Culture Condition Optimization And Ecological Function Of Nitrite-dependent Anaerobic Methane-oxidizing Bacteria

Nitrite-dependent anaerobic methane oxidation(N-DAMO)is a biological process that couples anaerobic oxidation of methane to the reduction of nitrite to dinitrogen.This bioprocess plays an important role in the global carbon and nitrogen cycles and is also a potential process in wastewater treatment.However,N-DAMO bacteria grow incredibly slowly,and their doubling times are as long as weeks to months.It is hard to get enough N-DAMO culture,which limits the further scientific research and engineering application.Although N-DAMO bacteria distributes widely,their abundance in some ecosystems are very low.The original primers are not sensitive enough to detected N-DAMO bacteria in such ecosystems.In this work,the environmental and nutritional conditions were improved to cultivate N-DAMO bacteria by short-and long-term experiments.The results were subsequently applied in the cultivation of N-DAMO bacteria in a bioreactor.A novel species of N-DAMO bacteria was obtained and the NC10-specific primers were improved based on the gene sequence of the novel species.Using the new primers and other technologies,the ecological function of N-DAMO bacteria in coastal wetlands was investigated.The major results are as follows:1)The effects of environmental conditions-temperature,pH and salinity-on the metabolic activity and growth rate of N-DAMO bacteria were investigated by a series of short and long-term experiments.The results of the short-term activity tests showed that all of the three environmental factors(temperature,pH and salinity)significantly impacted the metabolic activity of N-DAMO bacteria.The N-DAMO specific activity increased exponentially at low temperatures(<35℃),and sharply decreased at high temperatures(>35℃).The correlation between the N-DAMO specific activity and temperature could be described by the extended Arrhenius equation,and the optimal temperature for N-DAMO bacteria was approximately 35℃.As the pH increased,the N-DAMO specific activity increased at low pH(<7.5),but decreased at high pH(>7.5).The modified Antoniou equation was selected to fit the data,and the optimal pH of 7.6 was estimated from the best fitting.The freshwater N-DAMO bacteria were strongly inhibited by high salinities,and their activity almost disappeared at salinity of 20g NaCl·L-1.The results of the long-term bacterial cultivation indicated that temperature,pH, and salinity impacted the growth rate of N-DAMO bacteria.Temperature 35℃,pH 7.6,and salinity 0 g NaCl·L-1 were the optimal conditions for N-DAMO bacteria.N-DAMO bacteria were more active and grew faster under the optimal conditions.Notably,almost no N-DAMO activity could be detected at the start at salinity of 20 g NaCl·L-1,but the biomass regained N-DAMO activity after 90 days’ cultivation.The recovered N-DAMO activity was 0.029μmol CH4·h-1.The NC10 16S rRNA and pmoA genes were sequenced before and after 90 days of cultivation.The phylogenetic analyses revealed that the microbial community structure of N-DAMO bacteria did not change significantly.It indicated that the N-DAMO activity after 90 d was attributed to the salinity adaption of the original N-DAMO bacteria,not the growth of new halophilic N-DAMO bacteria.As far as we known,this is the first report of salinity adaption of N-DAMO bacteria.2)Short-and long-term tests were carried out to investigate the effects of iron (Fe),copper(Cu),zinc(Zn),molybdenum(Mo),cobalt(Co),manganese(Mn),and nickel(Ni)on the metabolic activity and growth rate of N-DAMO bacteria.Optimal concentrations of the trace elements were determined,and the results were applied to cultivate N-DAMO bacteria in an SBR reactor.The results of the short-term tests showed that ferrous ion(Fe(Ⅱ))and cupric ion (Cu(Ⅱ))impacted evidently on the N-DAMO activity.In the tested range(4～100 μmol·L-1),as the Fe(Ⅱ)concentration increased,N-DAMO activity first increased, then decreased slightly,and reached highest at approximately 20μmol·L-1.In the tested range(1～100μmol·L-1),as the Cu(Ⅱ)concentration increased,N-DAMO activity first increased slightly,then decreased considerably,and reached highest at approximately 10μmol·L-1.It indicated that high concentrations of Cu(Ⅱ)(25～100 μmol·L-1)would largely inhibit N-DAMO bacteria.Moreover,in the tested ranges, the effects of Zn,Mo,Co,Mn,and Ni on N-DAMO bacteria were not significant.The results of long-term orthogonal tests showed that Fe(II)and Cu(II)impacted significantly on N-DAMO bacteria.In the test ranges(4～20μmol·L-1 for