedehydrogenase (CODH), the trans-2-enoyl COA reductase is the key enzyme of hydration in betaoxidation, according to the metabolic pathways of anaerobic hydrocarbon degradation, it may bethrough the metabolism of fatty acid oxidation for subsequent metabolism.