Hydrogenotrophic denitrification and perchlorate reduction in ion exchange brines using membrane biofilm reactors

Halophilic (salt loving), hydrogenotrophic (H₂ oxidizing) denitrifying bacteria were investigated for treatment of nitrate <$>({\rm NO}_3^ ‐ )<$> and perchlorate <$>({\rm ClO}_4^ ‐ )<$> contaminated groundwater and ion exchange (IX) brines. Hydrogenotrophic denitrifying bacteria were enriched from a denitrifying wastewater seed under both halophilc and non‐halophilc conditions. The cultures were inoculated into bench‐scale membrane biofilm reactors (MBfRs) with an “outside in” configuration, with contaminated water supplied to the lumen of the membranes and H₂ supplied to the shell. Abiotic mass transfer tests showed that H₂ mass transfer coefficients were lower in brines than in tap water at highest Reynolds number, possibly due to increased transport of salts and decreased H₂ solubility at the membrane/liquid interface. An average <$>{\rm NO}_3^ ‐ <$> removal efficiency of 93% was observed for the MBfR operated in continuous flow mode with synthetic contaminated groundwater. Removal efficiencies of 30% for <$>{\rm NO}_3^ ‐ <$> and 42% for <$>{\rm ClO}_4^ ‐ <$> were observed for the MBfR operated with synthetic IX brine in batch operating mode with a reaction time of 53 h. Phylogenetic analysis focused on the active microbial community and revealed that halotolerant, <$>{\rm NO}_3^ ‐ <$> ‐reducing bacteria of the bacterial classes Gamma‐Proteobacteria and Sphingobacteria were the metabolically dominant members within the stabilized biofilm. This study shows that, despite decreased H₂ transfer under high salt conditions, hydrogenotrophic biological reduction may be successfully used for the treatment of <$>{\rm NO}_3^ ‐ <$> and <$>{\rm ClO}_4^ ‐ <$> in a MBfR. Biotechnol. Bioeng. 2009; 104: 483–491 © 2009 Wiley Periodicals, Inc.     

Kinetics of anaerobic methane oxidation coupled to denitrification in the membrane biofilm reactor

Anaerobic oxidation of methane coupled to denitrification (AOM-D) in a membrane biofilm reactor (MBfR), a platform used for efficiently coupling gas delivery and biofilm development, has attracted attention in recent years due to the low cost and high availability of methane. However, experimental studies have shown that the nitrate-removal flux in the CH₄-based MBfR (<1.0 g N/m²-day) is about one order of magnitude smaller than that in the H₂-based MBfR (1.1–6.7 g N/m²-day). A one-dimensional multispecies biofilm model predicts that the nitrate-removal flux in the CH₄-based MBfR is limited to <1.7 g N/m²-day, consistent with the experimental studies reported in the literature. The model also determines the two major limiting factors for the nitrate-removal flux: The methane half-maximum-rate concentration (K₂) and the specific maximum methane utilization rate of the AOM-D syntrophic consortium (k_max2), with k_max2 being more important. Model simulations show that increasing k_max2 to >3 g chemical oxygen demand (COD)/g cell-day (from its current 1.8 g COD/g cell-day) and developing a new membrane with doubled methane-delivery capacity (D_m) could bring the nitrate-removal flux to ≥4.0 g N/m²-day, which is close to the nitrate-removal flux for the H₂-based MBfR. Further increase of the maximum nitrate-removal flux can be achieved when D_m and k_max2 increase together.     

Bio-reductive dechlorination of 1,1,1-trichloroethane and chloroform using a hydrogen-based membrane biofilm reactor

A H(2)-based, denitrifying and sulfate-reducing membrane biofilm reactor (MBfR) was effective for removing 1,1,1-trichloroethane (TCA) and chloroform (CF) by reductive dechlorination. When either TCA or CF was first added to the MBfR, reductive dechlorination took place immediately and then increased over 3 weeks, suggesting enrichment for TCA- or CF-dechlorinating bacteria. Increasing the H(2) pressure increased the dechlorination rates of TCA or CF, and it also increased the rate of sulfate reduction. Increased sulfate loading allowed more sulfate reduction, and this competed with reductive dechlorination, particularly the second steps. The acceptor flux normalized by effluent concentration can be an efficient indicator to gauge the intrinsic kinetics of the MBfR biofilms for the different reduction reactions. The analysis of normalized rates showed that the kinetics for reductive-dechlorination reactions were slowed by reduced H(2) bio-availability caused by a low H(2) pressure or competition from sulfate reduction.     

