Variation Mechanism Of Denitrification Genes During Anaerobic Digestion With Livestock Waste

With the rapid development of the aquaculture industry,livestock manure has become the main source of agricultural pollution,and its reasonable disposal is an urgent problem to be solved.Anaerobic digestion?AD?is a microbially-mediated biotechnology employed for renewable energy production and livestock manure management.The digestate can be applied as organic fertilizer to the farmland to realize resource utilization of manure.Nitrogen is an important nutrient element necessar for crops.The anaerobic digestion process of manure can convert nitrogen into nitrogen-containing gas through denitrification.This may have an effect on anaerobic digestion process and reducing the fertilizer efficiency of digestate.Therefore,it is important to know the mechanism of nitrogen conversion and its influencing factors in anaerobic digestion for the anaerobic resource utilization of livestock manure.As antibiotics and heavy metals are widely used in livestock and poultry breeding to prevent diseases and promote growth.In the meantime,it causes a large amount of residue in the livestock manure which brings new challenges to the treatment and resource utilization of livestock manure.This study carried out a laboratory scale anaerobic digestion of livestock manure,to learn the effects of digestion parameters?temperature and total solid content?,residual pollutants?heavy metals and antibiotics?and additives on denitrification during anaerobic digestion.Denitrification gene abundance,microbial community and nitrogen transformation were analyzed,in order to reveal the mechanism of different factors on denitrification during anaerobic digestion.Aimed to reduce the nitrogen loss duing anaerobic digestion.The main results and conclusions obtained are as follows:?1?Thermophilic anaerobic digestion?55??significantly increased N₂ and N₂O emissions compared to mesophilic anaerobic digestion?35??,and reduced the abundance of the oxidized nitrous reductase nosZ gene.Pseudomonas,Enterococcus,Petrimonas and Bacillus are the major denitrifying bacterial,pH,temperature,and NH₄⁺-N are the main driver of denitrifying microbial changes.Temperature changes the co-occurrence of microbial communities and keystone species in the digestion process.Thermophilic anaerobic digestion increases the competition among microorganisms.In comparison,the microbial Network structure in mesophilic anaerobic digestion is more complicated,means the digestion system is more stable.?2?Different solid content changed the microbial community structure diversity and co-occurrence model in anaerobic digestion,which affected keystone species.The keystone species in wet digestion?total solid content=8%?were Alistipes,Christensenellaceae,Petrimonas,Flavobacterium and Syntrophomonadaceae,and the keystone species in dry digestion?total solids content=20%?are Lutispora,Methanobacterium,Anaerocella,CaproiciproandBacteroides.Methanobacteriaceae,Methanosarcinaceae,Methanocorpusculaceae,and Methanomicrobiaceae are the main methanogenic archaea in wet digestion and dry digestion,respectively.The main driving factor of microbial changes are VFAs and pH.The results of PICRUSt function prediction indicate that carbon source metabolism is annotated as the most important metabolic category.The functional metabolism of microbial communities in different solid content anaerobic digestions is different.The nitrogen related enzyme gene abundance in dry digestion is higher,while denitrification and ammoniation process related enzyme gene abundance is lower than wet digestion.?3?Copper can increase the N₂O/?N₂O+N₂?ratio during anaerobic digestion,which has an inhibitory effect on the reduction of N₂O to N₂,which leads to the accumulation of N₂O.Cu changed the microbial community composition during anaerobic digestion,and reduced the microbial community Chao1,Shannon and Simpson diversity index in the early stage of anaerobic digestion.This effect increased with the Cu concentration increased.During the digestion,the nirS and nirK genes showed different responses to different concentrations of Cu.High concentration of Cu inhibited the nosZ gene significantly.RDA analysis showed that microbial community changes had the highest interpretation rate of functional gene differences.pH,SCOD,Bio-Cu and NO₃^--N were the main driving factors for microbial community changes,indicating that Cu changed the denitrification genes at genetic level indirectly by affecting bacterial communities.?4?10 mg/kg tylosin increased N₂O emissions and reduced N₂ emissions,indicating that low concentrations of tylosin inhibited the N₂O consumption process more strongly than N₂O production,while 100 mg/kg Tylosin increases the emissions of N₂O and N₂.Enterococcus and Pseudomonas were the major denitrifying bacterial in the treatment of 10 mg/kg Tylosin in the late stage of digestion.The abundance of Halomonas in the treatment of 100 mg/kg Tylosin was significantly increased,indicating that the denitrification process have a different response to different concentrations of tylosin during anaerobic digestion.The co-occurrence of denitrification genes,antibiotic resistance genes,integrons genes,and microorganisms suggests that denitrification genes and antibiotic resistance genes may be located in the same potential host bacteria,and spread by integrons though Gene level transfer.This may increase ecological risk.IntI1 plays an important role in the transfer between genes.?5?Adding 5%zeolite to anaerobic digestion can effectively reduce the antibiotic resistance genes?sulfonamides,quinolones and macrolides?and reduce the bioavailable of heavy metals.The sequencing results showed that the zeolite increased the microbial community Chao1,Shannon and Simpson diversity index,changed the microbial community structure,and increased the abundance of Spirochaetes and Fibrobacteres in the digestion products.The results of qPCR showed that the addition of 5%zeolite significantly increased the abundance of nosZ in anaerobic digestion products,which may reduce the N₂O emission potential during the subsequent resource utilization of biogas residue.VFAs,pH,NO₃^--N and Bio-Cu are the main environmental factors that cause differences in denitrification genes.In conclusion,the temperature and solid content changed the microbial community co-occurrence pattern and keystone species during the digestion,nitrogen loss in the mesophilic anaerobic digestion was less,the metabolic activity of denitrification and ammoniation process was lower in the dry digestion than that in wet digestion;copper changed the microbial community structure,which in turn affect the denitrification gene,inhibit the N₂O reduction process;tylosin increased the horizontal transfer of denitrification genes and antibiotic resistance genes between species,resulting in higher nitrogen loss during anaerobic digestion;5%zeolite could reduce the risk of antibiotics and heavy metals effectively,and reduce N₂O potential emission.