Effect Of Different Nitrogen Forms On The Anaerobic Digestion System And Conversion Mechanism

The traditional nitrification–denitrification process is considered economically and environmentally unsustainable.It is thus imperative to explore alternatives to achieve the sustainable treatment of high nitrogen-containing organic wastewater.The development of a decarbonization and denitrification integrated process system based on anaerobic digestion technology is important in the field of wastewater treatment.In this study,the effect of different nitrogen(ammonia nitrogen,nitrate nitrogen,and nitrite nitrogen)stress intensities on the anaerobic digestion system was examined,and the process and operation performance were explored.Using 16S r RNA gene high-throughput sequencing technology,the succession of microbial diversity and the community structure was explored.Through metagenomics,the inhibitory mechanism of different nitrogen forms on the methanogenesis process and the nitrogen transformation mechanism in the anaerobic digestion system were explored.The major results are as follows:(1)Evaluation of performance and stability of different inoculaThe biogas produced by sewage sludge anaerobic digestion(SLAD),pig manure anaerobic digestion(PMAD),and water hyacinth residue anaerobic digestion(WHRAD)systems could be burned continuously on days 5,8,and 6,respectively.The R_maxof the SLAD system(2,175 m L/d)was significantly higher than that of the PMAD(1,196 m L/d)and WHRAD(1,468 m L/d)systems.The SLAD system had higher microbial community diversity and more functional modules,and parallel processing was realized.Therefore,the performances of the three inocula were in the following order:SLAD>WHRAD>PMAD.Moreover,a systematic inoculum evaluation method was established to provide theoretical and experimental evidence for biogas engineering operation.(2)Effect of ammonia nitrogen on anaerobic digestion systems evaluated using microbiological techniquesWith an increase in the ammonia nitrogen stress intensity,the microbial diversity gradually decreased.Under low ammonia nitrogen stress intensity conditions(NH₄⁺-N concentration<659.20±13.58 mg/L),the anaerobic oxidation of NH₄⁺-N to NO₃^--N and N₂occurred and methanogenesis was not inhibited.However,a high ammonia nitrogen stress intensity(NH₄⁺-N concentration of 5,660±458 mg/L)resulted in inhibition of the anaerobic oxidation of NH₄⁺-N to N₂and the methanogenesis process,and no anaerobic oxidation of NH₄⁺-N to NO₃^--N occurred.Ammonia nitrogen mainly inhibited the activity of Methanolinea.The genus unclassified?f??Nitrososphaeraceaecor responds to ammonia-oxidizing archaea that derive energy from the oxidation of NH₄⁺-N to NO₂^--N.Moreover,the genera norank?f??Anaerolineaceae,Bellilinea,Lactobacillus,and unclassified?f??Enterobacteriaceae were mainly involved in denitrification.The genus Bacillus could oxidize NO to NO₃^--N.(3)Effect of nitrate nitrogen on anaerobic digestion systems evaluated using microbiological techniquesWith an increase in the nitrate stress intensity,the microbial diversity gradually decreased,and the denitrification performance gradually increased.Under low nitrate nitrogen stress intensity conditions(COD/NO₃^--N>4.0),methanogenesis was not inhibited,simultaneous denitrification and methanogenesis occurred,and dissimilation nitrate reduction to ammonia(DNRA)did not occur.However,under high nitrate nitrogen stress intensity conditions(COD/NO₃^--N<4.0),DNRA occurred,methanogenesis was completely inhibited,and nitrate nitrogen mainly inhibited the activity of Methanosarcina.The genera Lactobacillus,Thiobacillus,Arenimonas,unclassified?f??Enterobacteriaceae,Bacillus,Limnobacter,Thermomonas,Pseudomonas,and Moheibacter were mainly involved in denitrification;the genus Cloacibacterium was mainly involved in the DNRA.(4)Effect of nitrite nitrogen on anaerobic digestion systems evaluated using microbiological techniquesWith an increase in the nitrite stress intensity,the microbial diversity gradually decreased and the performance of denitrification and anaerobic oxidation of NO₂^--N to NO₃^--N gradually increased.Under low nitrite nitrogen stress intensity conditions(NO₂^--N concentration=201±7.07 mg/L),the dissimilatory reduction of NO₂^--N to NH₄⁺-N occurred and methanogenesis was inhibited to a certain extent.However,high nitrite nitrogen stress intensity(1,882±98.99 mg/L NO₂^--N)completely inhibited methanogenesis,and nitrite nitrogen mainly inhibited Methanosaeta and Candidatus?Methanofastidiosum.The genera unclassified?f??Enterobacteriaceae,Lactobacillus,and unclassified?f??Bacillaceae were mainly involved in denitrification,Pseudomonas and Rhodopseudomonas were mainly involved in the NO₂^--N anaerobic oxidation to NO₃^--N,and Clostridium?sensu?stricto?1 was mainly involved in the dissimilatory NO₂^--N to NH₄⁺-N reaction.(5)Inhibition of methanogenesis by different nitrogen and nitrogen transformation mechanisms examined using metagenomicsIn the NH₄⁺-N system,when the ammonia nitrogen concentration was 5,660±458 mg/L,the hydrogenotrophic methanogenesis pathway(M00567),the acetateotrophic methanogenesis pathway(M00357),and the methylotrophic methanogenesis pathway(M00356 and M00563)were inhibited to some extent.In the NO₃^--N system,when COD/NO₃^--N was<4.0,the hydrogenotrophic methanogenesis pathway(M00567)and the methylotrophic methanogenesis pathway(M00356 and M00563)were completely inhibited,whereas the acetateotrophic methanogenesis pathway(M00357)was inhibited to some extent.In the NO₂^--N system,when the nitrite nitrogen concentration was 1,882±98.99 mg/L,the acetateotrophic methanogenesis pathway(M00357)and the methylotrophic methanogenesis pathway(M00356 and M00563)were mainly inhibited,whereas the hydrogenotrophic methanogenesis pathway(M00567)was weakly inhibited.The inhibition strength of the three types of inorganic reactive nitrogen compounds on the methanogenesis process were in the following order:NO₂^--N>NO₃^--N>NH₄⁺-N.When the concentration of ammonia nitrogen was less than 659.20±13.58 mg/L,the anaerobic oxidation of NH₄⁺-N to NO₃^--N occurred.Ammonia monooxygenase(EC1.14.99.39)was mainly enriched in the metabolic pathway of NH₄⁺-N anaerobic oxidation to hydroxylamine,hydroxylamine dehydrogenase(EC 1.7.2.6)was mainly enriched in the metabolic pathway of hydroxylamine anaerobic oxidation to NO₂^--N,and nitrite oxidoreductase(EC 1.7.99.-)was mainly enriched in the metabolic pathway of NO₂^--N anaerobic oxidation to NO₃^--N.When COD/NO₃^--N was>4.0,simultaneous denitrification and methanogenesis occurred.Nitrite oxidoreductase(EC 1.7.99.-)and nitrate reductase(EC 1.9.6.1)were mainly enriched in the metabolic pathway of the NO₃^--N dissimilatory reduction to NO₂^--N,nitrite reductase(EC 1.7.2.1)was mainly enriched in the metabolic pathway of NO₂^--N dissimilatory reduction to NO,nitric oxide reductase(EC 1.7.2.5)was mainly enriched in the metabolic pathway of NO dissimilatory reduction to N2O,and nitrous oxide reductase(EC 1.7.2.4)is mainly enriched in the metabolic pathway of N2O dissimilatory reduction to N₂.