The Community Of Denitrifying Bacteria In The Manure Of Meishan Pig And Its Nitrogen Removal Performance

This experiment selects conservation(60±3 days),finishing(170 ± 5 days)and pregnancy(pregnancy 70 ± 4 days)three physiological stages of Meishan pigs were collected every 10 head,fresh manure for composition analysis of different physiological stages of Meishan pig manure physicochemical properties and microbial community structure.On this basis,aerobic and anaerobic enrichment of pig manure were conducted to study the effects of different enrichment methods on the number and structure of denitrifying bacteria in pig manure.In order to further study the performance of denitrification and denitrifying bacteria,the denitrifying bacteria were isolated from pig manure,screening and denitrification ability the strongest strain,add it to the livestock wastewater treatment process,to explore its influence on the removal rate of nitrogen in wastewater treatment process,and finally through the whole genome sequencing technology,in-depth analysis of the strain to promote the mechanism of removal of livestock wastewater treatment process of nitrogen.The main contents are as follows:1 Physical and chemical characteristics and bacterial community structure of swine manure in different physiological stages in MeishanThis experiment selects the nursery phase(60±3 days)and finishing period(170±5 days)and pregnancy(pregnancy 70 ± 4 days)the 10 Meishan pig head,collected fresh fecal samples,analysis of the moisture content,organic matter,pH,total nitrogen,ammonium nitrogen,nitrate nitrogen,volatile fatty acid(VFA),and the use of PCR-DGGE technology to analyze the bacterial flora in feces.The results show that the physicochemical properties of pig manure,pig manure and fattening pigs was significantly higher than that of water content in breeding sows(P<0.05);conservation of pig manure pH was significantly lower than that of sows(P<0.05);fecal total nitrogen,nitrate nitrogen,ammonium nitrogen content in nursery pigs highest finishing pigs of reproductive female pigs lowest(P<0.05);the content of organic matter in the highest finishing pig manure,pig conservation of breeding sows was the lowest(P<0.05);and the organic matter and total nitrogen ratio in pig breeding sows,fattening pig manure of pig dung conservation lowest(P<0.05).The determination results showed that pig manure VFA piglets isobutyric acid content was significantly higher than that of fattening pigs(P<0.05),breeding sows were significantly lower than that of fattening pigs(P<0.05);breeding sows isovaleric acid was significantly lower than that in piglets and fattening pigs(P<0.05);nursery pigs valeric acid was the highest,followed by pig manure.The breeding sows was the lowest(P<0.05).There were no significant differences in total VFA,acetic acid,propionic acid and butyric acid content in pig manure between three physiological stages(P>0.05).PCR-DGGE analysis of bacterial diversity in swine manure showed that there were no significant differences in the number of DGGE bands and Shannon index of fecal bacteria between pigs,finishing pigs and breeding sows(P>0.05).Cluster analysis showed that the similarity between pig manure and fattening pig manure was more than 85%,while the pregnant pig dung samples were clustered individually.The results showed that the physicochemical properties and bacterial community composition of pig manure in Meishan were significantly different at different physiological stages.2 Effects of different enrichment methods on denitrifying ability,bacterial number and microflora of swine manure denitrifying bacteriaThis chapter selects Meishan finishing pig manure,with different concentrations of nitrate nitrogen by aerobic as substrate,1 d,3 d,7 d and 3 d anaerobic enrichment cultures,determination of nitrate nitrogen removal rate,denitrification rate and denitrifying bacteria,and the use of PCR-DGGE technology to analyze the microbial community structure,comparative study on the effect of different ways of pig manure enrichment of denitrifying bacteria flora and denitrification capacity.The analysis results show that the fifth generation of culture:when substrate concentration was 5 mM,7 d anaerobic treatment group nitrate removal rate significantly higher than other groups(P<0.05),aerobic 3 d treatment group was significantly lower than that of aerobic 1 d,3 d anaerobic treatment group(P<0.05);aerobic treatment of 1 d group denitrification rate significantly higher than other groups(P<0.05);aerobic denitrifying bacteria in 3 d treatment significantly higher than other groups(P<0.05).When substrate concentration was 25 mM,similar to the test results of nitrate nitrogen removal rate of 5 mmol/L and 1 d;aerobic and anaerobic treatment was 7 d denitrification rate was significantly higher than other groups(P<0.05),aerobic 3 d treatment group was significantly lower than that of 3 d anaerobic treatment group(P<0.05);anaerobic the 7 d group the amount of denitrifying bacteria was significantly higher than other groups(P<0.05),aerobic 1 d treatment group was significantly lower than that of aerobic 3 d,3 d anaerobic treatment group(P<0.05).The substrate concentration of 50 mM,3 d anaerobic treatment group nitrate removal rate significantly higher than other groups(P<0.05),aerobic 3 d treatment group was significantly lower than that of aerobic 1 d,7 d anaerobic treatment group(P<0.05);anaerobic