Bioreduction of para-chloronitrobenzene in a hydrogen-based hollow-fiber membrane biofilm reactor: effects of nitrate and sulfate

A continuous-stirred, hydrogen-based, hollow-fiber membrane biofilm reactor (HFMBfR) that was active in nitrate and sulfate reductions was shown to be effective for degradation or detoxification of para-chloronitrobenzene (p-CNB) in water by biotransforming it first to para-chloroaniline (nitro-reduction) and then to aniline (reductive dechlorination) with hydrogen (H₂) as an electron donor. A series of short-term experiments examined the effects of nitrate and sulfate on p-CNB bioreduction. The results obtained showed both higher nitrate and sulfate concentration declined the p-CNB bioreduction in the biofilm, and this suggests the competition for H₂ caused less H₂ available for the p-CNB bioreduction when the H₂ demand for the reductions was larger. Denitrification and sulfate reduction intermediates were thought to be potential factors inhibiting the p-CNB bioreduction. Analysis of electron-equivalent fluxes and reaction orders in the biofilm further demonstrated both denitrification and sulfate reduction competed more strongly for H₂ availability than p-CNB bioreduction. These findings have significant implications for the HFMBfR used for degrading p-CNB under denitrifying and/or sulfate reducing conditions.     

Effective biofilm control in a membrane biofilm reactor using a quenching bacterium (Rhodococcus sp. BH4)

The biofilm thickness in membrane biofilm reactors (MBfRs) is an important factor affecting system performance because excessive biofilm formation on the membrane surface inhibits gas diffusion to the interior of the biofilm, resulting in a significant reduction in the performance of contaminant removal. This study provides innovative insights into the control of biofilm thickness in O₂-based MBfRs by using the quorum quenching (QQ) method. The study was carried out in MBfRs operated at different gas pressures and hydraulic retention times (HRTs) using QQ beads containing Rhodococcus sp. BH4 at different amounts. The highest performance was observed in reactors operated with 0.21 ml QQ bead/cm² membrane surface area, 12 HRTs and 1.40 atm. Over this period, the performance increase in chemical oxygen demand (COD) removal was 25%, while the biofilm thickness on the membrane surface was determined to be 250 μm. Moreover, acetate and equivalent oxygen flux results reached 6080 and 10 640 mg·m⁻²·d⁻¹ maximum values, respectively. The extracellular polymeric substances of the biofilm decreased significantly with the increase of gas pressure and QQ beads amount. Polymerase chain reaction denaturing gradient gel electrophoresis results showed that the microbial community in the MBfR system changed depending on operating conditions and bead amount. The results showed that the QQ method was an effective method to control the biofilm thickness in MBfR and provide insights for future research.     

Community structure and function in a H2-based membrane biofilm reactor capable of bioreduction of selenate and chromate

Two different H₂-based, denitrifying membrane-biofilm reactors (MBfRs) initially reduced Se(VI) or Cr(VI) stably to Se⁰ or Cr(III). When the oxidized contaminants in the influent were switched, each new oxidized contaminant was reduced immediately, and its reduction soon was approximately the same or greater than it had been in its original MBfR. The precipitation of reduced selenium and chromium in the biofilm was verified by scanning electron microscopy and energy dispersive X-ray analysis. These results on selenate and chromate reduction are consistent with the interpretation that the H₂-based biofilm community had a high level of functional diversity. The communities’ structures were assessed by cloning analysis. Dechloromonas spp., a known perchlorate-reducing bacteria, dominated the clones from both reactors during selenate and chromate reductions, which suggests that it may have functional diversity capable of reducing selenate and chromate as secondary and dissimilatory acceptors.     

Electron-acceptor loadings affect chloroform dechlorination in a hydrogen-based membrane biofilm reactor

