| Biofloc technology（BFT）was a new approach that heterotrophic microorganisms used organic carbon sources to convert ammonia nitrogen into bacterial protein through assimilation by regulating the carbon-nitrogen ratio,thereby removing nitrogen in the water body and improving water quality.Recent studies have shown that complex denitrification processes also existed in biological floc,but its mechanism was unclear.In addition,the biofloc technology was mainly used in situ systems,but the in-situ biofloc aquaculture system（IBAS）was unstable and difficult to control.The separated biofloc aquaculture system（SBAS）can significantly improves system stability.In this experiment,IBAS was used as a control group,and SBAS was used as an experimental group.And it analyzed the effects of the two systems on the growth performance of Carassius auratus gibelio,the change of aquaculture water and nitrification functional genes.The main research contents were as follows:1.Influence of environmental factors on biological floc nitrogen removalUse batch experiments to explore the effect of different organic carbon sources（sodium acetate,glucose,trisodium citrate,sodium succinate,ethanol,sucrose）,carbon-to-nitrogen ratios（C/N）（10,13,16,19）and total suspended solids（TSS）（0g/L,1 g/L,2 g/L,3 g/L,4 g/L,5 g/L）on the removal rate of TAN,NO2--N and NO3--N.The experimental results shown that carbon source species had significant effect on nitrogen removal from biological flocs.The removal rate of TAN by sodium succinate was significantly higher than other carbon sources（P<0.05）.The removal rates of TAN were,from high to low,sodium succinate(0.075mg-N·L-1·min-1),ethanol(0.068 mg-N·L-1·min-1),and trisodium citrate(0.062mg-N·L-1·min-1),glucose(0.060 mg-N·L-1·min-1),sodium acetate(0.043mg-N·L-1·min-1),sucrose(0.025 mg-N·L-1·min-1).The removal rate of NO3--N gradually increased with the increase of C/N,and gradually increased from 1.74mg-N·L-1·h-1（C/N=10）to 2.21 mg-N·L-1·h-1（C/N=19）.The removal rate of inorganic nitrogen first increased and then decreased with the increase of TSS concentration in the range 0 g/L-5 g/L.The removal rates of TAN and NO3--N were the best when TSS was 3 g/L,which were 0.079 mg-N·L-1·min-11 and 0.021mg-N·L-1·min-1,and the removal rate of NO2--N was best when the TSS was 2 g/L(0.012 mg-N·L-1·min-1).So,sodium succinate was selected as organic carbon source,C/N was 19 and regulatory TSS 3 g/L for later experiments.2.Effects of Separated Biofloc Reactor on water quality and growth performance of Carassius auratus gibelioTaking the Carassius auratus gibelio as the breeding object and the In-situ biofloc aquaculture system（IBAS）as the control group,it studied the effect of Separate biofloc aquaculture system（SBAS）on the aquaculture water quality and the growth performance of Carassius auratus gibelio,and explored the water quality changes of the SBAS breeding tank and the biofloc reactor before and after adjusting the biofloc reactor TSS of the experimental group to 3 g/L.In this study,cassava flour was used as the organic carbon source,C/N was 19,and the experiment period was 50 days.The results shown that under high-density farming conditions,the final body weight and specific growth rate of SBAS crucian carp were significantly greater than IBAS.The survival rate of SBAS crucian carp was 93.55%,which was significantly higher than IBAS（58.06%）.Throughout the experiment,the concentrations of TAN（1.23±0.51 mg-N/L）and NO2--N（0.088±0.015 mg-N/L）in the SBAS breeding tank were stable,while the TAN and NO2--N concentrations of IBAS were reached the maximum values,17.04 mg-N/L and 8.38 mg-N/L,on day22,and then decreased to 2.26±0.78 mg/L and 0.13±0.012 mg/L,respectively.The TN concentration of IBAS was 156.23±28.43 mg/L,significantly higher than SBAS（70.75±19.49 mg/L）.After adjusting the TSS of the SBAS biofloc reactor to3 g/L,there was no significant change in TAN and NO2--N in the breeding tank,and the concentration of NO3--N increased from 3.96±2.96 mg/L to 10.93±1.38 mg/L.The removal rate of NO3--N was good in biofloc reactor,but the removal rate of TAN and NO2--N was not obvious.Nitrogen balance analysis of IBAS and SBAS shown that fish body protein accounted for 15.92%of SBAS total nitrogen output,which was significantly greater than 2.76%of IBAS,and SBAS biofloc biomass accounted for 31.89%of total nitrogen output,unlike IBAS（43.90%）.Significantly,gaseous nitrogen losses accounted for 50.07%and 51.13%of total nitrogen output of IBAS and SBAS,respectively.3.Denitrification mechanism of biofloc systemBefore and after adjusting the TSS of SBAS biofloc reactor to 3 g/L（0-23 d and24-49 d）,biofloc samples of IBAS and SBAS were collected and the total DNA was extracted.Then it determined abundance of functional genes in nitrate reduction（napA and narG）,nitrite reduction（nirS and nirK）and nitrous oxide reduction（nosZ）in the total DNA of biological floc samples by real-time quantitative PCR（qPCR）.The experimental results shown that IBAS and SBAS biological flocs contained these five functional genes.Before TSS regulation,the abundance of napA,narG and nirS genes of IBAS was significantly higher than that of SBAS（P<0.05）,and there was no significant difference between nirK and nosZ（P>0.05）;after regulating TSS,the abundance of narG and nirK of IBAS was significantly lower for SBAS（P<0.05）,there was no significant difference between napA and nosZ（P>0.05）,and nirS was significantly higher than SBAS.After regulating TSS,the abundance of napA,narG and nosZ genes in SBAS breeding tanks decreased,while the abundance of nirK and nirS genes increased from 2.89×10-55 and 1.70×10-33 to 4.96×10-33 and1.90×10-3,respectively.After regulating TSS,the abundance of napA,narG,nirS and nosZ genes in SBAS biofloc reactor decreased,and the abundance of nirK gene increased from 3.11×10-55 to 2.34×10-3. |
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