| The pig breeding industry is the most important industry in China’s livestock and poultry breeding industry.With the rapid development of the industry,a large number of high nitrogen swine wastewater is discharged into the environment,resulting in the increasingly prominent problem of nitrogen pollution in the water body,which has become the bottleneck of the sustainable and benign development of the industry.Excessive nitrogen concentration in water environment will stimulate algae overgrowth,leading to water quality decline,and even lead to the death of fish and other organisms,thereby endangering human health and ecosystem stability,and looking for efficient nitrogen removal technology to treat swine wastewater can alleviate the problem of nitrogen pollution.Biological nitrogen removal technology has the advantages of low cost,high efficiency,and no secondary pollution.With the discovery of microorganisms with different nitrogen removal functions,microbial nitrogen removal technology has been widely used.However,the microbial nitrogen removal process is complex,and the denitrification pathways of different microorganisms are very different.At present,many studies focus on mining efficient heterotrophic nitrification aerobic denitrification microorganisms.However,nitrogen as an essential element for microbial growth,microbial assimilation of nitrogen has been ignored.There are few studies on the synergistic effect between microbial heterotrophic ammonium assimilation and aerobic denitrification,and the nitrogen removal pathway has not been systematically studied from the molecular level;Secondly,heavy metal ions generally exist in wastewater,which will affect microbial nitrogen removal,and there are few studies on the effects of different heavy metal ions on heterotrophic ammonium assimilation aerobic denitrification microorganisms.The three-stage surface laminar flow constructed wetland planted with Myriophyllum aquaticum by the Institute of subtropical agricultural ecology,Chinese Academy of Sciences,located in Changsha County,Hunan Province,has been used to remove ammonia nitrogen from pig wastewater for a long time,and the effect is outstanding.Through N15mass balance analysis,it is found that there are a large number of nitrification and denitrification microorganisms in the Myriophyllum aquaticum wetland,which play a major role in the denitrification process,however,the characteristics and molecular mechanism of these denitrifying microorganisms in the wetland are not clear.If the key high-efficiency functional microorganisms can be screened from the wetland,the nitrogen removal effect of pig wastewater can be further improved.In this paper,an efficient denitrification strain was screened from Myriophyllum aquaticum constructed wetland.It was identified and named Rhodococcus erythropolis Y10.The nitrogen removal characteristics and pathways of strain Y10 were studied from the genomic and transcriptome levels,the nitrogen removal efficiency under the impact of different heavy metal ions,and the mechanism of the effect of heavy metal ions on its nitrogen removal.The following experimental results were obtained:（1）Strain Y10 can effectively remove ammonium nitrogen,nitrate nitrogen,and nitrite nitrogen.The strain Y10 was identified as Rhodococcus erythropolis by morphological,physiological,biochemical,and genetic analysis.The accession number of 16S r DNA was MZ823775.The whole-genome information of strain Y10 was obtained by the third-generation high-throughput sequencing.The length of the whole gene sequence was6839626 bp,among which the lengths of the chromosome sequence and plasmid sequence were 6411255 bp and 428371 bp,respectively,and the GC content was 62.45%and60.38%,respectively.The total length of the coding region in the genome is 6132171 bp,containing 7844 coding genes,with an average length of 781.77 bp,accounting for 89.66%of the whole genome.A total of 7271 functional genes were annotated by COG,of which the genes related to the basic metabolism,cell process and signal transduction,information storage and processing,and only used for function prediction and unclassified accounted for 49.071%,16.985%,16.6%,and 17.343%of the total genes,respectively.A total of8984 genes of strain Y10 were annotated through the GO database.The genes related to biological processes,cell components,and molecular functions accounted for 47.36%,9.38%,and 43.25%of the total genes,respectively.There were 11 gene islands in the strain genome,including 191 coding genes.