Metatranscriptomic Analysis Reveals the Coexpression of Hydrogen-Producing and Homoacetogenesis Genes in Dark Fermentative Reactors Operated at High Substrate Loads.

Microbial communities in dark fermentation continuous systems are affected by substrate type, concentration, and product accumulation (e.g., H2 and CO2). Metatranscriptomics and quantitative PCR (qPCR) were used to assess how high organic loading rates (OLR) from 60 to 160 g total carbohydrates (TC)/L-d modify the microbial community diversity and expression of key dark fermentative genes. Overall, the microbial communities were composed of H2-producing bacteria (Clostridium butyricum), homoacetogens (Clostridium luticellarii), and lactic acid bacteria (Enteroccocus gallinarum and Leuconostoc mesenteroides). Quantification through qPCR showed that the abundance of genes encoding the formyltetrahydrofolate synthetase (fthfs, homoacetogens) and hydrogenase (hydA, H2-producing bacteria) was strongly associated with the OLR and H2 production performance. Similarly, increasing the OLR influenced the abundance of the gene transcripts responsible for H2 production and homoacetogenesis. To evaluate the effect of decreasing the H2 partial pressure, silicone oil was added to the reactor at an OLR of 138 and 160 g TC/L-d, increasing the production of H2, the copies of genes codifying for hydA and fthfs, and the genes transcripts related to H2 production and homoacetogenesis. Moreover, the metatranscriptomic analysis also showed that lactate-type fermentation and dark fermentation simultaneously occurred without compromising the reactor performance for H2 production.

Chlorate addition enhances perchlorate reduction in denitrifying membrane-biofilm reactors.

Perchlorate is a widespread drinking water contaminant with regulatory standards ranging from 2 to 18 µg/L. The hydrogen-based membrane-biofilm reactor (MBfR) can effectively reduce perchlorate, but it is challenging to achieve low-µg/L levels. We explored chlorate addition to increase the abundance of perchlorate-reducing bacteria (PRB) and improve removals. MBfR reactors were operated with and without chlorate addition. Results show that chlorate doubled the abundance of putative PRB (e.g., Rhodocyclales) and improved perchlorate reduction to 23 ± 17 µg/L, compared to 53 ± 37 µg/L in the control. Sulfate reduction was substantially inhibited during chlorate addition, but quickly recovered once suspended. Our results suggest that chlorate addition can enhance perchlorate reduction by providing a selective pressure for PRB. It also decreases net sulfate reduction. KEY POINTS: • Chlorate increased the abundance of perchlorate-reducing bacteria • Chlorate addition improved perchlorate removal • Chlorate appeared to suppress sulfate reduction.

Carboxylates and alcohols production in an autotrophic hydrogen-based membrane biofilm reactor.

Microbiological conversion of CO2 into biofuels and/or organic industrial feedstock is an excellent carbon-cycling strategy. Here, autotrophic anaerobic bacteria in the membrane biofilm reactor (MBfR) transferred electrons from hydrogen gas (H2 ) to inorganic carbon (IC) and produced organic acids and alcohols. We systematically varied the H2 -delivery, the IC concentration, and the hydraulic retention time in the MBfR. The relative availability of H2 versus IC was the determining factor for enabling microbial chain elongation (MCE). When the H2 :IC mole ratio was high (>2.0 mol H2 /mol C), MCE was an important process, generating medium-chain carboxylates up to octanoate (C8, 9.1 ± 1.3 mM C and 28.1 ± 4.1 mmol C m-2 d-1 ). Conversely, products with two carbons were the only ones present when the H2 :IC ratio was low (<2.0 mol H2 /mol C), so that H2 was the limiting factor. The biofilm microbial community was enriched in phylotypes most similar to the well-known acetogen Acetobacterium for all conditions tested, but phylotypes closely related with families capable of MCE (e.g., Bacteroidales, Rhodocyclaceae, Alcaligenaceae, Thermoanaerobacteriales, and Erysipelotrichaceae) became important when the H2 :IC ratio was high. Thus, proper management of IC availability and H2 supply allowed control over community structure and function, reflected by the chain length of the carboxylates and alcohols produced in the MBfR.

