| ObjectiveThe purpose of this study was to investigate the bacteria species,abundance and distribution of sludge under different spatial distributions in the anaerobic hospital sewage treatment plant of a secondary hospital in Henggang,Shenzhen.The changes in bacterial community composition of drainpipe wall-attached sludge and floating sludge samples at different sewage depths in the anaerobic reaction tank were monitored.To establish a rapid method for identifying bacteria and monitoring microorganisms.Evaluate the sewage treatment capacity of the secondary general hospital sewage treatment plant.MethodsUsing an improved mechanical extrusion sampling device,15 sludge samples were sampled at multiple points in the anaerobic hospital wastewater plant located in Henggang,Shenzhen on April 11,2022.Combined with 16S rRNA gene V3-V4 region amplicon high-throughput gene sequencing technology,the species classification and the colony distribution analysis of sludge samples were performed.The hospital is equipped with an embedded sewage treatment system,the anaerobic reaction tank adopts an improved anaerobic-anoxic-oxidation(AAO)wastewater treatment process,the reaction tank is equipped with two inspection wells according to the distance to the inlet of the influent sewage,the sampling personnel adopted secondary protection,and by lifting the maintenance hole of the closer inspection manhole cover which is divided by the closer distance to the inlet of influent sewage in the anaerobic tank,and the inspection manhole cover was raised.The liquid level in the pool is 3 meters below the inspection manhole cover.At the inlet inside the anaerobic tank drainpipe,which was regarded as the initial point of the circumferential plane of the drainpipe wall in the anaerobic tank,the circular wall plane could be divided into 12 equal points with a 30degree angle per division.The sampling angle could be calculated by multiplying the value of the sampled specimen site name by 30 degrees.A total of 15 samples were collected and divided into A,B and C groups according to the position of the space plane where the samples were collected.The samples’names corresponded to the values of the corresponding sampling site and the sample composition of each group was labelled as follows:Six samples were collected by rotating the sewage pipe wall clockwise around the flat liquid surface,namely 1,2,3,10,11,and 12,divided into the liquid-level-group(Group A).Similarly,6 samples were collected from 1 meter below the liquid level and labelled 4,6,8,13,14,15,and divided into subsurface-sewage-group(Group B).The bulking floating sludge samples were labelled 5,7 and 9 respectively and were divided into floating-sludgegroup(Group C).Modified sampling devices were used throughout the entire sampling process.After sampling at each sampling point,the disposable sampling tube in the sampler was removed and replaced with a new sterile sampling tube,to achieve repeated sampling.After each specimen collection,they were transferred to the corresponding labelled sterile centrifuge tubes,all of which were placed in double-layer sealed plastic tubes and stored in a refrigerated transport box at-20℃ and sent to the laboratory for DNA extraction.Total DNA was extracted according to the iPure(?)DNA Extraction Kit instructions and 15 sludge samples were entered PCR amplification of the V3-V4 region of bacterial 16S rRNA gene,primer 341F/805R;PCR Amplification Using 16S r DNA V3-V4 Region Generic Citation:341F:(5’-CCTACGGGNGGCWGCAG-3’),805R:(5’GACTACHVGGGTATCTAATCC-3’)PCR Amplified using Illumina’s Mi Seq PE300 platform with 2%PCR products Electrophoresis detection of agarose gel(Shanghai Sangon Biotech Co.,Ltd.).The raw data obtained has been uploaded to the NCBI Sequence Read Archive(SRA)database(accession number:PRJNA935118).Cutadapt was used to remove primers and filter the original sequence,and the sequence was introduced into QIIME2 for DADA2 clustering and unifying rarefaction.16s rRNA gene V3-4 region was calculated by rescriptclassifying-sklearn,and annotated to the Silva database(Silva 138.1).The a diversity index,β diversity index and relative abundance at phylum and genus levels were calculated by QIIME2 and R software.The data were extracted by QIIME2R plug-in and imported into the R software package to evaluate the alpha diversity of the bacterial community and annotate the species.ResultsA total of 989951 valid sequences were obtained,belonging to 75 phyla,190 orders,408 orders,602 families,917 genera and 1850 species.After filtering and removing chimeras,409705 high-quality sequences were obtained,and the sequences with abundances less than 2 were unified and removed,and the sequences were statistically identified,and a total of 67 phyla,165 orders and 317 were identified Order,449 families,644 genera,and 1132 species.This study identified the dominant phylum and genera of bacteria in three groups of specimens.Among them,the top three in the dominant phylum of Group A were 27.32%of the Proteobacteria;Acidobacteriota phylum was 25.80%;the Bacteroides phylum was 9.27%.The top three dominant bacteria in Group B were 16.43%in the Proteobacteria phylum;the Bacteroides phylum was 14.43%;the Chloroflexi phylum was 11.62%.The top three dominant bacteria in Group C were 29.33%in the Acidobacteriota phylum;the Proteobacteria phylum was 27.53%;the phylum Bacteroides was 8.16%,and the dominant genus with the highest relative abundance in Group A was RB41,accounting for 7.54%;The dominant genus with the highest relative abundance in Group B was Methanosaeta,accounting for 8.99%;The dominant genus with the highest relative abundance in Group C was Sphingomonas,accounting for 7.33%.Pseudomonas was the dominant bacteria in samples 2,5,9 and 10,and its corresponding relative abundance was 1.26%,1.18%,1.16%and 1.05%,respectively.There was a significant difference in the abundance of Pseudomonas among the three groups(F=3.89,df1,df2,df2,12,P<0.05).The Chao1 indices of Group A,B,and C were 372.58±55.85,1132.92 ±55.13,and 363.87±76.91,respectively.The Shannon indices of these groups were 7.90±0.26,8.74±0.12,and 7.87±0.37,respectively.The Chao1 indices and Shannon index of Group A,B and C were tested by the SNK-q test,and there was no significant difference between Group A and Group C.However,there were significant differences between Group B and Group A and C.ConclusionsThe Chao1 index and Shannon index of sludge samples in Group B with deeper settling positions were significantly higher than those of sewage liquid level sludge samples and floating sludge samples.Compared with the upper sludge,the deeper the settling position is,the better the settling performance is,and the diversity and evenness of the bacterial community are higher.There is no significant difference in the composition of bacteria between floating sludge and liquid-level sludge at the phyla level,suggesting that they are homologous.With the bulking of floating sludge,the Acidobacteriota phylum becomes dominant,with Bacteroides as the main composition.The dominant Pseudomonas was not detected in the samples below the sewage level,but the detection rates of Pseudomonas in the floating sludge group and the sewage liquid plane group were 66.67%and 33.33%respectively(the relative abundance was greater than 1%as positive).The distribution of Pseudomonas in the three sampling spaces was significantly different.The results show that the number of biological sequences in the sludge samples collected by the sampling device is all effective sequence numbers,and the amount of effective sequence data is reliable,so it can be combined with 16Sr RNA gene amplification high-throughput sequencing analysis technology to analyze the species composition and diversity of sludge samples in the anaerobic biological reaction tank,especially suitable for microbial monitoring of floating matter.The improved sampling device and highthroughput sequencing technique of 16s rRNA gene V3-V4 region can be extended to evaluate the drug resistance of antibiotic-resistant bacteria in sludge and to detect the removal rate of drug-resistant bacteria regularly. |
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