Insight Into Anaerobic Benzene Degradation Under Denitrification:Denitrification-Anammox Synergy

Benzene is the most water-soluble,toxic,and recalcitrant substance of BTEX,therefore,controlling and mitigating benzene pollution in water bodies are of great significance to protecting the water environment.From a thermodynamic viewpoint,nitrate reduction is more sponteneous than sulfate reduction?high ATP yield?.Even through benzene degradation under denitrification has been reseasrched for many years,the degradation rate of benzene is very slow;the enrichment of the factional bacterias is difficult;as well as the mechanism is not understood fully.Therefore,further studies for benzene degradation under denitrification synergized with ANAMMOX?anaerobic ammonium oxidation?,the effect of benzene,toluene,phenol and benzoate on ANAMMOX activity,the effect of benzoate on benzene degradation under denitrification synergized with ANAMMOX and benzene degradation under oxygenic denitrification were investigated in this thesis.The achievements would provide important theoretical basis for the remendation of benzene contamination.The results were given as follows:?1?To evaluate the effect of anaerobic ammonium oxidation?ANAMMOX?on benzene degradation under denitrification,a sequencing batch reactor?SBR?under denitrification synergized with ANAMMOX?SBR-DenAna?for benzene degradation was established by inoculating anaerobic ammonium-oxidising bacteria?AnAOB?into a SBR under denitrification reactor?SBR-Den?for benzene degradation.The average rate of benzene degradation and the maximum first-order kinetic constant in SBR-DenAna were 2.34-and 1.41-fold those in SBR-Den,respectively,indicating that ANAMMOX improved the degradation of benzene under denitrification synergized with ANAMMOX.However,the average rate of benzene degradation decreased by 35%in the denitrification–ANAMMOX synergistic reactor when10 mg N·L^-1 NO₂^-was added;the rate recovered once NO₂^-was depleted,indicating that ANAMMOX might detoxify NO₂^-.Results from high-throughput sequencing analysis revealed that Azoarcus within the family Rhodocyclaceae might be associated with benzene degradation in the two SBRs.AnAOB affiliated with the family Candidatus Brocadiaceae were just detected in SBR-DenAna.?2?The inhibitory effects of benzene,toluene,phenol and benzoate in single and combination on Anammox activity were investigated by the short-term batch tests.The results indicated that the single compounds?benzene,toluene and phenol?had strong inhibition on Anammox activity,which could be well fitted with extended inhibition kinetic model.According to extended inhibition kinetic model,the IC₅₀ values and their95%confidence interval of benzene,toluene and phenol were 9.21?7.80-10.62?,35.28?30.16-40.40?and 124.55(113.51-135.59 mg·L^-1.However,the effect of benzoate on anammox activity was weak,which could be well fitted with Luong inhibition kinetic model.According to extended inhibition kinetic model,the IC₅₀ values and their 95%confidence interval of benzoate was 3833.12?3481.57-4184.67?mg·L^-1.Secondly,the inhibitions of bicomponent mixtures benzene with toluene,benzene with phenol and benzene with benzoate were additive;the inhibition of multicomponent mixture of benzene,toluene and phenol was partly additive,the possible reason was that benzene,toluene and phenol all belong to aromatic compounds with the similar structure and the similar mechanism of inhibition on microorganism;the joint inhibition of mixture of benzene,toluene,phenol and benzoate was synergistic,the mechanism need research further.?3?After 10mg·L^-1 of benzoate was added in the denitrification-Anammox synergy for benzene degradation,the average rate of benzene degradation reduced by 13%;the average rate of NO₃^- and NH₄⁺ increased by approximately 1-and 0.56-fold,respectively;the activity of Anammox increased approximately 1-fold.During SBR-DenAna for benzene degradation with added benzoate was operated for a long time,the ratio of gene copies of the genus Azoarcus and Dechoromonas,the family Peptococcaceae with the gene copies of total bacteria increased.Meanwhile the diversity of microoganisms in SBR-DenAna for benzene degradation was lower than that without benzoate added.Therefore,it was indicated that benzoate is conducive to the growth of AnAOB,which would improve the performance of the denitrification-Anammox synergy for benzene degradation.?4?After anaerobic benzene degradation under nitrate-reducing cultures?labeled benzene and unlabeled benzene?operated stably,putative nod gene fragments were amplified by nod-specific primers,cloned and sequenced.Phylogenetic analysis of the derived amino acid sequences indicates that the detected sequences cluster with unknown nor-related genes.Here,we present diversity analysis of nod genes in our enrichment culture as molarcule biological evidence to elucidate the pathways for benzene mineralization under oxygenic denitrification.