Microbial ecology in selenate-reducing biofilm communities: Rare biosphere and their interactions with abundant phylotypes.

Selenate (SeO42- ) reduction in hydrogen (H2 )-fed membrane biofilm reactors (H2 -MBfRs) was studied in combinations with other common electron acceptors. We employed H2 -MBfRs with two distinctly different conditions: R1, with ample electron-donor availability and acceptors SeO42- and sulfate (SO42- ), and R2, with electron-donor limitation and the presence of electron acceptors SeO42- , nitrate (NO3- ), and SO42- . Even though H2 was available to reduce all input SeO42- and SO42- in R1, SeO42- reduction was preferred over SO42- reduction. In R2, co-reduction of NO3- and SeO42- occurred, and SO42- reduction was mostly suppressed. Biofilms in all MBfRs had high microbial diversity that was influenced by the "rare biosphere" (RB), phylotypes with relative abundance less than 1%. While all MBfR biofilms had abundant members, such as Dechloromonas and Methyloversatilis, the bacterial communities were significantly different between R1 and R2. For R1, abundant genera were Methyloversatilis, Melioribacter, and Propionivibrio; for R2, abundant genera were Dechloromonas, Hydrogenophaga, Cystobacter, Methyloversatilis, and Thauera. Although changes in electron-acceptor or -donor loading altered the phylogenetic structure of the microbial communities, the biofilm communities were resilient in terms of SeO42- and NO3- reductions, because interacting members of the RB had the capacity of respiring these electron 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.

Accurate O2 delivery enabled benzene biodegradation through aerobic activation followed by denitrification-coupled mineralization.

Although benzene can be biodegraded when dissolved oxygen is sufficient, delivering oxygen is energy intensive and can lead to air stripping the benzene. Anaerobes can biodegrade benzene by using electron acceptors other than O2 , and this may reduce costs and exposure risks; the drawback is a remarkably slower growth rate. We evaluated a two-step strategy that involved O2 -dependent benzene activation and cleavage followed by intermediate oxidation coupled to NO3- respiration. We employed a membrane biofilm reactor (MBfR) featuring nonporous hollow fibers as the means to deliver O2 directly to a biofilm at an accurately controlled rate. Benzene was mineralized aerobically when the O2 -supply rate was more than sufficient for mineralization. As the O2 -supply capacity was systematically lowered, O2 respiration was gradually replaced by NO3- respiration. When the maximum O2 -supply capacity was only 20% of the demand for benzene mineralization, O2 was used almost exclusively for benzene activation and cleavage, while respiration was almost only by denitrification. Analyses of microbial community structure and predicted metagenomic function reveal that Burkholderiales was dominant and probably utilized monooxygenase activation, with subsequent mineralization coupled to denitrification; strict anaerobes capable of carboxylative activation were not detected. These results open the door for a promising treatment strategy that simultaneously ameliorates technical and economic challenges of aeration and slow kinetics of anaerobic activation of aromatics.

Effect of Supercritical Carbon Dioxide Extraction Parameters on the Biological Activities and Metabolites Present in Extracts from Arthrospira platensis.

