Nitrogen Retention Mechnism Of Microbial Agents In Swine Carcass Composting

Thermophilic composting is effective for the recycling of livestock and poultry carcasses as useful resource.Microorganisms are the main performers of material decomposition during composting,organic nitrogen from livestock and poultry carcasses can be converted into inorganic nitrogen that can be used by plants.In carcass composting piles,decomposition efficiency is low due to high protein and fat content in animal carcasses,so bacterial agents with high lipase and protease activity were inoculated into swine carcass composts to improve composting efficiency in previous experiment,but there was much nitrogen lost in the process.Nitrogen loss is a common phenomenon in the process of waste composting,it is an important means to control nitrogen conversion by adding exogenous nitrogen transforming bacteria.However,compared with manure,sludge and other organic waste composting,the uniformity of composting materials of animal carcasses is poor,and the dominant microbial species of composting are different due to the difference of pile matrix.At present,there are few reports on specific nitrogen retaining bacteria for animal carcass composting,and the mechanism of nitrogen retention is not clear.In this study,nitrogen transformation bacterial strains were screened from previous swine carcass composts,the nitrogen conservation mechanism of bacterial agents in dead pig tissues and sawdust composting system was explored through the changes of microbial diversity and nitrogen transformation functional genes during the composting process;Taking the key dominant strain obtained from composts as the research object,whole genome sequencing datas and different nitrogen metabolism pathways of strain in different nitrogen source media were analyzed to investigated the nitrogen transformation mechanism of the key dominant strain.The main results are displayed below:1.The effect of nitrogen transformation bacterial agent on nitrogen loss in swine carcass composting（1）The isolation and screen of nitrogen transformation bacterial agent.Three ammonifying bacterial strains,one nitrite bacterial strain and one nitrogen fixing bacterial strain were isolated and screened from previous swine carcass composts by specific medium.Based on the 16S rRNA sequence alignment result,3 ammonifying bacterial strains were identified as Bacillus cereus,Pseudomonas donghuensis and Bacillus licheniformis,1 nitrite strain and 1 nitrogen-fixer were determined as Klebsiella pneumoniae and Pseudomonas aeruginosa,respectively.（2）The effect of nitrogen transformation bacterial agent on physiochemical parameters in swine carcass composting process.The swine carcass composting system was established with sawdust and carcass tissue,which was divided into inoculant group（AM）and control group（CK）.In the AM group,ammonifiers were inoculated at the early stage of composting,and a nitrifier and a nitrogen fixer was inoculated at the middle stage of composting.The whole composting process lasted for 181 days,and experienced mesophilic period,thermophilic period,cooling period and mature period.The composting piles were turned on 92d.The highest temperature in the thermophilic period was higher than 57℃ in AM group and CK group,but the thermophilic period（>50℃）in the AM group was longer than that in CK group for 3 days.The total nitrogen content of composting product in AM group was significantly higher than that in CK group（P<0.01）.Compared with CK group,the ammonium nitrogen content in AM group increased significantly（P<0.05）after adding nitrite bacteria and nitrogen fixing bacteria in the middle stage of composting.（3）The effect of nitrogen transformation bacterial agent on microbial diversity and richness in swine carcass composting process.Amplicon sequencing results showed that Proteobacteria（62.7%）,Actinobacteria（23.4%）,Bacteroidetes（7.6%）and Firmicutes（4.1%）were the four most dominant phyla during the composting process.In AM group,the addition of ammonifying bacteria in the early stage of composting increased the diversity and richness of indigenous microorganisms,while the addition of nitrite bacterial strain and nitrogen fixing bacterial strain in the middle stage decreased the diversity and overall richness of microorganisms,but they increased the overall abundance of the top 20 dominant microorganisms.The correlation analysis between microbes and physicochemical indexes showed that Brevibacterium,Streptomyces and Ochrobactrum had a significant（P<0.05）and positive correlation with total nitrogen and ammonium content in both groups.Sphingobium,Sphingomonas,Rhizobium and Dongia were significantly（P<0.05）and negatively associated with total nitrogen and ammonium nitrogen content in both two groups.（4）The effect of nitrogen transformation bcatrial agent on nitrogen transformation in swine carcass composting process.Absolute quantitative PCR was used to detect the abundance changes of nitrogen conversion genes.Compared with CK group,the abundance of organic nitrogen degradation related genes aprA and nitrogen fixing gene nifH were significantly（P<0.01）increased in AM group at the early stage of composting,and the denitrification genes nar G,nirS,nirK,nor B and nos Z were significantly（P<0.05）increased at the middle stage of composting,while the abundance of Anammox 16S rDNA gene was significantly（P<0.01）inhibited.（5）The key nitrogen transformation bacterial strain in swine carcass composting.The microbial composition of the compost samples at the species level was determined by 16S rDNA sequencing technology.Pseudomonas aeruginosa had significantly（P<0.05 or P<0.01）positive correlation with total nitrogen and ammonium content after inoculating nitrite strain and nitrogen-fixing strain.Therefore,it was considered that Pseudomonas aeruginosa was the key dominant strain in the accumulation of total nitrogen and ammonium nitrogen during composting process,and it was named Pseudomonas aeruginosa LG2-2,the storage number was:CCTCC M2021366.2.Study on nitrogen transformation mechanism of Pseudomonas aeruginosa LG2-2（1）The analysis of nitrogen transformation function of Pseudomonas aeruginosa LG2-2.The whole genome of Pseudomonas aeruginosa LG2-2 was sequenced,KEGG results showed that there were some genes related to organic nitrogen degradation,denitrification,ammonia assimilation and dissimilatory nitrate reduction to ammonium（DNRA）.Detection of enzymes activity by Elisa kit showed that the strain also had Ammonia monooxygenase AMO and Hydroxylamine oxidoreductase HAO,indicating that the strain also performed nitrification.