Physiological And Metabolic Mechanism Of Extracellular Electron Shuttles On Methanosrcina Acetivorans C2A

Methane is an important greenhouse gas,whose greenhouse effect is 28-34 times that of equal mole carbon dioxide,and has a great impact on global climate change.Biomethane is the main source of atmospheric methane.Billions of tons of biomethane were released every year,accounting for around 70%of global methane emissions.Biomethane is mainly produced in the fermentation process of various natural and artificial anaerobic systems.Methanogens at the end of the anaerobic fermentation chain produce methane based on three methanogenic metabolic pathways:aceticlastic pathway,CO2 reduction pathway and methylotrophic pathway.In recent years,many studies have shown that some methanogenic archaea have the ability of extracellular electron transfer,which provides a new way to regulate methanogenesis.Extracellular electron transfer refers to the process of intracellular electron transfer to extracellular electron receptor(microbe)and its reverse reaction,the key of which is the extracellular electron acceptor or electron shuttles.The existence of extracellular electron transfer greatly affects the energy metabolism and methanogenesis of methanogens,and has become an important way to regulate global biomethane emmision.However,the mechanism of it affecting methanogenesis is rarely reported or unclear.Therefore,in this study,extracellular electron shuttles humic substance(HS)and iron-based nanomaterials(nano zerovalent iron,nZVI;sulfide nano zero-valent iron,SnZVI;nano-ferrous sulfide(nFeS)was used as test object to explore the specific mechanism of its metabolic effects on Methanosarcina acetivorans C2A.The main research contents and results of this paper are as follows:The effect of Fe-based nanomaterials on the growth of M.acetivorans was analyzed by measuring the growth parameters of cells during entire incubation time.The results showed that nZVI and SnZVI significantly inhibited the growth and methanogenesis of M.acetivorans in a concentration-dependent manner,while nFeS had little effect on the growth and methanogenesis of M.acetivorans.The intracellular reactive oxygen species(ROS)level,glutathione(GSH)level,scanning electron microscopy(SEM)and transmission electron microscopy(TEM)analysis showed that Fe-based nanomaterials could attach and penetrate into cells,resulting in increased intracellular ROS and GSH levels,causing oxidative stress of cells.By measuring the activity of methanol coenzyme M methyltransferase(Mta)and ATP synthase in cells,it was found that Fe-based nanomaterials inhibited the activity of methanogenesis and energy metablism-related enzymes.Finally,by measuring the concentration of Fe2+in the culture medium and the content of H2 in the system,it was found that M.acetivorans could accelerate the oxidative dissolution of Fe0 in nZVI.Based on the above results,the inhibition mechanism of Fe-based nanomaterials was proposed:Fe0 could occupy transmembrane H+gradient formed during the growth of M.acetivorans,oxidizing H+to H2 and Fe2+.The distruction of transmembrane H+gradient resulted in the decrease of ATP synthase activity and ATP synthesis,which ultimately inhibited the growth and methanogenesis of M.acetivorans.The effect of HS on the growth of M.acetivorans was analyzed by measuring the growth parameters of cells during entire incubation time.The results showed that HS can greatly promote the growth and methanogenesis of M.acetivorans.Through fourier transformation infrared spectroscopy(FTIR),SEM analysis,reduction experiments of quinone group and etc,it was found that M.acetivorans can reduce HS and quinone group is the main functional redox group involved in this process.Transcriptomic analysis showed that the expression of MA4284MA4315 gene cluster was overally upregulated in M.acetivorans reducing HS.The coding products of this gene cluster were cell surface quinone proteins(CSQs)located on the cell membrane and most of them contains pyrroloquinoline quinone(PQQ)as cofactors.After isolation and identification,PQQ was found to exist in the membrane components of M.acetivorans,and the content of PQQ in the membrane was significantly increased after cultured with HS.Through the extracellular Fe(Ⅲ)reduction experiment,it was found that the cells with upregulated CSQs had higher electron transfer rate,and the electron transfer rate increased 410 times after the addition of methanophenazine(MP),which is a membrane-bound quinone analogue.These results indicated that CSQs and MP play an important role in the extracellular electron transfer.Based on the above phenomenon,a HS-based extracellular respiration of M.acetivorans was proposed:CSQs couple with the redox process of MP,transfering electrons derived from reducing equivalents to extracellular HS via PQQ,then reduces quinone groups in HS and finally completes extracellular respiration.This process can generate additional transmembrane H+gradient,enhance ATP synthesis and promote growth and methanogenesis of M.acetivorans.