Arthrospira platensis was used to obtain functional extracts through supercritical carbon dioxide extraction (SFE-CO2). Pressure (P), temperature (T), co-solvent (CX), static extraction (SX), dispersant (Di) and dynamic extraction (DX) were evaluated as process parameters through a Plackett-Burman design. The maximum extract yield obtained was 7.48 ± 0.15% w/w. The maximum contents of bioactive metabolites in extracts were 0.69 ± 0.09 µg/g of riboflavin, 5.49 ± 0.10 µg/g of α-tocopherol, 524.46 ± 0.10 µg/g of ß-carotene, 1.44 ± 0.10 µg/g of lutein and 32.11 ± 0.12 mg/g of fatty acids with 39.38% of palmitic acid, 20.63% of linoleic acid and 30.27% of γ-linolenic acid. A. platensis extracts had an antioxidant activity of 76.47 ± 0.71 µg GAE/g by Folin-Ciocalteu assay, 0.52 ± 0.02, 0.40 ± 0.01 and 1.47 ± 0.02 µmol TE/g by DPPH, FRAP and TEAC assays, respectively. These extracts showed antimicrobial activity against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and Candida albicans ATCC 10231. Overall, co-solvent was the most significant factor for all measured effects (p < 0.05). Arthrospira platensis represents a sustainable source of bioactive compounds through SFE using the following extraction parameters P: 450 bar, CX: 11 g/min, SX: 15 min, DX: 25 min, T: 60 °C and Di: 35 g.

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

Remediation of selenate (SeO4 (2-) ) contamination through microbial reduction is often challenging due to the presence of sulfate (SO4 (2-) ), which can lead to competition for the electron donor and the co-production of toxic H2 S. Microbial reduction of SeO4 (2-) in the presence of SO4 (2-) was studied in two hydrogen-based membrane biofilm reactors (MBfRs). One MBfR was initiated with SO4 (2-) -reducing conditions and gradually shifted to SeO4 (2-) reduction. The second MBfR was developed with a SeO4 (2-) -reducing biofilm, followed by SO4 (2-) introduction. Biofilms within both MBfRs achieved greater than 90% SeO4 (2-) reduction, even though the SeO4 (2-) 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 SeO4 (2-) loading. Specifically, high SeO4 (2-) 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). SeO4 (2-) 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 SeO4 (2-) or SO4 (2-) -reducing conditions attained high SeO4 (2-) removal rates even though their microbial community composition was quite distinct. Biotechnol. Bioeng. 2016;113: 1736-1744. © 2016 Wiley Periodicals, Inc.

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 ß-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.

Pyrosequencing analysis yields comprehensive assessment of microbial communities in pilot-scale two-stage membrane biofilm reactors.

We studied the microbial community structure of pilot two-stage membrane biofilm reactors (MBfRs) designed to reduce nitrate (NO3(-)) and perchlorate (ClO4(-)) in contaminated groundwater. The groundwater also contained oxygen (O2) and sulfate (SO4(2-)), which became important electron sinks that affected the NO3(-) and ClO4(-) removal rates. Using pyrosequencing, we elucidated how important phylotypes of each "primary" microbial group, i.e., denitrifying bacteria (DB), perchlorate-reducing bacteria (PRB), and sulfate-reducing bacteria (SRB), responded to changes in electron-acceptor loading. UniFrac, principal coordinate analysis (PCoA), and diversity analyses documented that the microbial community of biofilms sampled when the MBfRs had a high acceptor loading were phylogenetically distant from and less diverse than the microbial community of biofilm samples with lower acceptor loadings. Diminished acceptor loading led to SO4(2-) reduction in the lag MBfR, which allowed Desulfovibrionales (an SRB) and Thiothrichales (sulfur-oxidizers) to thrive through S cycling. As a result of this cooperative relationship, they competed effectively with DB/PRB phylotypes such as Xanthomonadales and Rhodobacterales. Thus, pyrosequencing illustrated that while DB, PRB, and SRB responded predictably to changes in acceptor loading, a decrease in total acceptor loading led to important shifts within the "primary" groups, the onset of other members (e.g., Thiothrichales), and overall greater diversity.

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 (ClO(4)(-) ) at ~10 mg/L, an unusually high concentration. To enhance ClO(4)(-) removal, we either increased the H2 pressure or decreased the electron-acceptor surface loading. The ClO(4)(-) 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 /m(2) day. We then decreased the acceptor surface loading stepwise from 0.49 to 0.07 g H2 /m(2) day, and the ClO(4)(-) removal improved to 99.6%, giving an effluent ClO(4)(-) concentration of 41 µg/L. However, the tradeoff was that sulfate (SO(4)(2-) ) reduction occurred, reaching 85% conversion at the lowest acceptor surface loading (0.07 g H(2) /m(2) day). In two steady states with the highest ClO(4)(-) 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 ClO(4)(-) concentration below 41 µg/L.

