Nitrification/Denitrification Function And Its Conversion In Carbon Regulated Microbial Suspended Growth Aquaculture Systems

The application of microbial suspended growth systems in the field of industrial aquaculture wastewater treatment has changed from the activated sludge pool in the early recirculating aquaculture system（RAS）to a new form,biofloc technology（BFT）.In order to give full application to the advantages of BFT in controlling water quality,providing natural food,ecological disease prevention,recycling resources,and to overcome its disadvantages when achieving the reutilization of aquaculture water,in situ nitrifying suspended growth aquaculture systems and RAS based on ex situ suspended growth bioreactors（SGBRs）were constructed to study their nitrification/denitrification function and its conversion under carbon regulation.Details of the study are as follows:In the water reusing in situ nitrifying suspended growth aquaculture systems,extra carbohydrate was added into the treatment group,while it was without extra carbohydrate in the control group.The results of 56-day culture experiment showed that there were no peak values of total ammonia nitrogen（TAN）and nitrite（NO₂^--N）during the experiment period,they were stable at low levels,and the concentration of NO₂^--N in the control group was significantly lower than that of treatment group.The concentrations of nitrate（NO₃^--N）and soluble reactive phosphate(PO₄^3--P)in the control group continually accumulated to more than 800 mg/L and 60 mg/L,respectively,which were significantly higher than that in the treatment group.The final stocking denisity of tilapia（GIFT Oreochromis niloticus）in the treatment group and control group could be as high as 26.04±2.04 kg/m³ and 24.32±1.04 kg/m³,respectively（P>0.05）.Denitrifying bacteria,such as Flavobacterium sp.and Thauera sp.could be important species of bacteria in the aquaculture systems.Nitrogen removal was carried out by adding extra carbohydrate into the tailwater of in situ nitrifying suspended growth aquaculture systems,where the initial concentration of total nitrogen（TN）and NO₃^--N were 306.62 mg/L and 264.29 mg/L,respectively.The results showed that the denitrification process was heterotrophic denitrification.When the initial extra organic carbon to nitrate addition ratio（C/NO₃^--N）was≥2,the concentration of TN and NO₃^--N could be decreased to less than 91.25±14.84 mg/L and6.42±0.30 mg/L,respectively,with NO₂^--N closed to zero and achieving partial phosphorus removal.When C/NO₃^--N was 4.16,the highest nitrogen recovery efficiency（23.08 mg-N/g-C）could be obtained.However,dissimilatory nitrate reduction to ammonia occurred during the denitrification process,the final residual concentration of TAN could be more than 10 mg/L,and it was increased with the raising of C/NO₃^--N.In the tailwater of in situ nitrifying suspended growth aquaculture systems,the relative abundance of denitrifying bacteria,the gene copy numbers of nar G,nir S and nos Z could reach to 3.39±0.32%,（0.31±0.08）×10⁸ copies/m L,（0.30±0.08）×10⁷copies/m L and（1.71±0.39）×10⁵ copies/m L,respectively.In the initial stage of conversion to denitrifying suspended growth systems,the original denitrifying bacteria,function genes,carbohydrate addition and dissolved oxygen（DO）played a major role in the initial process of partial complete denitrification.After the beginning of denitrification,the relative abundance and gene copy number of nitrifying bacteria had been reduced to very low levels;after four days,the copy number of key denitrifying functional genes successively reached to peak values.Six days later,the denitrification process ended,the relative abundance of nitrifying bacteria and the number of key gene copies decreased to the very low levels in the denitrifying suspended growth system.Aerated group and unaerated group were installed to study the conversion process of denitrifying suspended growth systems to nitrifying suspended growth systems.The results showed that it was a reestablishment process of nitrification,functional microbial composition and key genes.After the water conversing to nitrifying suspended growth systems,the relative abundance of nitrifying bacteria and the number of key gene copies were the same levels as that of the initial tailwater of in situ nitrifying suspended growth aquaculture systems,which indicated that the treated water can be re-utilized for another nitrifying aquaculture cycle according to the microbiological function.Besides,the alkalinity produced during the denitrification process could be used during the process of nitrification reestablishment.The nitrification reestablishment process of aerated group was much fast than that of unaerated group,and there were peak values of the relative abundance of nitrifying bacteria and the number of key gene copies in the aerated group’s process.The aerated group took 10 days to reestablish the nitrification,namely 