Chloroform (CF) can undergo reductive dechlorination to dichloromethane, chloromethane, and methane. However, competition for hydrogen (H₂), the electron-donor substrate, may cause poor dechlorination when multiple electron acceptors are present. Common acceptors in anaerobic environments are nitrate (NO₃⁻), sulfate (SO₄²⁻), and bicarbonate (HCO₃⁻). We evaluated CF dechlorination in the presence of HCO₃⁻ at 1.56 e⁻ Eq/m²-day, then NO₃⁻ at 0.04–0.15 e⁻ Eq/m²-day, and finally NO₃⁻ (0.04 e⁻ Eq/m²-day) along with SO₄²⁻ at 0.33 e⁻ Eq/m²-day in an H₂-based membrane biofilm reactor (MBfR). When the biofilm was initiated with CF-dechlorination conditions (no NO₃⁻ or SO₄²⁻), it yielded a CF flux of 0.14 e⁻ Eq/m²-day and acetate production via homoacetogenesis up to 0.26 e⁻ eq/m²-day. Subsequent addition of NO₃⁻ at 0.05 e⁻ Eq/m²-day maintained full CF dechlorination and homoacetogenesis, but NO₃⁻ input at 0.15 e⁻ Eq/m²-day caused CF to remain in the reactor's effluent and led to negligible acetate production. The addition of SO₄²⁻ did not affect CF reduction, but SO₄²⁻ reduction significantly altered the microbial community by introducing sulfate-reducing Desulfovibrio and more sulfur-oxidizing Arcobacter. Dechloromonas appeared to carry out CF dechlorination and denitrification, whereas Acetobacterium (homoacetogen) may have been involved with hydrolytic dechlorination. Modifications to the electron acceptors fed to the MBfR caused the microbial community to undergo changes in structure that reflected changes in the removal fluxes.     

Managing microbial communities in membrane biofilm reactors

Membrane biofilm reactors (MBfRs) deliver gaseous substrates to biofilms that develop on the outside of gas-transfer membranes. When an MBfR delivers electron donors hydrogen (H₂) or methane (CH₄), a wide range of oxidized contaminants can be reduced as electron acceptors, e.g., nitrate, perchlorate, selenate, and trichloroethene. When O₂ is delivered as an electron acceptor, reduced contaminants can be oxidized, e.g., benzene, toluene, and surfactants. The MBfR’s biofilm often harbors a complex microbial community; failure to control the growth of undesirable microorganisms can result in poor performance. Fortunately, the community’s structure and function can be managed using a set of design and operation features as follows: gas pressure, membrane type, and surface loadings. Proper selection of these features ensures that the best microbial community is selected and sustained. Successful design and operation of an MBfR depends on a holistic understanding of the microbial community’s structure and function. This involves integrating performance data with omics results, such as with stoichiometric and kinetic modeling.

     

Kinetics of microbial bromate reduction in a hydrogen-oxidizing, denitrifying biofilm reactor

Bromate (BrO(3)(-)) is an oxidized contaminant produced from bromide (Br(-)) during ozonation and advanced oxidation of drinking water. Previous research shows that denitrifying bioreactors can reduce bromate to innocuous bromide. We studied a hydrogen-based, denitrifying membrane-biofilm reactor (MBfR) for bromate reduction, and report the first kinetics for a hydrogen-based bromate reduction process. A mixed-culture MBfR reduced up to 1,500 microg/L bromate to below 10 microg/L with a 50-min hydraulic residence time. Kinetics were determined using short-term tests on a completely mixed MBfR at steady state with an influent of 5 mg N/L nitrate plus 100 microg/L bromate. Short-term tests examined the impact of pH, nitrite, nitrate, and bromate on bromate reduction rates in the MBfR. Kinetic parameters for the process were estimated based on the short-term bromate tests. The q(max) for bromate reduction was 0.12 mg BrO(3)(-) x mg(x)(-1) x day(-1), and the K was 1.2 mg BrO(3)(-)/L. This q(max) is 2-3 times higher than reported for heterotrophic enrichments, and the K is the first reported in the literature. Nitrite and nitrate partially inhibited bromate reduction, with nitrite exerting a stronger inhibitory effect. Bromate was self-inhibitory at concentrations above 15 mg/L, but up to 50 mg/L of bromate had no inhibitory effect on denitrification. The optimum pH was approximately 7. We also examined the performance of an MBfR containing pure culture of the denitrifying bacterium Ralstonia eutropha. Under conditions similar to the mixed-culture tests, no bromate reduction was detected, showing that not all denitrifying bacteria are active in bromate reduction. Our results suggest the presence of specialized, dissimilatory bromate-reducing bacteria in the mixed-culture MBfR.     

