Aura Ontiveros-Valencia

Interactions between Nitrate-Reducing and Sulfate-Reducing Bacteria Coexisting in a Hydrogen-Fed Biofilm

To explore the relationships between denitrifying bacteria (DB) and sulfate-reducing bacteria (SRB) in H(2)-fed biofilms, we used two H(2)-based membrane biofilm reactors (MBfRs) with or without restrictions on H(2) availability. DB and SRB compete for H(2) and space in the biofilm, and sulfate (SO(4)(2-)) reduction should be out-competed when H(2) is limiting inside the biofilm. With H(2) availability restricted, nitrate (NO(3)(-)) reduction was proportional to the H(2) pressure and was complete at a H(2) pressure of 3 atm; SO(4)(2-) reduction began at H(2) ≥ 3.4 atm. Without restriction on H(2) availability, NO(3)(-) was the preferred electron acceptor, and SO(4)(2-) was reduced only when the NO(3)(-) surface loading was ≤ 0.13 g N/m(2)-day. We assayed DB and SRB by quantitative polymerase chain reaction targeting the nitrite reductases and dissimilatory sulfite reductase, respectively. Whereas DB and SRB increased with higher H(2) pressures when H(2) availability was limiting, SRB did not decline with higher NO(3)(-) removal flux when H(2) availability was not limiting, even when SO(4)(2-) reduction was absent. The SRB trend reflects that the SRBs metabolic diversity allowed them to remain in the biofilm whether or not they were reducing SO(4)(2-). In all scenarios tested, the SRB were able to initiate strong SO(4)(2-) reduction only when competition for H(2) inside the biofilm was relieved by nearly complete removal of NO(3)(-).

Using a two-stage hydrogen-based membrane biofilm reactor (MBfR) to achieve complete perchlorate reduction in the presence of nitrate and sulfate.

We evaluated a strategy for achieving complete reduction of perchlorate (ClO(4)(-)) in the presence of much higher concentrations of sulfate (SO(4)(2-)) and nitrate (NO(3)(-)) in a hydrogen-based membrane biofilm reactor (MBfR). Full ClO(4)(-) reduction was achieved by using a two-stage MBfR with controlled NO(3)(-) surface loadings to each stage. With an equivalent NO(3)(-) surface loading larger than 0.65 ± 0.04 g N/m(2)-day, the lead MBfR removed about 87 ± 4% of NO(3)(-) and 30 ± 8% of ClO(4)(-). This decreased the equivalent surface loading of NO(3)(-) to 0.34 ± 0.04-0.53 ± 0.03 g N/m(2)-day for the lag MBfR, in which ClO(4)(-) was reduced to nondetectable. SO(4)(2-) reduction was eliminated without compromising full ClO(4)(-) reduction using a higher flow rate that gave an equivalent NO(3)(-) surface loading of 0.94 ± 0.05 g N/m(2)-day in the lead MBfR and 0.53 ± 0.03 g N/m(2)-day in the lag MBfR. Results from qPCR and pyrosequencing showed that the lead and lag MBfRs had distinctly different microbial communities when SO(4)(2-) reduction took place. Denitrifying bacteria (DB), quantified using the nirS and nirK genes, dominated the biofilm in the lead MBfR, but perchlorate-reducing bacteria (PRB), quantified using the pcrA gene, became more important in the lag MBfR. The facultative anaerobic bacteria Dechloromonas, Rubrivivax, and Enterobacter were dominant genera in the lead MBfR, where their main function was to reduce NO(3)(-). With a small NO(3)(-) surface loading and full ClO(4)(-) reduction, the dominant genera shifted to ClO(4)(-)-reducing bacteria Sphaerotilus, Rhodocyclaceae, and Rhodobacter in the lag MBfR.