17 days beginning from the denitrification treatment,the concentration of NO₃^--N increased to 19.86±2.93 mg/L,TAN and NO₂^--N were closed to zero,and there was no significant difference of the above pollutants between the two groups when the nitrification was reestablished.A closed RAS containing three pilot-scale ex situ SGBRs for the water treatment of pollutants was constructed.These SGBRs（CN12,CN8 and CN4）were designed to control extra carbon addition at carbon/nitrogen ratios（C/N）of 12,8,and 4,respectively.It was determined that the culture tanks removed TAN and NO₂^--N by nitrification,and the SGBRs removed NO₃^--N by denitrification and also PO₄^3--P.Concentrations of NO₃^--N and PO₄^3--P in the effluent decreased,as the controlled C/N of the SGBR increased.In CN12,the influent concentrations of NO₃^--N（35.41±1.22mg/L）and PO₄^3--P（4.60±0.67 mg/L）were reduced to 7.70±3.58 mg/L and 0.30±0.16mg/L,respectively.Thus,this proposed system had the ability to control simultaneously TAN,NO₂^--N,NO₃^--N and PO₄^3--P.The survival rate of Anguilla marmorata could be98%,with a final stocking density of 56.46±0.51 kg/m³.A RAS containing ex situ denitrifying SGBRs was installed,the concentrations of NO₃^--N and PO₄^3--P in the aquacultural water could be controlled below 7.2 mg/L and1.2 mg/L,respectively.Besides,the alkalinity produced by the SGBRs could meet the requirement of RAS.Denitrifying bacteria was the primary species in the denitrifying SGBRs.In the initial stage of conversion to a nitrifying SGBR,the original nitrifying bacteria could quickly contribute to the nitrification water treatment efficiency after stopping adding carbon source;subsequently,physicochemical conditions with low C/N and high DO increased the relative abundance of nitrifying bacteria,and nitrification was gradually enhanced.After stopping adding carbon source,the concentrations of dissolved total nitrogen（DTN）and NO₃^--N in the ex situ denitrifying SGBRs was continually increased,which was higher than that of influent after the 40th hour;before the 40th hour,the reactor maintained the original treatment efficiency of PO₄^3--P,and then the concentration of PO₄^3--P in effluent increased rapidly,and exceeded the concentration of influent after the 84th hour.Consequently,the nitrifying SGBR had advantage of NO₂^--N treatment,while there was no phosphorus removal efficiency.The survival rate of Jade Perch（Scortum barcoo）in the experimental system was99.32±0.01%,and the final stocking density of the aquaculture tanks was 21.73±0.03kg/m³.When a RAS was installed with ex situ nitrifying SGBRs,the concentrations of TAN and NO₂^--N in the aquaculture water could be controlled at levels of 2 mg/L and0.5 mg/L,respectively;however,the concentrations of NO₃^--N and PO₄^3--P increased linearly to more than 100 mg/L and 40 mg/L,respectively.In the ex situ SGBRs,with a main water treatment efficiency of nitrification,denitrifying bacteria and nitrifying bacteria were both the dominant functional bacteria,these relative abundances could be about 6%and 1.3%,respectively.After beginning to adding carbon source regularly,in the initial stage of conversion to a denitrifying SGBR,the original denitrifying bacteria could contribute to the denitrifying water treatment efficiency quickly;subsequently,the microbiological function of denitrification was gradually enhanced under physicochemical conditions with higher C/N and lower DO.The concentrations of NO₃^--N,PO₄^3--P and alkalinity in the reactor were lower than those in the influent after16th hour,thus the concentrations of NO₃^--N and PO₄^3--P in the aquaculture water could be reduced subsequently.When a SGBR converted to denitrifying one,it had phosphorus removal efficiency,while the NO₂^--N treatment efficiency was decreased.The survival rate of Jade Perch in the experimental system was 96.55±1.01%,and the final stocking density of the aquaculture tanks was 24.98±0.96 kg/m³.In conclusion,the conversion process of in situ denitrifying suspended growth systems to nitrifying suspended growth systems was a reestablishment process of nitrification,functional microbial composition and key genes.But beyond that,there were simultaneous nitrifying and denitrifying microbiological function in the other in situ and ex situ microbial suspended growth systems.During the initial conversion process between nitrification and denitrification,nitrifying or denitrifying microbiological function could make the systems change the water treatment efficiency rapidly with carbon regulation.Subsequently,the corresponding microbial function was enhanced under relevant physicochemical conditions.The suspended growth systems with a main water treatment efficiency of nitrification had an advantage of NO₂^--N treatment,while there was no phosphorus removal efficiency.The alkalinity produced from the denitrifying suspended growth systems could be subsequently used by nitrification.