Consortium diversity of a sulfate‐reducing biofilm developed at acidic pH influent conditions in a down‐flow fluidized bed reactor

Sulfate reduction is an appropriate approach for the treatment of effluents with sulfate and dissolved metals. In sulfate‐reducing reactors, acetate may largely contribute to the residual organic matter, because not all sulfate reducers are able to couple the oxidation of acetate to the reduction of sulfate, limiting the treatment efficiency. In this study, we investigated the diversity of a bacterial community in the biofilm of a laboratory scale down‐flow fluidized bed reactor, which was developed under sulfidogenic conditions at an influent pH between 4 and 6. The sequence analysis of the microbial community showed that the 16S rRNA gene sequence of almost 50% of the clones had a high similarity with Anaerolineaceae. At second place, 33% of the 16S rRNA phylotypes were affiliated with the sulfate‐reducing bacteria Desulfobacca acetoxidans and Desulfatirhabdium butyrativorans, suggesting that acetotrophic sulfate reduction was occurring in the system. The remaining bacterial phylotypes were related to fermenting bacteria found at the advanced stage of reactor operation. The results indicate that the acetotrophic sulfate‐reducing bacteria were able to remain within the biofilm, which is a significant result because few natural consortia harbor complete oxidizing sulfate‐reducers, improving the acetate removal via sulfate reduction in the reactor.     

Characterization and quantification of class 1 integrons and associated gene cassettes in sewage treatment plants

Three hydrogen-based membrane biofilm reactors (MBfR) biologically reduced nitrate and perchlorate in a synthetic ion-exchange (IX) brine. Inocula from different natural saline environments were employed to initiate the three MBfRs. Under high-salinity (3%) conditions, bioreduction of nitrate and perchlorate occurred simultaneously, and the three MBfRs from the different inocula exhibited similar removal fluxes for nitrate and perchlorate. Clone libraries were generated from samples of the biofilms in the three MBfRs and compared to those of their inocula. When H₂ was the sole exogenous electron donor under high-salinity conditions, MBfR-driven community shifts were observed with a similar pattern regardless of inoculum. The following 16S rRNA gene phylogenetic analysis showed the presence of novel perchlorate-reducing bacteria in the salt-tolerant mBfR communities. These findings suggest that autohydrogenotrophic and high-salinity conditions provided strong selective pressure for novel perchlorate-reducing populations in the mBfRs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A chalcone with potent inhibiting activity against biofilm formation by nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi), an important human respiratory pathogen, frequently causes biofilm infections. Currently, resistance of bacteria within the biofilm to conventional antimicrobials poses a major obstacle to effective medical treatment on a global scale. Novel agents that are effective against NTHi biofilm are therefore urgently required. In this study, a series of natural and synthetic chalcones with various chemical substituents were evaluated in vitro for their antibiofilm activities against strong biofilm‐forming strains of NTHi. Of the test chalcones, 3‐hydroxychalcone (chalcone 8 ) exhibited the most potent inhibitory activity, its mean minimum biofilm inhibitory concentration (MBIC₅₀) being 16 μg/mL (71.35 μM), or approximately sixfold more active than the reference drug, azithromycin (MBIC₅₀ 419.68 μM). The inhibitory activity of chalcone 8 , which is a chemically modified chalcone, appeared to be superior to those of the natural chalcones tested. Significantly, chalcone 8 inhibited biofilm formation by all studied NTHi strains, indicating that the antibiofilm activities of this compound occur across multiple strong‐biofilm forming NTHi isolates of different clinical origins. According to antimicrobial and growth curve assays, chalcone 8 at concentrations that decreased biofilm formation did not affect growth of NTHi, suggesting the biofilm inhibitory effect of chalcone 8 is non‐antimicrobial. In terms of structure–activity relationship, the possible substituent on the chalcone backbone required for antibiofilm activity is discussed. These findings indicate that 3‐hydroxychalcone (chalcone 8 ) has powerful antibiofilm activity and suggest the potential application of chalcone 8 as a new therapeutic agent for control of NTHi biofilm‐associated infections.     

Biofilm model calibration and microbial diversity study using Monte Carlo simulations

Mathematical models are useful tools for studying and exploring biological conversion processes as well as microbial competition in biological treatment processes. A single‐species biofilm model was used to describe biofilm reactor operation at three different hydraulic retention times (HRT). The single‐species biofilm model was calibrated with sparse experimental data using the Monte Carlo filtering method. This calibrated single‐species biofilm model was then extended to a multi‐species model considering 10 different heterotrophic bacteria. The aim was to study microbial diversity in bulk phase biomass and biofilm, as well as the competition between suspended and attached biomass. At steady state and independently of the HRT, Monte Carlo simulations resulted only in one unique dominating bacterial species for suspended and attached biomass. The dominating bacterial species was determined by the highest specific substrate affinity (ratio of µ/K_S). At a short HRT of 20 min, the structure of the microbial community in the bulk liquid was determined by biomass detached from the biofilm. At a long HRT of 8 h, both biofilm detachment and microbial growth in the bulk liquid influenced the microbial community distribution. Biotechnol. Bioeng. 2013; 110: 1323–1332. © 2012 Wiley Periodicals, Inc.     