Effects of multiple electron acceptors on microbial interactions in a hydrogen-based biofilm

To investigate interactions among multiple electron acceptors in a H2-fed biofilm, we operated a membrane biofilm reactor with H2-delivery capacity sufficient to reduce all acceptors. ClO4(-) and O2 were input electron acceptors in all stages at surface loadings of 0.08 ± 0.006 g/m(2)-d (1.0 ± 0.7 e(-) meq/m(2)-d) for ClO4(-) and 0.51 g/m(2)-d (76 e(-) meq/m(2)-d) for O2. SO4(2-) was added in Stage 2 at 3.77 ± 0.39 g/m(2)-d (331 ± 34 e(-) meq/m(2)-d), and NO3(-) was further added in Stage 3 at 0.72 ± 0.03 g N/m(2)-d (312 ± 13 e(-) meq/m(2)-d). At steady state for each stage, ClO4(-), O2, and NO3(-) (when present in the influent) were completely reduced; measured SO4(2-) reduction decreased from 78 ± 4% in Stage 2 to 59 ± 4% in Stage 3, when NO3(-) was present. While perchlorate-reducing bacteria (PRB), assayed by qPCR targeting the pcrA gene, remained stable throughout, sulfate-reducing bacteria (SRB), assayed by the dsrA gene, increased almost 3 orders of magnitude when significant SO4(2-) reduction occurred in stage 2. The abundance of denitrifying bacteria (DB), assayed by the nirK and nirS genes, increased in Stage 3, while SRB remained at high numbers, but did not increase. Based on pyrosequencing analyses, β-Proteobacteria dominated in Stage 1, but ε-Proteobacteria became more important in Stages 2 and 3, when the input of multiple electron acceptors favored genera with broader electron-accepting capabilities. Sulfuricurvum (a sulfur oxidizer and NO3(-) reducer) and Desulfovibrio (a SO4(2-) reducer) become dominant in Stage 3, suggesting redox cycling of sulfur in the biofilm.

A biofilm model to understand the onset of sulfate reduction in denitrifying membrane biofilm reactors

This work presents a multispecies biofilm model that describes the co‐existence of nitrate‐ and sulfate‐reducing bacteria in the H2‐based membrane biofilm reactor (MBfR). The new model adapts the framework of a biofilm model for simultaneous nitrate and perchlorate removal by considering the unique metabolic and physiological characteristics of autotrophic sulfate‐reducing bacteria that use H2 as their electron donor. To evaluate the model, the simulated effluent H2, UAP (substrate‐utilization‐associated products), and BAP (biomass‐associated products) concentrations are compared to experimental results, and the simulated biomass distributions are compared to real‐time quantitative polymerase chain reaction (qPCR) data in the experiments for parameter optimization. Model outputs and experimental results match for all major trends and explain when sulfate reduction does or does not occur in parallel with denitrification. The onset of sulfate reduction occurs only when the nitrate concentration at the fibers outer surface is low enough so that the growth rate of the denitrifying bacteria is equal to that of the sulfate‐reducing bacteria. An example shows how to use the model to design an MBfR that achieves satisfactory nitrate reduction, but suppresses sulfate reduction. Biotechnol. Bioeng. 2013; 110: 763–772.

Phylogenetic analysis of nitrate- and sulfate-reducing bacteria in a hydrogen-fed biofilm

Using two membrane biofilm reactors in which hydrogen (H₂) was the only exogenous electron donor, we studied the microbial community structure of biofilms composed primarily of denitrifying bacteria (DB) and sulfate-reducing bacteria (SRB). In steady-state EDvSS, H₂ availability was restricted and varied. In steady-state EAvSS, the input nitrate (NO₃⁻) concentration was varied relative to a fixed sulfate (SO₄²⁻) concentration. SRB co-existed with DB, even when SO₄²⁻ reduction was absent due to restricted H₂ availability. UniFrac and principal coordinate analysis indicated that H₂ availability and electron-acceptor loadings framed the microbial community structure, with H₂ availability having a greater impact. In EDvSS, restricted H₂ availability favored heterotrophic DB (i.e. Burkholderiales) compared with autotrophic DB (e.g. Hydrogenophilales and Rhodocyclales). In EAvSS, SO₄²⁻ reduction lowered the relative abundance of some DB (e.g. Hydrogenophilales), and the biofilm was colonized by Desulfovibrionales and Bacteroidales. Reinforcing the impact of H₂ availability, EAvSS showed a higher microbial diversity and more even distribution among microbial groups than did EDvSS. Thus, the biofilm community in a H₂-fed biofilm with DB and SRB became more heterotrophic when the H₂ availability was constrained, while low NO₃⁻ loading allowed more SO₄²⁻ reduction, causing a shift to more SRB.