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 H(2)-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 H(2) as their electron donor. To evaluate the model, the simulated effluent H(2), 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 fiber's 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.

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, ß-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.

Identifying biodegradation pathways of cetrimonium bromide (CTAB) using metagenome, metatranscriptome, and metabolome tri-omics integration.

Traditional research on biodegradation of emerging organic pollutants involves slow and labor-intensive experimentation. Currently, fast-developing metagenome, metatranscriptome, and metabolome technologies promise to expedite mechanistic research on biodegradation of emerging organic pollutants. Integrating the metagenome, metatranscriptome, and metabolome (i.e., tri-omics) makes it possible to link gene abundance and expression with the biotransformation of the contaminant and the formation of metabolites from this biotransformation. In this study, we used this tri-omics approach to study the biotransformation pathways for cetyltrimethylammonium bromide (CTAB) under aerobic conditions. The tri-omics analysis showed that CTAB undergoes three parallel first-step mono-/di-oxygenations (to the α, ß, and ω-carbons); intermediate metabolites and expressed enzymes were identified for all three pathways, and the ß-carbon mono-/di-oxygenation is a novel pathway; and the genes related to CTAB biodegradation were associated with Pseudomonas spp. Four metabolites - palmitic acid, trimethylamine N-oxide (TMAO), myristic acid, and betaine - were the key identified biodegradation intermediates of CTAB, and they were associated with first-step mono-/di-oxygenations at the α/ß-C. This tri-omics approach with CTAB demonstrates its power for identifying promising paths for future research on the biodegradation of complex organics by microbial communities.

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 SRB's 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)(-).

Increased expression of antibiotic-resistance genes in biofilm communities upon exposure to cetyltrimethylammonium bromide (CTAB) and other stress conditions.

Quaternary ammonium compounds (QAC, e.g., cetyltrimethylammonium bromide, (CTAB)) are widely used as surfactants and disinfectants. QAC already are commonly found in wastewaters, and their concentration could increase, since QAC are recommended to inactivate the SARS-CoV-2 (COVID-19) virus. Exposure of bacteria to QAC can lead to proliferation of antibiotic resistance genes (ARG). In particular, O2-based membrane biofilm reactors (O2-MBfRs) achieved excellent CTAB biodegradation, but ARG increased in their biofilms. Here, we applied meta-transcriptomic analyses to assess the impacts of CTAB exposure and operating conditions on microbial community's composition and ARG expression in the O2-MBfRs. Two opportunistic pathogens, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, dominated the microbial communities and were associated with the presence of ARG. Operating conditions that imposed stress on the biofilms, i.e., limited supplies of O2 and nitrogen or a high loading of CTAB, led to large increases in ARG expression, particularly for genes conferring antibiotic-target protection. Important within the efflux pumps was the Resistance-Nodulation-Division (RND) family, which may have been active in exporting CTAB from cells. Oxidative stress appeared to be the key factor that triggered ARG proliferation by selecting intrinsically resistant species and accentuating the expression of ARG. Our findings suggest that means to mitigate the spread of ARG, such as shown here in a O2-based membrane biofilm reactor, need to consider the impacts of stressors, including QAC exposure and stressful operating conditions.

Antimonate removal by diatomite modified with Fe-Mn oxides: application and mechanism study.

