Functional Microbial Community Structure And Regulation Of Denitrifying Sulfide Removal Process For Wastewater Treatment

Wastewater produced by pharmacy,textile printing,paper mills that conclude nitrogen and sulfur are increasing year by year.It is becoming an urgent need to prevent secondary contamination.Sulfur?sulfide,sulfate,thiosulfate...?should be removed before wastewater discharge.It is reported that some microorganisms could convert sulfide to elemental sulfur by using nitrate as electron donor,as is named denitrifying sulfide removal.The elemental sulfur generated by this method took on colloidal property and could be easily recovered by precipitation.To better separate elemental sulfur from wastewater,sulfate reduction?SR?combined with denitrifying sulfide removal?DSR?and ammoxidation process was proposed by our lab.Based on the above,this study discovered the sulfide removal rate mediated by functional microorganisms in simulated wastewater that existed in Continuous Stirred Tank Reactor?CSTR?,expanded granular sludge bed?EGSB?and anaerobic baffled reactor?ABR?,investigated microbial metabolic mechanism in these reactors,and aimed at providing basis in practical wastewater treatment.Until now,little is known about the detailed functions of bacteria in these bioreactors,especially the bacteria that could simultanelously removal carbon,nitrogen,sulfur.Thus,three reactors with substrates of acetate?heterotrophy?,bicarbonate?autotrophy?,and acetate mixed bicarbonate?mixotrophy?were operated over 200 days.The samples were collected from water inlet and then sequenced by high-throughput sequencing platforms to clarify bacterial special function and further supply theoretical basis for DSR process.The results showed the diversity of denitrifying and sulfide-oxidizing bacteria were significantly influenced by different nutrient conditions during long-term operation system of DSR.The results demonstrated the key bacteria of DSR process are Thauera,Azoarcus,Thiobacillus,Pseudomonas,Arcobacter,Geobacter,and Sulfurimonas.Thauera and Arcobacter were the dominant genera in mixotrophic culture,while the functional genera in this condition were focused on Geobacter and Sulfurimonas.The predominant bacteria in autotrophic and heterotrophic culture was affiliated in Thiobacillus,Azoarcus and Pseudomonas,respectively.When bioreactor was operated under mixotrophic condition,the elemental recoveries were relatively higher than autotrophic and hetertrophic condition.However,the bacterial mechanism under different sulfide/nitrate ratios was unknown.Here,phylogenetic and functional bacterial community for elemental sulfur?S0?recovery and nitrate?NO3-?removal were investi gated with the switched S2-/NO3-molar ratio ranged from 5/2 to 5/9.We found that thediversity of denitrifying and sulfur oxidizing bacteria were also affected by electron acceptor/donor ratio in mixtrophic condition.When S2--S/NO3--N?S/N?was 5:6,elemental sulfur generation rate achieved the highest value of 84.4%,with the bacterial genera predominated with Thauera,Enterobacter,Thiobacillus and Stappia,and the sqr gene highly expressed.With S2-/NO3-ratio of 5/2,autotrophic S2-oxidization genera,Arcobacter,Desulfobulbus and Thermovirga,were dominated and NO3-reduction activity was low,confirmed by the low expressed nir K gene.In contrast,S2-/NO3-ratio switched to 5/8 and 5/9 introduced diverse heterotrophic nitrate reduction and S0 over oxidization genera in accompanied with the highly expressed nir K genes.The results identified that the S/N ratio could influence elemental sulfur generation in CSTR and the optimized sulfide/nitrate ratio was 5:6 for DSR process in this study.Influent loading is another factor affected DSR process.Since EGSB reactor could sustain a high influent loading,it was used here to investigated how did influent loading effect DSR.The results showed that elemental sulfur was not existed in effluent under low loading?COD/nitrate/sulfide loading of 1.05/0.60/0.95 kg d-1m-3,HRT of 24h?.On the contrary,the concentration of sulfate in effluent was 182 mg/L.When influent loads increased,COD removal experienced a fluctuate and then returned to a high rate?90%?as the loading was not changed.Sulfide was completely removed in both loadings.The results showed Anaerolineaceae was the predominant genus in low loadingcondition,meanwhile,the discrepancy of bacteria structure was distinct.In contrast,the structure of bacteria was similar and the predominant genera wereaffiliate with Thauera and Azoarcus with high loading.Meanwhile,gene copies of nir K,sqr and sox were all boost as loads increase,suggesting influent loading was an important factor that affected final production of sulfide oxidation in DSR process.It indicted that relative higher loaing was a key factor for DSR process operation.And then,DSR units were also affected by residual sulfate which was not fully utilized in SR units.Thus,two kinds of sulfur compound?sulfide,sulfate?were found in DSR unit.So we investigated whether elemental sulfur generation couldbe affected by residual sulfate and tried to explain the microbial menchanism in a sulfide and sulfate-mixed system.When influent COD/SO₄^2-ratio was 1,the concentration of sulfate,thiosulfate and elemental sulfur in effluent were relatively high,with the value of 139.6mg/L,48.2mg/L,and 101.8mg/L,respectively.When influent COD/SO₄^2-ratio was increased to 3,methane production was obviously enhanced.The total gas of 11.8 L was collected from bioreactor,in which methane could account for 53%.Meanwhile,the concentration of sulfide was up to 270.1 mg/L,but elemental sulfur was less than20.3 mg/L.When influent COD/SO₄^2-ratio was recovered to 1,there was no methane detected in biogas.The gas production was also decreased,and elemental sulfur generation was restored.The results of high-throughput sequencing and digital PCR showed the mechanism in simultaneous desulfurization and denitrification process: First,formic acid and CO2 were produced from anaerobic digestion through Anaerolineaceae,Desulfobulbus,Sulfurovum.In addition,H2 was produced from Anaerolineaceae.In methanogenesis,Methanobacterium converted formic acid,H2 to methane,and Methanosaeta used direct electron transfer pathway to produce methane.In denitrification,Sulfurovum and Anaerolineaceae converted nitrate to nitrogen.In sulfur cycle,sulfide was reduced by Desulfobulbus from sulfate,elemental sulfur was generated by Sulfurovum with nitrate.Sulfurovum aslo played a key role in oxidizing elemental sulfur/sulfide to sulfate.Based on the above studies,ABR was employed as the optimal reactor configuration for DSR process.Simultaneous removal of COD,SO₄^2-and NO3-and recovery of elemental sulfur?S0?were evaluated in a four-compartment anaerobic baffled reactor?ABR?with separated functional units of sulfate reduction?SR?and denitrifying sulfide removal?DSR?.To improve elemental sulfur recovery rate,NO3-was injected into the third unit to make sure that sulfate-reducing bacteria and denitrifying sulfide oxidation bacteria were separated in different units.Optimal SO₄^2--S/NO3--N ratio was evaluated as 5:5,with a substantial improvement of S0 recovery maintained at 79.1%.The results of 454 pyrosequencing and q PCR confirmed the reaction that occurred in the first two units?sulfate reduction?and the last two units?sulfide oxidation?.In this paper,we clarified the microbial diversity and functional genes expression of DSR process by using CSTR,EGSB,and ABR.The results of performance using different influent parameters provides us theoretical foundation for practical application.