Palladium Recovery in a H2-Based Membrane Biofilm Reactor: Formation of Pd(0) Nanoparticles through Enzymatic and Autocatalytic Reductions

Recovering palladium (Pd) from waste streams opens up the possibility of augmenting the supply of this important catalyst. We evaluated Pd reduction and recovery as a novel application of a H2-based membrane biofilm reactor (MBfR). At steady states, over 99% of the input soluble Pd(II) was reduced through concomitant enzymatic and autocatalytic processes at acidic or near neutral pHs. Nanoparticulate Pd(0), at an average crystallite size of 10 nm, was recovered with minimal leaching and heterogeneously associated with microbial cells and extracellular polymeric substances in the biofilm. The dominant phylotypes potentially responsible for Pd(II) reduction at circumneutral pH were denitrifying β-proteobacteria mainly consisting of the family Rhodocyclaceae. Though greatly shifted by acidic pH, the biofilm microbial community largely bounced back when the pH was returned to 7 within 2 weeks. These discoveries infer that the biofilm was capable of rapid adaptive evolution to stressed environmental change, and facilitated Pd recovery in versatile ways. This study demonstrates the promise of effective microbially driven Pd recovery in a single MBfR system that could be applied for the treatment of the waste streams, and it documents the role of biofilms in this reduction and recovery process.

Perchlorate reduction from a highly contaminated groundwater in the presence of sulfate-reducing bacteria in a hydrogen-fed biofilm.

We used a hydrogen (H2)‐based biofilm to treat a groundwater contaminated with perchlorate (ClO4−) at ∼10 mg/L, an unusually high concentration. To enhance ClO4− removal, we either increased the H2 pressure or decreased the electron‐acceptor surface loading. The ClO4− removal increased from 94% to 98% when the H2 pressure was increased from 1.3 to 1.7 atm when the total acceptor surface loading was 0.49 g H2/m2 day. We then decreased the acceptor surface loading stepwise from 0.49 to 0.07 g H2/m2 day, and the ClO4− removal improved to 99.6%, giving an effluent ClO4− concentration of 41 µg/L. However, the tradeoff was that sulfate (SO42−) reduction occurred, reaching 85% conversion at the lowest acceptor surface loading (0.07 g H2/m2 day). In two steady states with the highest ClO4− reduction, we assayed for the presence of perchlorate‐reducing bacteria (PRB), denitrifying bacteria (DB), and sulfate‐reducing bacteria (SRB) by quantitative polymerase chain reaction (qPCR) targeting characteristic reductases. The qPCR results documented competition between PRB and SRB for space within the biofilm. A simple model analysis for a steady‐state biofilm suggests that competition from SRB pushed the PRB to locations having a higher detachment rate, which prevented them from driving the ClO4− concentration below 41 µg/L. Biotechnol. Bioeng. 2013;110: 3139–3147.

Hydrogen‐fed biofilm reactors reducing selenate and sulfate: Community structure and capture of elemental selenium within the biofilm

Remediation of selenate (SeO42−) contamination through microbial reduction is often challenging due to the presence of sulfate (SO42−), which can lead to competition for the electron donor and the co‐production of toxic H2S. Microbial reduction of SeO42− in the presence of SO42− was studied in two hydrogen‐based membrane biofilm reactors (MBfRs). One MBfR was initiated with SO42−‐reducing conditions and gradually shifted to SeO42− reduction. The second MBfR was developed with a SeO42−‐reducing biofilm, followed by SO42− introduction. Biofilms within both MBfRs achieved greater than 90% SeO42− reduction, even though the SeO42− concentration ranged from 1,000–11,000 μg/L, more than 20–200 times the maximum contaminant level for drinking water (50 μg/L). Biofilm microbial community composition, assessed by 16S rRNA gene‐based amplicon pyrosequencing, was distinct between the two MBfRs and was framed by alterations in SeO42− loading. Specifically, high SeO42− loading resulted in communities mainly composed of denitrifying bacteria (e.g., Denitratisoma and Dechloromonas). In contrast, low loading led to mostly sulfate‐reducing bacteria (i.e., Desulfovibrio) and sulfur‐oxidizing bacteria (i.e., Sulfuricurvum and Sulfurovum). SeO42− was reduced to elemental selenium (Se°), which was visualized within the biofilm as crystalloid aggregates, with its fate corresponding to that of biofilm solids. In conclusion, microbial biofilm communities initiated under either SeO42− or SO42−‐reducing conditions attained high SeO42− removal rates even though their microbial community composition was quite distinct. Biotechnol. Bioeng. 2016;113: 1736–1744.

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.