denitrification rate of 7 d treatment group was significantly higher than that in other groups(P<0.05),aerobic 3 d treatment group was significantly lower than that of aerobic 1 d,3 d anaerobic treatment group(P<0.05);aerobic treatment was 1 d the amount of denitrifying bacteria was lower than other groups(P<0.05).Culture shows DGGE map,when the substrate concentration is 50 mmol/L,aerobic treatment was 1 d DGGE band number was significantly higher than that of 7 d anaerobic treatment group(P<0.05);diversity index analysis showed that:when the substrate concentration is 25 mmol/L,aerobic 1 d treatment group was significantly higher than that of good diversity index oxygen is 3 d and 7 d anaerobic treatment group(P<0.05),when the substrate concentration was 50 mmol/L,1 d index of aerobic treatment group was significantly higher than that of variety 7 d anaerobic treatment group(P<0.05).The correlation showed that nitrate nitrogen removal rate was positively correlated with denitrification rate and denitrifying bacteria number(P<0.05).This study shows that when the substrate concentration,denitrifying bacteria in pig manure on nitrate nitrogen removal rate was higher than that of anaerobic aerobic denitrification rate;under anaerobic conditions with increasing substrate concentration increased;nitrate nitrogen removal rate and denitrification rate,denitrification bacteria showed a significant positive correlation(P<0.05)pig manure;advantage denitrifying bacteria as Pseudomonas sp.(Pseudomonas sp.)and bacteridium(Bacterium sp.).3 Study on isolation,culture and denitrification of denitrifying bacteriaThe purpose of this study was to screen denitrifying bacteria with strong ability to remove nitrogen from pig manure and to add them to the process of livestock wastewater denitrification,and to investigate the effect of denitrification on the denitrification capacity of livestock wastewater.Test selection,conservation,pregnancy in Meishan fattening pig manure,through aerobic and anaerobic denitrifying bacteria were screened,determine the ability of denitrification and molecular identification;then the denitrification capacity of the strongest strains added to livestock wastewater treatment process,to explore the influence of nitrogen removal rate.Test a total of 6 strains of aerobic denitrifying bacteria,16S rRNA gene sequence was identified as Pseudomonas sp.20_BN Pseudomonas stutzeri DSM(99%),10701(99%),Pseudomonas stutzeri strain 1BS5(99%),Myroides odoratimimus strain YRL08 Comamonas denitrificans strain(99%),14(99%),Pseudomonas xanthomarina strain H5(99%);3 strains of anaerobic denitrifying bacteria,16S rRNA gene sequence was identified as Pseudomonas sp.HY-24(92%),Pseudomonadaceae bacterium X2(99%),Pseudomonas sp.M-08(99%).Comparison of the nitrate nitrogen removal rate of all the strains found that nitrate removal rate was above 88%,the strain HFJ2a(Myroides odoratimimus strain YRL08,99%)nitrate nitrogen removal rate as high as 94.80%strains;denitrification ability comparison showed that HFJ2a strain denitrification ability strongest.Therefore,the strain HFJ2a was added to livestock wastewater treatment process to study its effect on nitrogen removal rate of wastewater treatment process.The test results showed that after 4 h pH treatment group was significantly higher than that of bacteria without bacteria began to experiment group(P<0.05);treatment group stage aeration oxygen content with bacteria was significantly lower than that without bacteria group(P<0.05);after 2 d treatment group with bacteria removal rate of ammonia nitrogen was significantly higher than the group without bacteria test(P<0.05);with bacteria treatment group 1 d,3 d,5 d and 7 d N2O emissions were significantly lower than those without bacteria group(P<0.05).This study showed that the pig manure in the denitrifying bacteria added to livestock wastewater treatment technology can significantly improve the removal rate of ammonia,and reduce emissions in the process of Nitrous Oxide.4 Whole genome sequencing studies the mechanism of HFJ2a strains promoting nitrogen removal in livestock wastewater treatment processOn the basis of the above study,the use of Illumina Miseq sequencing platform for genome sequencing,HFJ2a assembly,gene prediction and functional annotation,in-depth analysis of the strain mechanism to promote livestock sewage treatment process of nitrogen removal performance.A total of 4384240 sequences(1096060000 bases)were obtained by sequencing,and 4348481 usable sequences were obtained after mass filtration.The average depth of sequencing was 227X.After SOAPdenovo assembly,57 scaffolds were obtained,the genome length was 4504971 bp,and the content of GC was 45.88%.The result of gene prediction showed that the strain contained 4926 genes,with a total length of 3937719 bp,accounting for 87.4%of genome and 799 BP of gene.KEGG database annotation revealed that 186 metabolic pathways were involved in 2437 genes,which participate in the denitrification pathway genes the conversion of nitrate to nitrite narH(2),narG(2),narl(1)and narJ(2)gene;NO is transformed into N2O norB(1)gene.No genes related to converting nitrite to NO and transforming N2O into N2 were detected in the strain.The results showed that the strain mainly promoted the transformation of nitrate to nitrite in livestock wastewater treatment process,so as to improve the nitrogen removal performance of livestock wastewater treatment process.