Responses Of Microbial Communities In Lake And Soil Environments To Inputs Of Nitrogen And Phosphorus

Microorganisms are involved in biogeochemical cycles of various earth elements.They are also playing important roles in pollutant biodegradation,plant growth,and ecosystem maintenance.Iron-reducing bacteria(FeRB)are well known for its ability of extracellular electron transfer.They utilize Fe(?,Mn(?),Azo dye,and nitrobenzene as electron acceptor,and reduce them,thus showing great potential in metal cycling and pollutant degradation.Two of the important representatives are Geobacter and Shewanella.With the development of industry,agriculture,and urbanization,excessive nitrogen,phosphorus and other nutrients are released into the environment and accumulate in soils and waters.The eutrophication in lakes,rivers,and other aquatic ecosystems become critical.These changes exert great influences on the microbial community therein.In order to control the eutrophication aggravation and achieve sustainable development of agriculture,it is of primiary importance to understand the responses and mechanisms of microbial communities to eutrophication and nitrogen fertilization.In this work,the community structures of FeRB,represented by Geobacter and Shewanella genera,in eutrophic aquatic ecosystems were investigated.The responses of the microbial communities to nitrogen fertilization in agricultural soils and mechanisms of responses were also explored.The main contents and results of this dissertation are as follows.1.Design and application of new specific primer sets to Shewanella genus.Shewanella,as a model genus of FeRB,shows great potential in bioremediation and electricity generation.Primers with better accuracy and higher coverage are essential to evaluate their roles in various environments,in consideration of the increasing number of Shewanella species.First,de-novo specific new primer sets(640F/815R and 211F/815cR)to Shewanella genus were designed based on the conserved Shewanella 16S rRNA gene sequences.Second,the newly designed primer sets were compared with previous primer sets(783F/1245R,211F/1259R and 120F/220R)in specificity and coverage by in silica tests and clone library analyses of natural samples.Both the results of virtual and actual tests indicated that the newly designed primer sets 640F/815R and 211F/815cR outperformed previous primer sets in specificity and coverage for Shewanella species.When the new primer sets were applied to investigate the distribution pattern of Shewanella in natural environments,more abundant and diverse Shewanella species were found than previous reports in freshwater ecosystems.Moreover,Shewanella species in freshwater ecosystems were different from those in marine environments.The abundance and diversity of Shewanella species in wastewater treatment plants(WWTPs)were largely affected by the process and operating conditions.Pearson correlation analysis results indicated that the abundance of Shewanella was significantly correlated with mixed liquor suspended solids(MLSS)and oxidation-reduction potential(ORP)in WWTPs.However,the abundance of Shewanella was significantly correlated with nutrients level in sediment samples.These results reveal that the distributions of Shewanella in different environments were affected by different environmental factors,which need to be further verified by more investigations and microbe-isolation studies.In a conclusion,the new primers facilitate the investigation of the distribution,abundance,and diversity of Shewanella genus and evaluation of their potential roles in diverse natural and engineered environments.2.Distribution,abundance and diversity of FeRB in eutrophic Chaohu Lake sediments.FeRB are widely distributed in various anaerobic environments.However,the abundance and community diversity distribution pattern in eutrophic freshwater ecosystems remain largely unknown.In this research,the abundance and diversity of Geobacter,Shewanella,and total FeRB in eutrophic Chaohu Lake sediments were investigated through quantitative PCR,clone library analysis and high-throughput sequencing.FeRB were found to be widely distributed in the sediments of the heavily polluted Chaohu Lake ecosystem.The abundance of Geobacter,Shewanella,and total FeRB were higher in the heavily polluted zone than in the mildly polluted zone.The abundance of Geobacter was at least one order of magnitude higher than Shewanella in cold seasons.The biodiversity of Geobacter was much higher than that of Shewanella in the lake sediments.Three Shewanella-related operational taxonomic units were detected while sixty-one Geobacter-related operational taxonomic units were grouped into three phylogenetic clades.Desulfuromonas and Geobacter were identified as the main members of FeRB in the lake sediments.Pearson correlation,CCA and RDA analyses results indicated that the abundances and community structures of Geobacter,Shewanella,and