（2）The KEGG database was used to predict and analyze the nitrogen metabolism pathway of strain Y10.It was found that there were three nitrogen metabolism pathways in strain Y10,including heterotrophic ammonium assimilation,dissimilatory nitrate reduction to ammonium（DNRA）,and aerobic denitrification.Due to the deletion of ammonia monooxygenase encoding gene amo and hydroxylamine reductase encoding gene hao in the genome of the strain,it was speculated that strain Y10 could not perform heterotrophic nitrification.When ammonium was the only nitrogen source,the results of nitrogen balance calculation and key enzyme activity test showed that strain Y10 transformed all ammonia nitrogen into biological nitrogen for its growth through heterotrophic ammonium assimilation,that is,ammonium→biological nitrogen.The influencing factors of heterotrophic ammonium assimilation of strain Y10 were studied.It was determined that the nitrogen removal effect of strain Y10 was the best when trisodium citrate was used as carbon source,the C/N ratio was 15,the initial p H was 7,the temperature was 30℃,rotating speed was 120 rpm.When nitrate or nitrite is the only nitrogen source,strain Y10can carry out DNRA pathway（nitrate→nitrite→ammonium→biological nitrogen）and aerobic denitrification pathway（nitrate→nitrite→gaseous nitrogen）.When ammonia nitrogen and nitrate nitrogen or nitrite nitrogen exist at the same time,strain Y10 can carry out heterotrophic ammonium assimilation and aerobic denitrification at the same time.Increasing ammonia nitrogen is conducive to the heterotrophic ammonium assimilation of the strain to expand the biomass of the strain and promote the aerobic denitrification of nitrate nitrogen or nitrite nitrogen.（3）From the transcriptome level,Rhodococcus erythropolis Y10 cultured under different nitrogen sources（ammonium,nitrate,and glutamine）was used as the experimental object to sequence its transcriptome,and through bioinformatics analysis,to mine differentially expressed genes.Analyze the differential gene expression between ammonia nitrogen group and glutamine group,nitrate nitrogen group and glutamine group,and nitrate nitrogen group and ammonia nitrogen group,and explore the metabolic pathways of ammonia nitrogen and nitrate nitrogen in strain Y10.Some key enzyme coding genes of ammonium and nitrate metabolism in strain Y10 were found:glutamyl ammonia synthase coding gene gln A,glutamate synthase coding genes glt B and glt D,nitrate transporter coding gene nrt,assimilation nitrate reductase coding gene nar B,assimilation nitrite reductase coding genes nir B and nir D,periplasmic nitrate reductase coding gene nap A,dissimilatory nitrite reductase coding genes nir S and nir K,nitric oxide reductase coding gene nor B and nitrous oxide reductase coding gene nos Z.This result verified that there were three nitrogen metabolism pathways in Rhodococcus erythropolis Y10:heterotrophic ammonium assimilation,DNRA,and aerobic denitrification.（4）The effects of four common heavy metal ions Cu（II）,Zn（II）,Cd（II）,and Cr（VI）in aquaculture wastewater on the biological nitrogen removal process of strain Y10 were studied.The experimental data showed that the effects of four heavy metal ions on the aerobic denitrification of strain Y10 were stronger than those of heterotrophic ammonium assimilation.The low concentration（≤0.5 mg/L）of metal ions had little effect on the heterotrophic ammonium assimilation of strain Y10,while the high concentration（>0.5mg/L）of heavy metal ions had an obvious inhibitory effect on strain Y10.With the gradual increase in the concentration of heavy metal ions,the inhibitory degree of heterotrophic ammonium assimilation was gradually strengthened.Heavy metal ions have a greater impact on the aerobic denitrification of strain Y10.In addition to low concentrations（0.5mg/L）copper ions have a certain promotion effect on aerobic denitrification,high concentrations（>0.5 mg/L）copper and all test concentrations of the other three heavy metal ions have different degrees of inhibition on the aerobic denitrification of strain Y10,and the inhibition increases significantly with the increase of heavy metal ions.In addition,the influence mechanism of heavy metal ions on the biological nitrogen removal of strain Y10 was discussed with copper ions as an example.The transcriptome results showed that low concentration（0.5 mg/L）of copper ions could affect the nitrogen removal process of strain Y10 by affecting the expression of related nitrogen metabolism genes in the nitrogen removal pathway,and the aerobic denitrification process of strain Y10 was more sensitive to metal copper ions than the heterotrophic ammonium assimilation process. |
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