Acetylene reduction and hydrogen metabolism by a cyanobacterial/sulfate‐reducing bacterial mat ecosystem

We report a study of nitrogenase activity (acetylene reduction) and hydrogen gas metabolism in intact smooth cyanobacterial mats from Hamelin Pool, Shark Bay, Western Australia. The predominant cyanobacterial population in these mats is Microcoleus chthonoplastes. The mats had a significant capacity for nitrogen fixation, predominantly attributable to the photosyn‐thetic component. By physical and chemical perturbation we revealed an active hydrogen metabolism within the mats. Most of the H₂ formation was attributed to fermentative processes, whereas hydrogen was consumed in light‐dependent, together with oxygen‐ and sulfate‐dependent respiratory processes. It was concluded that H₂ formed by fermentative bacteria in the dark drives a significant proportion of sulfate reduction in the mats, but there was little H₂ transfer from the cyanobacteria to the sulfate‐reducing bacteria. Thus photosynthetically produced H₂ gas is unlikely to significantly alter the previously measured carbon: sulfur ratio relating photosynthesis to sulfate reduction.     

Biofilm form and function: carbon availability affects biofilm architecture, metabolic activity and planktonic cell yield

Aims: To investigate carbon transformation by biofilms and changes in biofilm architecture, metabolic activity and planktonic cell yield in response to fluctuating carbon availability. Methods and Results: Pseudomonas sp. biofilms were cultured under alternating carbon‐replete and carbon‐limited conditions. A shift to medium without added carbon led to a 90% decrease in biofilm respiration rate and a 40% reduction in planktonic cell yield within 1 h. Attached cell division and progeny release were shown to contribute to planktonic cell numbers during carbon limitation. Development of a significantly enlarged biofilm surface area during carbon limitation facilitated a rapid increase in whole‐biofilm metabolic activity, cell yield and biomass upon the re‐introduction of carbon after 8 days of limitation. The cumulative number of planktonic cells (>10¹⁰ CFU) released from the biofilm during the cultivation period contained only 1·0% of the total carbon available to the biofilm, with 6·5% of the carbon retained in the biofilm and 54% mineralized to CO₂. Conclusions: Biofilm‐derived planktonic cell yield is a proliferation mechanism. The rapid response of biofilms to environmental perturbations facilitates the optimal utilization of resources to promote both proliferation and survival. Biofilms function as efficient catalysts for environmental carbon transformation and mineralization. Significance and Impact of the study: A greater understanding of the relationship between biofilm form and function can inform strategies intended to control and/or promote biofilm formation.     

Top‐down effects of a lytic bacteriophage and protozoa on bacteria in aqueous and biofilm phases

Lytic bacteriophages and protozoan predators are the major causes of bacterial mortality in natural microbial communities, which also makes them potential candidates for biological control of bacterial pathogens. However, little is known about the relative impact of bacteriophages and protozoa on the dynamics of bacterial biomass in aqueous and biofilm phases. Here, we studied the temporal and spatial dynamics of bacterial biomass in a microcosm experiment where opportunistic pathogenic bacteria Serratia marcescens was exposed to particle‐feeding ciliates, surface‐feeding amoebas, and lytic bacteriophages for 8 weeks, ca. 1300 generations. We found that ciliates were the most efficient enemy type in reducing bacterial biomass in the open water, but least efficient in reducing the biofilm biomass. Biofilm was rather resistant against bacterivores, but amoebae had a significant long‐term negative effect on bacterial biomass both in the open‐water phase and biofilm. Bacteriophages had only a minor long‐term effect on bacterial biomass in open‐water and biofilm phases. However, separate short‐term experiments with the ancestral bacteriophages and bacteria revealed that bacteriophages crash the bacterial biomass dramatically in the open‐water phase within the first 24 h. Thereafter, the bacteria evolve phage‐resistance that largely prevents top‐down effects. The combination of all three enemy types was most effective in reducing biofilm biomass, whereas in the open‐water phase the ciliates dominated the trophic effects. Our results highlight the importance of enemy feeding mode on determining the spatial distribution and abundance of bacterial biomass. Moreover, the enemy type can be crucially important predictor of whether the rapid defense evolution can significantly affect top‐down regulation of bacteria.     

