Performance And Mechanisms For Bioaugmentation Of Solid-phase Denitrification Using Diaphorobacter Polyhydroxybutyrativorans SL-205

Nitrate pollution of water bodies have been becoming one of the major environmental concerns worldwide and it seriously impacts on human health.Recently,nitrogen removed by biological means had been found to be a widely adopted for waste water treatment.The additionally soluble carbon sources,such as methanol,ethanol,and acetic acid,are required for treating the nitrate-contaminated water with a low C/N ratio using the conventional heterotrophic denitrification process.These soluble carbon sources have the risks of insufficient doses or overdosing that deteriorates the effluent quality;therefore,a sophisticated process control and continuous monitoring is demanded.To overcome these disadvantages,a novel type of denitrification termed as "solid-phase denitrification(SPD)" has recently been developed.It has been successfully applied to the removal of nitrate from groundwater,aquaculture wastewater.However,the process still has the problems of long start-up time and low denitrification efficiency.In the present study,we used a PHBV-degrading denitrifer Diaphorobacter polyhydroxybutyrativorans strain SL-205 as the inoculum to enhance the removal of nitrate from contaminated groundwater in a SPD reactor packed with PHBV[poly(3-hydroxybutyrate-co-3-hydroxyvalerate)]as carbon source.The dynamic changes of microbial community and the expression level of denitrification function genes during the operation of the reactor were analyzed by molecular biology technique.The following main results were obtained.Firstly,by detecting the denitrification performance of the strain SL-205.The results showed that the removal efficiencies of nitrate reached up to over 99%by strain SL-205 using sodium acetate,sodium succinate and PHBV as carbon sources under the anoxic conditions.The optimum addition dosage of PHBV was 2.0g/L with 315mg/L of the initial nitrate nitrogen.And the optimal tempeture was 30 ?,and the optimal pH was 7.0.The strain SL-205 can utilize nitrate and nitrite for denitrification and ammonium for nitrification using poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV)as the carbon source under aerobic conditions.SL-205 removed 99.11%of NH4+-N,95.02%of NO3--N,and 84.13%of NO2--N,with average removal rates of 1.73 mg NH4+-N/(L·h),6.10 mg NO3--N/(L·h),and 4.95 mg NO2--N/(L·h),suggesting that SL-205 has a potential for nitrogen removal from wastewater.Moreover,hydroxylamine oxidoreductase,nitrate reductase,and nitrite reductase were detected,further indicating that strain SL-205 underwent heterotrophic nitrification coupled with aerobic denitrification.Expression of four denitrification genes in SL-205 under anoxic conditions was systematically analyzed by means of droplet digital PCR(ddPCR),aiming to preliminarily discuss molecular mechanism of denitrification by SL-205 from gene level.Results manifested that gene type of nitrate reductase,nitrite reductase and nitric oxide reductase was narG,nirS and cnorB,respectively,and there was nosZ in SL-205.With the increase of dissolved oxygen,the expression of narG decreased,but the concentration of dissolved oxygen was 2.0 mg/L,the expression of narG was still 1128 copies/mL,and the dissolved oxygen was increased again,and narG almost did not express.It indicates that the expression level of narG is not sensitive to the inhibitory effect of dissolved oxygen.This conclusion provides a theoretical basis for aerobic denitrification of strain SL-205.The expression of irS increased with the increase of dissolved oxygen,indicating that the concentration of dissolved oxygen promoted the expression of nirS.On the contrary,the expression level of cnorB and nosZ decreased with the increase of dissolved oxygen.The results showed that the concentration of hyperoxia could inhibit the expression of the two genes,and the oxygen restriction or microaerobic environment may be more beneficial to the gene expression.The denitrification performance of bioreactor with different nitrate loading was investigated.Results indicated that the biaoaugmentation with strain SL-205 was conducive to the rapid startup of the reactor,which exhibited a better denitrification performance.The nitrate load in influent increased to 1.6 g/(L·d),the nitrate removal rate was more than 97%,the denitrification rate was 1.56 g/(L ·d),and there was no nitrite accumulation.In the study of the expression level of denitrification related genes in the process of operation,it was found that the expression of narG in biological augmentation reactor was higher than that in activated sludge reactor for the 66 d and the 91 d.The expression of nitrite reductase gene in the bioreactor was higher than that in the activated sludge reactor.The reason of no nitrite accumulation in the bioreactor was explained from the molecular level.The biofilm on PHBV surface of bioaugmentation reactor was dense and complete,indicating that microbial activity was high.High throughput sequencing technique was used to further analyze the dynamic changes of microbial community structure.Proteobacteriae,Firmicutes and Bacteroidetes were the dominant bacteria in PHBV reactor.The dominant strains of bioreactor were Diaphorobacter,Dechloromonas and Acidovorax,while those in activated sludge reactor were Diaphorobacterium,Dechloronasti,Stenotrophomonas and Thauera.The abundance of denitrifying bacteria Diaphorobacter and Acidovorax,which can degrade PHBV in bioreactor,was higher than that in activated sludge reactor.From the principal component analysis and veen analysis,it can be concluded that the change of nitrate loading in the influent will also affect the microbial community structure.