In this study, diatomite coated with Fe-Mn oxides (DFMO) was synthesized through calcination. The adsorption of antimonate (Sb(V)) by DFMO was studied, and environmental factors affecting the adsorption were investigated. The components of DFMO were identified as γ-Fe2O3, γ-MnO2, and SiO2, in the presence of diatomite covered with nanoscale metal oxides. Batch experiments were carried out to evaluate the antimonate adsorption performance in aqueous solution. Results showed that maximum Sb(V) adsorption capacity of DFMO reached 10.7 mg/g at pH 4, corresponding to 22.2 mg/g per unit metal oxides. Antimonate adsorption occurred on heterogenous surface, following the Freundlich and Pseudo-second order model. Overall, antimonate adsorption was favored at acidic condition due to low point of zero charge. However, when treating electroplating wastewater, neutral pH condition exhibited a higher efficiency than acidic pH, because co-existing ions in electroplating wastewater significantly affects antimony adsorption. Further investigation showed that among different potential co-existing ions, fluoride can strongly inhibit the adsorption of antimonate at 5 mg/L under pH 4. Density functional theory (DFT) analysis confirmed that adsorption energy on DFMO follows: HF < F- < Sb(OH)6-, indicating that fluoride is easier to bind with DFMO compared to antimonate, especially under pH 3.5 at which fluoride exists as HF. Moreover, the competitive adsorption of fluoride toward antimonate indicated the necessity of pre-treatment like neutralization and precipitation before adsorption process.

Hydrogenotrophic Microbial Reduction of Oxyanions With the Membrane Biofilm Reactor.

Oxyanions, such as nitrate, perchlorate, selenate, and chromate are commonly occurring contaminants in groundwater, as well as municipal, industrial, and mining wastewaters. Microorganism-mediated reduction is an effective means to remove oxyanions from water by transforming oxyanions into harmless and/or immobilized forms. To carry out microbial reduction, bacteria require a source of electrons, called the electron-donor substrate. Compared to organic electron donors, H2 is not toxic, generates minimal secondary contamination, and can be readily obtained in a variety of ways at reasonable cost. However, the application of H2 through conventional delivery methods, such as bubbling, is untenable due to H2's low water solubility and combustibility. In this review, we describe the membrane biofilm reactor (MBfR), which is a technological breakthrough that makes H2 delivery to microorganisms efficient, reliable, and safe. The MBfR features non-porous gas-transfer membranes through which bubbleless H2 is delivered on-demand to a microbial biofilm that develops naturally on the outer surface of the membranes. The membranes serve as an active substratum for a microbial biofilm able to biologically reduce oxyanions in the water. We review the development of the MBfR technology from bench, to pilot, and to commercial scales, and we elucidate the mechanisms that control MBfR performance, particularly including methods for managing the biofilm's structure and function. We also give examples of MBfR performance for cases of treating single and co-occurring oxyanions in different types of contaminated water. In summary, the MBfR is an effective and reliable technology for removing oxyanion contaminants by accurately providing a biofilm with bubbleless H2 on demand. Controlling the H2 supply in accordance to oxyanion surface loading and managing the accumulation and activity of biofilm are the keys for process success.

Enhancing denitrification using a novel in situ membrane biofilm reactor (isMBfR).

The insufficient supply of electron donor in surface water contaminated with nitrate leads to its incomplete reduction in natural or constructed wetlands. Although the addition of organic matter (represented as chemical oxygen demand, COD) can stimulate N removal by denitrification, direct supplementation of COD creates unacceptable risks to effluent quality. An alternative for stimulating denitrification is supplying hydrogen gas (H2) as an inorganic electron donor. We evaluate an innovative means to do H2-based denitrification of surface waters in a wetland setting: the in-situ membrane biofilm reactor (isMBfR), in which H2 is delivered to a biofilm of denitrifying bacteria on demand based on the presence of nitrate. We carried out a proof-of-concept study in which an upper "photo zone" and a lower "MBfR root zone" were combined to remove nitrate and COD from simulated surface water. Employing mass-balances for H2, COD, nitrate, and oxygen, we documented nearly complete removals of nitrate and COD, except when the H2 supply was intentionally shut off. All nitrate removal was accomplished in the "MBfR root zone," where H2 delivery supplemented the COD supply (as needed) and provided the large majority of electron equivalents to reduce nitrate to N2.