Investigation Of Novel Process And Enrichment Of Key Microorganisms Involved In Nitrogen-cycling From Mangrove Sediments

As a typical wetland ecosystem in the land-sea interface,mangroves were one of the most productive ecosystems.It possessed active elements cycles of carbon,nitrogen,sulfur and abundant microbial resources.Meanwhile,mangrove habitats possess rich sulfur and organic matter with multiple land derived inputs,and characterized as periodic aerobic and anaerobic environments,making them unique habitats of great ecological value.Anaerobic Ammonium Oxidation(Anammox)bacteria,N2ODependent Anaerobic Methane Oxidation(N2O-AMO)process and Ammonia Oxidizing Archaea(AOA)were newly discovered microorganisms or process that can participate in carbon and nitrogen cycles in recent years.The discoveries and researches of those microorganisms were of great significance to the improvement of the theory of the global carbon and nitrogen cycle.However,the current reports about them in mangroves are lack or mainly focus on in-situ investigations and lacked the understanding of their specific physiological and biochemical properties.In the previous work of our lab,it was found that the habitat-specific terrestrial AOA clade contributed the main nitrification activity in the mangroves surface sediments.The mangrove sediments at different depths possessed active NO3--,SO42--and NO2--AMO and Anammox activities,and started the initial enrichment of Anammox derived from mangrove sediments.Based on these results,the Zhangjiang Estuary mangrove sediments were took as the research object,the in situ environmental factors and N2OAMO potential rate were investigated,and N2O-AMO enrichment system was constructed.The specific AOA groups of mangrove sediments were enriched and purified to explore their physiological properties.New Anammox enrichment reactors were conducted.The pure culture or high abundance enrichment could improve our understanding of new nitrogen cycle microorganisms in mangroves.The main findings are as follows:1.The environmental factors in the vertical sections of mangrove sediments from different habitats in Zhangjiang Estuary were measured and analyzed.Results showed that the properties of surface and deep sediments were significantly different,and the contents of various environmental factors changed with the increase of sediment depth.With the increase of depth,the contents of water content,total carbon(TC),total nitrogen(TN),NH4+,Fe2+,salinity,redox oxidation-reduction potential(Eh)and SO42decreased,while the contents of NO2-,carbonyl sulfide(COS),total sulfur(TS)and pH increased.Low Eh and high concentration of various electron acceptors(such as NO2-,SO42-,Fe3+)reflected the strong reducibility of mangrove sediments.The contents of TC,TN and TS confirmed that the mangrove sediments in Zhangjiang Estuary were carbon and sulfur rich but nitrogen deficient.It provided the material basis for the active metabolism of carbon,nitrogen,sulfur and their coupling in mangrove sediments.In addition,the contents of H2S,CO2 and CH4 didn’t change with the depth,but the extremely high concentrations hinted that mangrove sediments were important sources of H2S,CO2 and CH4.Surprisingly,the low N2O content in mangrove sediments hinted that mangroves were important N2O sinks.2.Based on 13CH4 isotope tracing experiments,it was the first time that N2O-AMO activity was found to decrease along with the sediments depth in Aegiceras corniculatum(AC)habitat in Zhangjiangkou mangrove wetland,with potential rates of 14.3-40.9 nmol 13CO2·g-1·d-1.Correlation analysis showed that the activity was significantly positively correlated with salinity,Eh,S0,NC10 phylum abundance,TC,N2O,SO42-and TN,while negatively correlated with depth,pH and NO2-.Simultaneous removal of CH4 and N2O was found in enrichment cultures derived from AC habitat sediments.The subsurface sediments(10-20 cm)enrichment cultures possessed higher AMO activity than that in the surface(0-10 cm)sediments.It was found that the the abundance of NC10 phylum overlapped with N2O-AMO activity,suggesting that N2OAMO might be drove by NC10 phylum.3.In order to further explore the physiological characteristics of AOA that contributed to the main nitrification activity in mangrove sediments under artificial nitrogen input.The enrichment of marine and terrestrial mangrove AOA was initiated by different pH media with marine or freshwater salinity.The enriched marine and terrestrial AOA were Nitrosapumilus and Nitrosophaera,respectively,which showed a tendency to alkaline or acid environment,with relative abundance of 22%and 45%,respectively,after 30 days initial enrichments.After 170 days of subculture,the ammonia oxidation activity of AOA increased by 7 times,while the relative abundance only increased to 48%,and another AOA group,Nitrosocosmicus,appeared.Finally,AOA were purified by filtrations,multiple antibiotics treatments and two times of endpoint dilutions.Absolute quantitative PCR verified that the purity of purified AOA culture reached 99.98%.Its 16S rRNA and amoA genes were 98.24 and 93.83%similar to the soil AOA model strain Nitrososphaera viennensis EN76,respectively.The optimal initial NH4+concentration of AOA was 1 mM,and the oxidation of ammonia nitrogen was completed after 7 days.Compared with N.viennensis EN76,this AOA strain was more tolerant to ammonia,and its activity was completely inhibited by 10 mM initial NO2-.Phylogenetic analysis and specific physiological properties indicated that it should be the representative strain of mangrove sediment habitat specific AOA group,temporarily named Candidatus Nitrososphaera mangrovi 86-2.4.To further enrich Anammox,columnar or ampullaceous continuous reactors with salinity of ocean(3.3%),sediment(2%)or fresh water(0.2%)were conducted.After nearly 180 days,the freshwater and marine salinity enrichment reactors only showed denitrification activity,while the sediment salinity columnar(SeFCR)and ampullaceous(SeFBR)reactors showed simultaneous removal of NO2-and NH4+after several activity fluctuations,with the ratios of 1.50 and 1.32,respectively,showing stable anammox activity.Compared with the nitrogen removal efficiency(6.97 mgN·L-1·d-1)in the sequencing batch reactor(SBR),the nitrogen removal efficiency of SeFBR and SeFCR increased to 10.10 and 29.21 mg-N·L-1·d-1,respectively,which were 1.5 and 4.2 times of that in SBR.Through high-throughput sequencing,it was found that only Scalindua genus anammox bacteria existed in the inoculum and the non-woven fabric fragments,and no anammox sequence was found in the washout fluid of the reactor.Therefore,the activity of anammox was further improved in the constructed continuous non-woven reactors.