Bio-reduction Of Selenate And Nitrate Using Hydrogen/methane As The Electron Donor

Natural systems and human activities caused series selenium?Se?pollution in water bodies.Selenate(SeO₄^2-)and selenite?SeO32-?are the main forms in water,and they are hexavalent and tetravalent,respectively.Both of these oxyanions have high water solubility and are toxic to organisms.On the contrary,elemental Se?Se0?is un-soluble and can be removed by filtration and centrifugation.Microbial reduction of SeO₄^2-is an efficient and simple method to control the Se-contamination in water body,in which the microorganisms reduce SeO₄^2-to SeO32-and further to Se0,using a variety of electron donors.Recently,the byproducts of anaerobic digestion,hydrogen?H₂?and methane?CH₄?,have been applied in wastewater bio-remediation.In the process,microorganisms use H₂ and CH₄ as the electron donor,to reduce oxidized pollutants,e.g.nitrate and selenate.The membrane biofilm reactor?MBfR?is found a great example for H₂ and CH₄ application because gas could be slowly delivered through the walls of hollow fiber membranes and serves as electron donors,overcoming the shortages of low gasous solubility and slow bacterial growth.This thesis studied the SeO₄^2-reduction process in the H₂/CH₄ based MBfR,and explored the interactions between NO₃^-and SeO₄^2-reductions under different loadings.Microbial community structure of biofilm samples were analyzed by high-throughput sequecing.A pair of primers targeting selenate reducing bacteria?SeRB?was designed to analyze the abundance of SeRB.Based on these findings,the mechanisms involved in SeO₄^2-reduction and the effect of NO₃^-on SeO₄^2-reduction in the H₂/CH₄ based MBfR were explored and discussed.1)SeO₄^2-bio-reduction in the H₂/CH₄ based MBfRIn the H₂-based MBfR,the SeRB and/or hydrogenotrophic denitrifying bacteria?DB?utilized H₂ as the electron donor to mediate SeO₄^2-bio-reduction.SeRB,e.g.,Thauera selenatis performed SeO₄^2-reduction using specific selenate reductase.DB,e.g.,Hydrogenophaga reduce SeO₄^2-to SeO32-,and finally to Se0 by nitrate reductase.In the CH₄-based MBfR,selenate was reduced via aerobic oxidation of methane couple to selenate reduction?OM-SeR?and anaerobic oxidation of methane couple to selenate reduction?AOM-SeR?,The OM-SeR might be accomplished by a synergistic mechanism,or be conducted by methanotrophs when they oxidized CH₄.The AOM-SeR was conducted by the synergy of archaea and bacteria,while the Archaea,e.g.,ANME activate CH₄ through reverse-methanogenisis,and deliver electrons to SeRB,the SeRB then reduce SeO₄^2-to SeO32-and further to Se0 using the electrons generated by archaea.The final SeO₄^2-reduction product was nano-type Se0,identified by scanning electron microscopy coupled to energy dispersive X-ray analysis?SEM-EDS?and X-ray photoelectron spectroscopy?XPS?.The Se0 consists of nanospheres ranging in diameter from 300 to 500 nm,and has excellent optical propties.The Se0 has potancial application prospect for nanotechology.2)The interactions of SeO₄^2-and NO₃^-reductions in H₂/CH₄ based MBfRIn H₂-based MBfR,When SeO₄^2-was supplied as the only electron acceptor in the influent,40%of SeO₄^2-was reduced to SeO.SeO₄^2-reduction rate decreased to<10%when NO₃^-was added at high level of concentrations?10 mg N/L?,in which H₂ supply was limited and NO₃^-was not completely reduced.When the input NO₃^-returned to zero,SeO₄^2-reduction was recovered,and 60%of SeO₄^2-was conversed to Se0 at steady state.When the concentration of influent NO₃^-was re-introduced at ?5.0 mg N/L,SeO₄^2-reduction remained stable at 60%,while NO₃^-was completely reduced.In CH₄-based MBfR,When SeO₄^2-was the only electron acceptor,100%of the influent SeO₄^2-was reduced to Se0.The introduction of NO₃^-didn't impact SeO₄^2-removal,but<25%of SeO₄^2-was only reduced to SeO32-,with complete NO₃^-reduction.When the influent contained high loading of NO₃^-?10 mg N/L?,CH₄ supply was limited,the two oxyanions were simultaneously reduced,but the removal percentage of SeO₄^2-and NO₃^-declined to 60%and 70%,respectively.3)Quantitative detection of SeRB targeting the selenate reductase gene and the microbial community structure analysisA primer set targeting the selenate reductase gene?serA?for detection of SeRB was desigend.This primer set was designed based on the sequences of serA gene from Dechloromonas sp.A34?GeneBank accession AC V70151?and Thauera selenatis?Q9S1H0?.The primer sequences were serAF?5'-CCGCTCAAGTCCTATCCCTAC-3'?and serAR?5'-ATACTCGCTCACCTGCTCCTC-3'?.Successful PCR amplification was achieved by using the designed primer set and the template from H₂-based MBfR biofilms.The sequences of cloned serA genes from the biofilm are very closely related to that of SeRB Thauera selenatis and DB Dechloromonas.The qPCR assay targeting serA gene further revealed that these two genus were the key microorganisms for Se042" reduction in the H₂-based MBfR biofilm.Further analysis by pyrosequencing revealed that Hydrogenophaga,a common autotrophic denitrifier,predominated in the biofilm of H₂-based MBfR,implying its importance in SeO₄^2-reduction.Besides,this genus has a positive relationship with NO₃^-flux.Community analysis revealed that introducing a high concentration of NO₃^-had a great and long-lasting impact on selenate-reducing microbial community.In the CH₄-based MBfR,Methylomonas??-Proteobacteria?predominated the biofilm.Considering Methylomonas was able to perform CH₄ oxidation and NO₃^-reduction by itself,and part of nitrate reductases?Nar?might have the potential to reduce SeO₄^2-,the first mechanism was proposed:Methylomonas coupled CH₄ oxidation and SeO₄^2-reduction directly.The detection of a high abundance of known SeRB such as Comamonadaceae and Azospira raised the possible second mechanism:methanotrophs activated CH₄ and released metabolic intermediates that were electron donors for a synergistic SeRB.