Fe(Ⅱ)and 1～10μmol·L-1 for Cu(Ⅱ)),the growth of N-DAMO bacteria was accelerated by the increase of the concentrations of Fe(Ⅱ)or Cu(Ⅱ).This result was subsequently confirmed by long-term single factor tests.Moreover,in the tested ranges,the influence of other trace elements(Zn,Mo,Co,Mn,and Ni)was lower than the random error of the experiments.Therefore,the concentrations of Fe(Ⅱ)and Cu(Ⅱ)in the improved medium were set as 20 and 10μmol·L-1,respectively,and those of other trace elements were not changed.The improved medium was applied in an SBR reactor to cultivate N-DAMO bacteria.The number and activity of N-DAMO bacteria increased after the application of the improved medium,and the doubling time was shorten to one third of the original one.Especially,many big and dense aggregates of N-DAMO bacteria were observed after the usage of the improved medium.A comparison of the increase in the number of N-DAMO bacteria and the cell number in one aggregate showed that the aggregates were formed mainly by the adhesion of many single cells,not by the growth of one cell.3)A novel species of N-DAMO bacteria was obtained.NC10-specific primers were improved according to the sequence of the novel species,and a new PCR protocol with high sensitivity was developed.The complete 16S rRNA gene sequence of the novel species was obtained by amplifying with the bacterial universal primer set 8F/1492R.According to the NCBI genbank,this novel species distributes widely in Chinese habitats,and it might be ecologically significant.Some defects were found in the widely used primers,and the primers have been improved in this work.Primer 202F has a strongly bind at the wrong positions 552～563,which results in nonspecific amplifications.Primer qP1F has a base mismatch at the 3’ end with the sequence of the novel species.In this work,a new primer 1051F was designed and a degenerate 3’ end was introduced into primer qP1F.A new nested PCR protocol with high specificity and sensitivity was developed to amplify NC10 phylum 16S rRNA genes.The limit of detection of the new protocol was approximately 103 copies·g-1 dry sediment,much lower than that of the original one(105 copies·g-1 dry sediment).Moreover,a degenerate 3’ end was introduced into qPCR primer qP1F.More copy numbers were obtained with the improved primers, and the results agreed well with the N-DAMO specific activities.4)Using the molecular biological methods and isotope tracer technology,the ecological function of N-DAMO bacteria in coastal wetlands was determined.Chemical profiles of sedimentary methane and electron acceptors in coastal sediments were determined.Methane was at high concentrations(up to 32μmol·L-1) in the deeper sediments(>3 cm),and at low concentrations(<0.3μmol·L-1)in the upper sediments(<3 cm).The depths of oxygen penetration in the sediments were less than 1 cm,while those of nitrate/nitrite were 2～4 cm.Sulfate was at high levels in all the tested sediments.The Chemical profiles of methane and nitrate/nitrite were overlapped in the subsurface sediments,indicating that N-DAMO process might occur there.The rates of methane production and consumption at different depth of sediments were determined.Net methane production was found in the surface and deep sediments,and net methane consumption was determined in the subsurface sediments (0.5～2 cm).Aerobic MO,DAMO(including N-DAMO and nitrate-AOM),and sulfate-AOM occurred mainly in the surface,subsurface,and deep sediments, respectively,and their activities were 0.5～1.6,1.4～3.2 and 0.7～3.0 nmol CH4·day-1·mL-1 wet sediment,respectively.The microbial community structures of methanotrophic bacteria and archaea in the coastal wetlands were determined.Layered microbial community of methanotrophic bacteria and archaea was discovered in the tested sediments.Aerobic methane-oxidizing bacteria(MOB)were mainly in the surface sediments.DAMO microorganisms(including N-DAMO bacteria and DAMO archaea)were mainly in the subsurface sediments,and N-DAMO bacteria,affiliated to NC10 phylum, dominated the DAMO microbial community.Anaerobic methanotrophic archaea responsible for sulfate-AOM were largely in the deep sediments.Moreover,the results of qPCR also showed that N-DAMO bacteria thrived in the subsurface,more abundant than MOB.These results indicated that N-DAMO was an important sink of sedimentary methane,and its contribution ratio was estimated to be 23～58%.Notably,due to the existence of N-DAMO bacteria in the subsurface sediments,the intact sediment cores became atmospheric methane sink,with methane-oxidizing rate of 0.10-0.18 nmol CH4·day-1·cm-2.