Study On Effects Of Temperature And Salinity On The Methanogen McrA Gene Transcription And Community In Anaerobic Sludge

Anaerobic biological treatment technology possesses the advantages of high organic loading,energy recycling,low energy consumption and sludge production,which make it one of the most important technologies for the sustainable treatment of industrial and municipal wastewater in the future.However,sensitivity of anaerobic process to environmental variations limits its widespread application,especially under psychrophilic or high salinity environment.Methanogens are the core microorganisms in anaerobic process and the mcrA gene(encoding the ?-subunit of coenzyme M reductase)is the unique functional gene which catalyzes the final step of methyl coenzyme M reduction.Presently,the effects of environment variations on mcrA gene transcription activity still lack.This thesis focuses on the responses of mcrA gene trancription activity and methanogeic community facing low temperature and high salinity,providing underlying information for microbial mechanism of methanogens.Research results are as follows:(1)The effects of low temperature shock on performance and physiology features(viability,ATP and CoM)of digesters fed with three carbon sources(acetate,formate and methanol)were studied.The results showed that carbon sources determined the resistivity potential of anaerobic sludge to low temperature under the same temperature shock condition.After low temperature shock of 15?,formate as carbon source showed the best anaerobic performance in the earlier period,however both performance and sludge activity declined gradually afterwards.At 25?,similar performance of formate set with its 35? after short term cultivation was found.Differently,acetate and methanol sets exhibited abrupt inhibition and then slow recovery during low temperature shock of 25? and 15?.On day 70 at 15?,the highest effluent COD of formate set(372.3 mg/L)and the highest decreasing percentage of SMA(92.1%)was found compared to other carbon sources;while the lowest effluent COD(215.3 mg/L)at 25? occurred within formate set.All SMA of three carbon sources decreased significantly(p<0.05)after temperature decreased to 25? or 15?.At 35?,viability of anaerobic sludge exhibited significant differences:methanol set(67.1%-73.4%)>acetate set(52.7%-57.9%)>formate set(44.0%-51.4%).After temperature decreased to 25? or 15?,significant decrease of the viability of formate set to 39.8%(25?)and 33.2%(15?)on day 70 after experiencing a stable period with no obvious difference of the viability of acetate set observed;methanol set exhibited a decrease of viability and recovered later.Largest CoM decrease was found in acetate set,with the decrease percentage of 22.0%(25?)and 60.8%(15?).(2)The effects of low temperature shock on mcrA gene/transcript abundance were studied.At 35?,mcrA gene abundance of three carbon sources showed differences by orders of magnitudes,in order of formate set(3.12×109copies/gVSS·h)>acetate set(1.07×108copies/gVSS·h)>methanol set(9.46×106copies/gVSS·h).After cultivation to day 70,formate showed the highest decreasing amplitude of mcrA gene abundance(89.0%)and methanol set exhibited highest decreasing amplitude of transcript abundance(89.1%)at 25?.The abundance of mcrA gene decreased most in formate set(98.3%)and transcript abundance decreased most in methanol set(98.0%)at 15%.The PCA analysis showed that acetotrophic and methylotrophic methanogens shared the same responses of transcription to temperature decrease,which was absolutely different from the hydrogenotrophic methanogens.mcrA transcript abundance was always correlated to SMA(p<0.01)during the process of temperature decrease,while mcrA gene abundance only correlated to SMA(p<0.01)at the end of trial(day 70).(3)The effects of low temperature shock on methanogens community of three carbon sources were studied.The results showed that the abundane of hydrogenotrophic Methanobacteriumincreased in all sets of three carbon sources as temperature decreased to 15?,indicating strong resistance to low temperature.Afterwards,the abundance of Methanobacterium increased continuously in formate set while decreased in acetate and methanol sets.As temperature decrease to 25?,the abundance of Methanobrevibacter increased gradually in formate sets.Methanosaetaperdominated in acetate sets,whose abundance increased continuously after temperature decreasing to 25? and recovered after a slight drop after temperature decreased to 15?.RDA analysis showed that hydrogenotrophic methanogens contributed most to the mcrA gene and transcript abundance.(4)The effects of salinity on performance and physiology features of anaerobic digestions with formate as sole carbon source at 35? were studied.The results showed that NH4Cl had obvious inhibition on the removal of organic matter while NaCl only had a restricted inhibition.Contrary to NH4Cl and NaCl,MgCl2 had promoting effect on anaerobic digestion.NH4Cl significantly inhibited SMA and the decreased magnitude of low,medium and high salinity sets were 46.4%,71.1%and 78.2%,respectively.All three salinity sets of MgCl2 showed promoting effects on SMA,rising by 43.9%-55.5%.The results showed that the viability of NH4Cl and NaCl high salinity sets on day 80 decreased by 47.5%and 28.4%.NH4CI had suppressive effect on ATP,with the decreased magnitude of high salinity up to 51.5%.The ATP concentration of MgCl2 high salinity set was significantly higher than control set.The suppressive magnitudes of high concentration NH4Cl and MgCl2 on coenzyme M were up to 39.9%and 34.1%.In all three MgCl2 sets with different salinity,the amounts of coenzyme M all increased in the medium term while decreased to the same level of control set later.(5)The effects of salinity on mcrA gene/transcript abundance with formate as sole carbon source at 35? were studied.The results showed that the abundance of mcrA gene in control set remained at 9.03×109-1.13×1010copies/gVSS.High salinity NH4Cl had significant inhibition on mcrA gene abundance,with the decreased magnitude up to 72.6%.The abundance of mcrA gene in NaCl set remained stable and it only decreased to 1.81×109 copies/gVSS on day 80.MgCl2 had a promoting effect on mcrA gene abundance in the early term while showed suppressive effect on the later term.The abundance of mcrA transcript of all three NH4Cl sets with different salinity decreased by two orders of magnitude.The mcrA transcript abundance decreased by 89.9%in NH4Cl high salinity set on day 32.The mcrA gene abundance of MgCl2 sets were slightly lower than control set.PCA analysis showed that ATP and viability had significant positive correlation with SMA,while mcrA gene/trnascript abundance had no correlation with SMA.(6)The effects of salinity on methanogens community with formate as sole carbon source at 35? were studied.Methanobacterium was the predominant methanogen in all samples,followed by Methanosaeta and Methanolobus.Methanosaeta increased and Methanobacterium decreased as a respond to high salinity.RDA analysis indicated that Methanobacterium was correlated to mcrA gene/transcript abundance.Methanobacterium was the predominant methanogen and it contributed most to the quality of mcrA gene and transcript.