（2）Transformation pathway of organic nitrogen by Pseudomonas aeruginosa LG2-2.In the medium with organic nitrogen source,organic nitrogen was converted to ammonium by Pseudomonas aeruginosa LG2-2.A small amount of nitrate mixed in the culture medium was converted to ammonium by DNRA reaction.One part of the ammonium nitrogen was transformed into nitrogen-containing gas through short-cut nitrification and denitrification,and the other part was absorbed by bacteria through ammonia assimilation.（3）Transformation pathway of nitrate nitrogen by Pseudomonas aeruginosa LG2-2.Under the condition that nitrate was the sole nitrogen source,K¹⁵NO₃was used to investigate the nitrate transformation pathway,the results showed that the transformation pathway of nitrate included DNRA,denitrification and amminia assimilation.Quantitative analysis of nitrogen transformation functional genes showed that under the condition of high nitrate concentration,one part of nitrate was converted to N₂ through denitrification,the other part was reduced to ammonium,and the ammonium was assimilated by cells.After nitrate concentration declined to be low level,ammonium produced from dissimilatory nitrate reduction was converted by short-cut nitrification and denitrification and ammonia assimilation.（4）Transformation pathway of nitrite nitrogen by Pseudomonas aeruginosa LG2-2.In the nitrite medium,the conversion rate of nitrite in the medium was 100%,in which 80.99%of nitrite was converted to nitrogen-containing gas through denitrification,and the rest was converted to ammonium,the ammonium could be assimilated by the strain LG2-2.（5）Transformation pathway of ammonium nitrogen by Pseudomonas aeruginosa LG2-2.Under the condition of ammonium salt as the only nitrogen source,¹⁵N stable isotope labeling technique and quantification of enzyme activity and nitrogen transformation gene was used to investigate the ammonium transformation pathway,the results showed that ammonium nitrogen could be transformed by short-cut heterotrophic nitrification and aerobic denitrification and ammonia assimilation.The strain LG2-2 could convert ammonium to nitrogen gas through short-cut nitrification and denitrification in high ammonium concentration condition,while the strain LG2-2preferred to uptake ammonium nitrogen through assimilation pathway in low ammonium concentration condition.（6）Transformation pathway of ammonium and nitrate nitrogen simultaneously by Pseudomonas aeruginosa LG2-2.In the SND medium with ammonium and nitrate,the strain LG2-2 could convert ammonium and nitrate simultaneously.According to the quantitative results of nitrogen transformation genes,it was speculated that nitrate was transformed through denitrification,and ammonium was transformed through shortcut nitrification and denitrification and ammonia assimilation.After ammonium and nitrate concentration declined to the lowest level,the strain LG2-2 uptaked nitrogen mainly through nitrate dissimilatory reduction to ammonium and ammonia assimilation.（7）Transformation pathway of ammonium and nitrite nitrogen simultaneously by Pseudomonas aeruginosa LG2-2.When the nitrogen sources in the medium contained ammonium and nitrite simultaneously,nitrite was converted mainly by denitrification,and ammonium nitrogen was converted mainly by ammonia assimilation.When the inorganic nitrogen in the medium was completely transformed,the ammonium nitrogen and total nitrogen content began to increase with the decrease of biomass,which were related to the degradation of dead bacteria.（8）Transformation pathway of ammonium,nitrite and nitrate nitrogen simultaneously by Pseudomonas aeruginosa LG2-2.When the nitrogen sources in the medium contained nitrate,ammonium and nitrite simultaneously,nitrate and nitrite were transformed mainly by denitrification and ammonium was transformed mainly by assimilation.The utilization priority of three nitrogen resources by the strain LG2-2was ammonium>nitrite>nitrate.（9）The effect of nitrite on nitrogen transformation rate of Pseudomonas aeruginosa LG2-2.When the strain LG2-2 was cultured in the medium containing mixed nitrogen source,nitrite could promote the transformation of ammonium and nitrate.The expression level of gene napA and nirS and NIR enzyme activity decreased as the nitrite concentration increased.It was speculated that high concentration of nitrite could inhibit denitrification genes expression and enzymes activity,and prolong the lag period of strain LG2-2.（10）The antibacterial ability of Pseudomonas aeruginosa LG2-2.Antibacterial experiment showed that Pseudomonas aeruginosa LG2-2 could suppress the growth of gram-positive and gram-negative bacteria.Therefore,Pseudomonas aeruginosa LG2-2 might be the main reason for the decrease of microbial diversity and abundance in the composting process,this strain might also inhibit the growth of anammox bacteria.Main conclusions:（1）The nitrogen transforming microbial agents could change the diversity and richness of endogenous microorganisms in the composting process.Ammonifiers had increased composting temperature and prolonged thermophilic phase.Nitrite bacterium and nitrogen fixer increased the production of total nitrogen and ammonium.（2）Pseudomonas aeruginosa LG2-2 was the key strain to regulate the nitrogen transformation in composting process,which mainly possessed three nitrogen conversion pathways:short-cut heterotrophic nitrification-aerobic denitrification,DNRA and ammonia assimilation.In the composting process,the inoculated Pseudomonas aeruginosa LG2-2 coud promote the conversion of organic nitrogen to ammonium nitrogen,and promote the conversion of nitrate and nitrite to ammonium through DNRA pathway.Pseudomonas aeruginosa significantly inhibited the growth of anammox bacteria and further promote the accumulation of ammonium nitrogen.