Comparsion Of Microbial Communities Diversity In Three Sediment With BIPES

Most micro-organisms as the community structure widespread distrbute in the natural environment, they drive basic elements of life, biogeochemical cycling, and degrate of various pollutants. Sediment is a special realm in aquatic environments with high microbial diversity. Sediment is a special realm in aquatic ecosystem. The biomass and taxa richness of microbes in sediment are much higher than that in the corresponding water bodies. For the purpose of understanding the potential effect of climate change to biogeochemical cycle by microorganisms, as well as exploring the microbial resources, scientists have carried out extensive survey about the sediment microbial diversity, especially in marine environments. In addition, rhizospheric and sediment microbes have been suggested to be the major biological components that contribute to the productivity of intertidal mangrove ecosystem. many groups including us have isolated a large number of xenobiotic-degrading bacteria from intertidal wetland sediments, which aroused the scientist’s interest in looking for microbial resources in the intertidal. Comparing to marine environments, the freshwater sediment has received less attention. Recent meta-analysis showed that the freshwater environments had a higher bacterial and archaeal diversity in inland freshwaters than the marine environments. Nevertheless, PCR conditions, sequencing methods, and16S rRNA short variable regions being sequenced might affect the meta-analysis for a-diversity, In the present study, we determined a total of2.3million tags from three types of sampling sites of freshwater sediment, intertidal wetland sediment, and marine sediment along the Pearl River in south China, and a detailed comparison of the bacterial diversity and indicator bacterial groups in each type of sediments were revealed.ObjectiveComparison of inter-tidal mangrove wetlands, freshwater lake and marine sediments, three types of sediment microbial diversity; through the analysis of intertidal sediments bacteria and archaeal community structure suggest that the intertidal zone has a special ecological system.Material and MethodsA total of35samples were taken from three types of sediments along Pearl River varied from an upstream reservoir (F), a costal intertidal wetland(W), and marine sediment(M) in the harbor in August2009. We used0.25g of each sample for DNA extraction using a PowerSoil DNA kit (MO BIO); used the barcoded967F (CNACGCGAAGAACCTTANC) and1046R (CGACAGCCATGCANCACCT) primers to amplify the bacterial16S rRNA V6fragments; used the barcoded958F (AATTGGANTCAACGCCGG) and1048AR (CGRCGGCCATGCACCWC) primers to amplify the archaea bacterial16S rRNA V6fragments. Amplified PCR products were detect by agarose gel electrophoresis, followed by BandScan scan software to do the relative quantification.then we determine the volume of each sample to add to mixing and by the Invitrogen fluorescence quantitative instrument for absolute quantification. After the above steps, we can determine the volume of each group to sent to sequncing according to the concentration and the sequencing depth which we have planed. Finally, we sent the samples to BGI company for PE75sequencing by the Solexa GAII sequencing instrument. After sequencing, the data analysis pipeline was similar to that reported in our BIPES method paper. Then using the UCHIME for screening chimeras, after the set of quality control steps, we used a Two-Stage-Clustering (TSC) algorithm to cluster tags into OTUs. Finally, the pipeline generated three Excel worksheet which containing these OTUs classified information.Result1We obtained2,318,609reads that spanned the V6hyper-variable region of the16S rRNA gene for the35samples using the BIPES method. The clean sequences were screened for the presence of chimeras using the UCHIME,2,311,761clean reads remained in the final dataset. Finaly, we obtained96037OTUs by TSC. The sequencing depth of per sample range from40000to100000reads, among which W12reached174678reads.Our present result obviously demonstrated that the a-diversity of bacterial community in intertidal sediment was between the other two. Rarefaction curves with normalized and unnormalized sequence numbers showed that the upstream freshwater sediment had the steepest rarefaction curves with the highest taxa richness, while the marine sediment had the lowest ones. We used the relative abundances of the phylotypes (0.03distance OTU) to analyze the community similarity among the35samples by primer6.0. The clustering analysis separated the communities into two main groups, and the PCA analysis was similar to clustering result.According to the phylum level distribution pattern, the intertidal sediment community showed an intermediate type between the other two, which was similar to that of the a-diversity result. Then plotting all35samples using the principal component analysis (PCA), the three types of communities could be separated well. indicating that the three sediments had significantly different bacterial phyla. For instance, three Proteobacteria groups (γ-,ε-,δ-and NA) were enriched in the marine and intertidal sediments, while the Acidobacteria and Nitrospira were enriched in freshwater sediment.The genus level distribution pattern. Within the top ten genera in each type of sediment, the intertidal sediment played as a transition between the freshwater sediment and marine sediment, which was similar to above results. The PCA result showed that samples from each type of sediment clustered much tighter than that in the phylum level result. Moreover a large number of genera related with sulfur cycling (Desulfobulbus, Desulforhopalus, Desulfobacterium, Desulfuromonas, Desulfofustis) were found to be enriched in the intertidal wetland sediment. Further more, within the five determine environmental factors, the three factors related with high nutrient concentration (TOM, TKN and TP) were correlated with the specific bacterial community living in the intertidal wetland.We divided OTUs according to their abundance level and performed PCA analysis at different abundances. From the most abundant (OTUs with abundances higher than0.1%) to the rarest (OTUs with only2reads in all35samples) OTUs, bacterial communities from the three types of sediments could be obviously separated, demonstrating that bacterial communities from the dominant to the rarest were