Autotrophic Denitrification Based Iron-dependent Biocarriers For Low C/N Wastewater

Nitrate contaminant is one of the important factors for eutrophication in water bodies,and it has become a serious environmental problem in water systems.Biological denitrification process is a relatively economical and efficient method.However,nitrate removal efficiency is limited by insufficient organic carbon source in raw water.Therefore,autotrophic denitrification is becoming a hot spot.In order to establish novel denitrification technologies for low carbon nitrogen ratio(C/N)wastewater and reveal diversity and metabolic function of autotrophic denitrifying bacteria communities,a novel combined autotrophic and heterotrophic denitrification system was developed in this study.DNA based stable isotope probing(DNA-SIP)was applied to identify autotrophic and heterotrophic denitrifying bacteria in this denitrification system.Further,the continuous operation reactor of autotrophic denitrification was performed.Autotrophic denitrifying bacteria were identified,and its metabolism functions of carbon and nitrogen were demonstrated.The main results in this study are as follows:(1)A continuous combined autotrophic and heterotrophic denitrifying reactor was developed.The NO3-N and TN removal efficiencies were 95.2%and 91.3%in this reactor with C/N of 1 and pH of 8.2,which was superior to heterotrophic denitrification with C/N of 5.The novel iron based autotrophic and heterotrophic denitrification DNA-SIP systems were developed,and NO3-N were removed completely and total nitrogen(TN)removal efficiencies were 87.2-92.8%when C/N were 0.5 in these systems.The relative abundance of dominant bacteria Thauera,Arenimonas,Comamonas,Rubellimicrobium and Thermomonas were 21.7%,11.8%,8.1%,7.8%,and 6.0%by high throughput sequencing of 16S rRNA,respectively.The carbon source from NaHCO3 and CH3C.OONa were labeled by 13C in DNA-SIP incubation.Ultracentrifugation results showed that Thauera and Comamonas were heterotrophic denitrifying bacteria,Thermomonas and Thiobacillus were autotrophic denitrifying bacteria,and Arenimonas and Rubellimicrobium were amphitrophic denitrifying bacteria,respectively.(2)Furthermore,a completely autotrophic denitrification process was developed under pure inorganic conditions.The NO3-N and TN removal efficiencies were 69.4-91.1%and 55.5-77.7%when pH was 9.1-9.6,respectively.Autotrophic denitrifiers mainly included Sideroxydans(12.4%),Thiobacillus(5.6%)and Gallionella(4.9%).When pH was decreased to 8.2,The NO3-N and TN removal efficiencies changed to 94.2%and 89.1%,which were superior to heterotrophic denitrification with C/N of 5.Thiobacillus became absolutely dominant denitrifier in the system with the relative proportion of 27.0%.The nitrogen decreased gradually along with bioreactor,while little change for microbial community.The results of C/N influence research showed the NO2-N accumulation was decreased when C/N in influent increased.And the relative abundance of heterotrophic denitrifier Thauera increased,while autotrophic denitrifying bacteria decreased with increasing C/N.(3)Thiobacillus was identified as autotrophic denitrifier in the CEAD-C by 13C-DNA-SIP incubation.Carbon fixation in Thiobacillus was performed using Calvin cycle by cbbL and cbbM clone libraries.Abundant copies of denitrifying genes nirS and nosZ and carbon fixation genes cbbL and cbbM were found in the CEAD-C by qPCR.Metagenome results showed that the dominant autotrophic denitrifier Thiobacillus possesses metabolism pathway of complete denitrification and carbon fixation by Calvin cycle.