Solid support membrane‐aerated catalytic biofilm reactor for the continuous synthesis of (S)‐styrene oxide at gram scale

Catalytic biofilms minimize reactant toxicity and maximize biocatalyst stability in selective transformations of chemicals to value‐added products in continuous processes. The scaling up of such catalytic biofilm processes is challenging, due to fluidic and biological parameters affording a special reactor design affecting process performance. A solid support membrane‐aerated biofilm reactor was optimized and scaled‐up to yield gram amounts of (S)‐styrene oxide, a toxic and instable high value chemical synthon. A sintered stainless steel membrane unit was identified as an optimal choice as biofilm substratum and for high oxygen mass transfer. A stable expanded polytetrafluoroethylene (ePTFE) membrane was best suited for in situ substrate delivery and product extraction. For the verification of scalability, catalytic biofilms of Pseudomonas sp. strain VLB120ΔC produced (S)‐styrene oxide to an average concentration of 390 mM in the organic phase per day (equivalent to 24.4 g L_aq^–1 day^–1). This productivity was gained by efficiently using the catalyst with an excellent product yield on biomass of 13.6 g_product g_biomass^–1. This product yield on biomass is in the order of magnitude reported for other continuous systems based on artificially immobilized biocatalysts and is fulfilling the minimum requirements for industrial biocatalytic processes. Overall, 46 g of (S)‐styrene oxide were produced and isolated (purity: 99%; enantiomeric excess [ee]: >99.8%. yield: 30%). The productivity is in a similar range as in comparable small‐scale biofilm reactors highlighting the large potential of this methodology for continuous bioprocessing of bulk chemicals and biofuels.     

The role of sulfate‐reducing bacteria in the deposition of sedimentary uranium ores

The formation of many important sediment‐hosted uranium ore deposits is thought to have resulted from the reduction of relatively soluble uranyl ion—U(VI)—to insoluble uranium (IV) oxides and silicates by aqueous sulfide species. This study focused on the influence that the sulfate‐reducing bacteria Desulfovibrio desulfuricans (ATCC 7757) has on this process. Preliminary studies showed that bacterial growth was not inhibited by concentrations of uranyl ion up to 100 mg U per liter. More detailed studies showed that sulfate‐reducing bacteria have an influence on uranyl ion removal beyond the simple production of the aqueous sulfide reductant. Comparative studies of bacterial cultures containing high densities of the sulfate reducers with bacterial cell‐free but otherwise identical media showed that the bacteria themselves enhance uranium removal from solution. At pH 8.0, no reaction was observed in H₂S‐bearing cell‐free media, whereas at the same H₂S concentration, the uranyl ion decreased markedly in the presence of the bacteria. At pH 7.0, some uranium removal occurred in the absence of bacteria, but it was much more rapid in their presence. We postulate that these effects are due to the ability of bacterial cell walls to adsorb uranium. Adsorption to surfaces is known from independent studies to enhance uranium reduction, and evidently this two‐step adsorption‐reduction mechanism is occurring in our experiments. We conclude that sulfate‐reducing and other bacteria may play a significant role in the geochemical cycling of uranium.     

Quantitative detection of selenate-reducing bacteria by real-time PCR targeting the selenate reductase gene

We designed a primer set to target selenate reductase (SerA) for detecting selenate reducing bacteria (SeRB). Our serA gene-based PCR primer set has high specificity in that it and positively amplified some SeRB, but not denitrifying bacteria (DB). Phylogenetic analysis of serA clone sequences of environmental samples from selenate-reducing membrane biofilm reactor (MBfR) biofilms showed that these sequences were closely grouped and had high similarity to selenate reductase gene sequences from SeRB Thauera selenatis and DB Dechloromonas; however, they were distant to other genes from dimethylsulfoxide (DMSO) enzyme family. Constructing a standard curve targeting the serA gene, we found that the good linearity for the qPCR assay when applied it to quantify SeRB in MBfR biofilms, and the gene copies of SeRB correlated well to the selenate removal percentages. Our results demonstrated the feasibility of using the serA gene-based PCR primer set to detect and quantify SeRB in environmental samples.