specific to their habitats. Meanwhile, we could observe that the higher the OTU abundance, the higher the PC1+PC2explanation, indicating that bacteria at different abundances might have different level of selection from environmental factors.The LEfSe statistical analysis reveal Bacterial groups with statistical differences in three types of freshwater, intertidal, and marine sediments. The freshwater sediment with Acidobacteria, Nitrospira, Verromicrobia, α-and β-Proteobacteria as indicators. The intertidal sediment with high primary productivity and eutrophic microbes, such as Chloroflexi, Bacillariophyta, γ-and ε-Proteobacteria, Bacteroidetes, Firmicutes, and Actinomycetales. The marine sediment with γ-and some8-Proteobacteria orders, which were mainly involved with sulphate reduction in anaerobic conditions.2We obtained839,616reads that spanned the V6hyper-variable region of the16S rRNA gene for the13samples using the BIPES method. After using the UCHIME screened for chimeras837,041clean reads remained in the final dataset. With an average of64388reads per sample range from47583to80427reads. After that, we normalized all samples to40000reads by random resample for the following OTU analysis. Rarefaction curves with normalized showed that the mature mangrove sediment had the steepest rarefaction curves with the highest taxa richness, while the mudflat sediment had the lowest ones. We used the relative abundances of the phylotypes (0.03distance OTU) to analyze the community similarity among the13samples. The clustering analysis separated the communities into two main groups Group1contained communities from the mudflat sediment, the Spartina sediment, and the mangrove edge sediment; Group2contained the bulk and rhizosphere sediment from the mature mangrove stand. The two groups were further divided into five subgroups containing samples from each type of sediment. The clustering patterns at all four different abundance levels (1000and up,100-999,10-99, and2-9) were generally the same as that of the total OTU datasetAccording to Phylum distribution, there were16phyla or proteobacterial classes that were found to have an average abundance of greater than1%across all of the samples. Three phyla, γ-Proteobacteria,δ-Proteobacteria, and Planctomycetes, composed more than50%of the proteobacterial classes in every type of sediment. By comparing the group1and group2communities,6of the16most abundant phyla showed statistical differences (t-test, P<0.05). namely Planctomycetes, Firmicutes, Chlorobi,s-Proteobacteria, Nitrospira,and LentisphaeraeThe most abundant OTUs in the wetland sediment. In the analyzed wetland sediment,9of the10most abundant OTUs were from proteobacteria, particularly y- and δ-Proteobacteria. Statistical analysis showed that8out of the10most abundant OTUs had significant differences among the five types of sediment habitats (one-way ANOVA test,P<0.05).OTUs with significant differences among the five habitats. We divided OTUs into four different abundance levels as described in the previous section.And the OTU abundance data for the13samples were normalized using the mean and the standard deviation of the OTU abundance data in each row (Value=[(Value)-Mean(Row x)]/[Standard deviation(Row x)]. The heatmap revealed OTUs with significant differences among the five types of analyzed sediment (P<0.05). At every abundance level, we discerned that OTUs showed higher relative abundances in certain sediment types, indicating that these bacterial groups may be selectively enriched in specific habitats.OTUs in the major phyla with significant differences between the two groups (Student’s t-test, P<0.05) revealed different habitats have different selective advantage.OTUs specific to the mudflat and rhizosphere sediment. The most prominent bacterial groups were Desulfobacteraceae, Desulfuromonas, Acidobacteria, Clostridiales, and Ectothiorhodospiracea in mudflat; while Planctomycetaceae, Hyphomicrobium (Rhizobiales), Pseudomonadales, Nitrospira, and Caldilineaceae rich in d rhizosphere sediment.3We obtained129,830reads that spanned the V6hyper-variable region of the16S rRNA gene for the12samples using the BIPES method. After using the UCHIME screened for chimeras129,480clean reads remained in the final dataset. With an average of10,790reads per sample range from2,088to30,084reads. Finaly, we obtained3404OTUs by TSC.Rarefaction curves with normalized and unnormalized sequence numbers showed that the seaward edge of mangrove stand sediment had the steepest rarefaction curves with the highest taxa richness, while the mature mangrove sediment had the lowest ones. The phenomenon was opposite to bacterial a-diversity.β-diversity result of the archaea in the Intertidal sediment showed that the Group F samples clustering close, and the remaining three groups of sample cluster is more dispersed, which may be caused by our sequencing depth not enough or samples difference vary little.According to the phylum level distribution pattern, the archaea community distribution is consistent at four samples sites in Intertidal wetlands, mainly composed of Euryarchaeota and Crenarchaeota. Moreover there is a small part of Nanoarchaeota exists in group M and F.According to the class level distribution pattern, Crenarchaeota has four taxa and the domain class is Thermoprotei; while Euryarchaeota has eight taxa, Methanomicrobia is the domain class, followed by Methanobacteria, Thermoplasmata, Halobacteria, TMEG, Methanococci, Thermococci and MGⅡ.Conclusion1Freshwater sediment has the highest bacterial richness and eveness, the intertidal was the medium one, while the marine sediment was the lowest.2The intertidal sediment covered with the plants bacterial a diversity higher than without plants covered.3On the contratry, intertidal sediment covered with the plants archaea a diversity lower than without plants covered.4The freshwater sediment had the highest diversity, with Acidobacteria, Nitrospira, Verromicrobia, α-and β-Proteobacteria as indicators. The marine sediment had the lowest diversity enriched with γ-and some δ-Proteobacteria orders, which were mainly involved with sulphate reduction in anaerobic conditions.The intertidal sediment had the medium diversity with high primary productivity and eutrophic microbes, such as Chloroflexi, Bacillariophyta, γ-and ε-Proteobacteria. And the archaea microbial community composed by Crenarchaeota (Thermoprotei) and Euryarchaeota (Methanomicrobia).