total FeRB were significantly correlated with the carbon,nitrogen,and phosphorus contents.rather than the iron contents.Nutrients were likely to be the most important factors governing the distribution,abundance,and biodiversity of FeRB in the highly eutrophic freshwater lake.The high relationship between the abundance and diversity of FeRB and eutrophication degree reveals that FeRB may have a great potential in alleviating the eutrophication and contamination of aqueous environments.3.Impacts of nitrogen fertilization and pH change resulting from fertilization on bacteria and fungal communities in Switchgrass field soil.Many studies have been conducted to analyze the influences of mineral nitrogen on microbial communities.However,the influences and mechanisms of bacterial communities and fungal communities to elevated nitrogen fertilizer level and the change of pH remain unclear.Thus,two Switchgrass N Rate Experiments,located at the KBS(Michigan,USA)and ARL(Wisconsin,USA),were conducted to explore the influences.The comparison between short-term fertilization at ARL and long-term fertilization at KBS made it easier to distinguish the effect of pH from the effect of nitrogen fertilizer.High-throughput amplicon sequencing of 16S rRNA gene and ITS2 gene was applied to comprehensively analyze the bacterial and fungal communities respectively.At ARL site,there was no significant change of soil pH,and the diversities and compositions of bacterial community were very stable across the nitrogen fertilizer gradient(0,56,196 kg N/ha).At KBS site,we found that bacterial diversities,structures,and compositions were strongly affected by interactive effects of high fertilizer(196 kg N/ha)and low soil pH.The low fertilizer loading(56 kg N/ha)without significantly changed pH showed no significant effect on the bacterial communities.Therefore,pH was the key factor to shift bacteria communities,which was consistent with previous studies.By comparison,the fungal communities were less affected by pH,wheras the fungal community structure and composition were more susceptible to nitrogen fertilizer.This work provided a deeper understanding of the response mechanisms of the microbial communities to nitrogen fertilization and useful information for the sustainable development of agriculture.4.Impacts of nitrogen fertilization on the microbial community at different Switchgrass growth stages,and reveal the microbial metagenome and microbial molecular ecological networks.Previous studies focused on the response of microbial community structure and composition to nitrogen fertilization,while the effects of nitrogen fertilization on microbial metagenome and microbial molecular ecological networks need to be further explored.Information about the effects of plant developmental stage on microbial communities under the treatment of nitrogen fertilization is also lacking.We thus tried to answer these questions in the present study.By combining high-throughput amplicon sequencing,shotgun metagenome sequencing,and microbial network analysis,we found that microbial community structures were relatively stable through the growth of Switchgrass.There were no significant differences on microbial communities at different developmental stages at the same nitrogen fertilizer loading.However,a high fertilizer loading(196 kg N/ha)increased the variation of microbial communities between the growth stages of Switchgrass at KBS site.Microbial network analysis indicated that microbial communities were more resistant to changes in low nitrogen fertilizer loading(56 kg N/ha),while the microbial communities in high fertilizer rate were more susceptible to disturbance.We found that the interactions within fungi reduced with increased nitrogen fertilizer loading.The negative interactions within the microbial community were also reduced,resulting in a reduction of overall network complexity.Shotgun sequencing data annotation indicated that a high nitrogen fertilizer loading promoted many metabolic processes,especially carbohydrate-related metabolism at the KBS site.However,the high nitrogen loading also inhibited many functional genes,particularly signal transducton genes.Annotation results of nitrogen cycling genes indicated that a high nitrogen fertilizer loading inhibited nitrogen fixation,promoted the ammonia-oxidizing genes of archaea,and showed no significant effects on the genes involved in the denitrification process.Thus,the nitrogen fertilization loading affected microbial communities in various ways.Variations in community structures and physiological metabolisms may cause changes in the microbial interaction network,thereby weakening the resistance of microbial ecosystem to external environmental changes.This work helps us to deeply understand responses of microbial communities to elevated nitrogen levels and its mechasism and facilitates the establishment of sustainable agriculture.