Bromate and Nitrate Bioreduction Coupled with Poly-ß-hydroxybutyrate Production in a Methane-Based Membrane Biofilm Reactor.

This work demonstrates bromate (BrO3-) reduction in a methane (CH4)-based membrane biofilm reactor (MBfR), and it documents contrasting impacts of nitrate (NO3-) on BrO3- reduction, as well as formation of poly-ß-hydroxybutyrate (PHB), an internal C- and electron-storage material. When the electron donor, CH4, was in ample supply, NO3- enhanced BrO3- reduction by stimulating the growth of denitrifying bacteria ( Meiothermus, Comamonadaceae, and Anaerolineaceae) able to reduce BrO3- and NO3- simultaneously. This was supported by increases in denitrifying enzymes (e.g., nitrate reductase, nitrite reductase, nitrous-oxide reductase, and nitric-oxide reductase) through quantitative polymerase chain reaction (qPCR) analysis and metagenomic prediction of these functional genes. When the electron donor was in limited supply, NO3- was the preferred electron acceptor over BrO3- due to competition for the common electron donor; this was supported by the significant oxidation of stored PHB when NO3- was high enough to cause electron-donor limitation. Methanotrophs (e.g., Methylocystis, Methylomonas, and genera within Comamonadaceae) were implicated as the main PHB producers in the biofilms, and their ability to oxidize PHB mitigated the impacts of competition for CH4.

Enrichment of denitratating bacteria from a methylotrophic denitrifying culture.

Denitratation (nitrite produced from nitrate), has the potential applications in wastewater treatment by combining with ANAMMOX process. The occurrence of denitratation has been shown to be effected qualitatively by various parameters in the environment. A more quantitative understanding can be obtained using enrichment cultures in lab-scale experiments, yet information on the enrichment of functional microorganisms responsible for denitratation is lacking. In this study, a stable denitratation-dominated culture was obtained from methylotrophic denitrifying culture. The results showed that, besides the substitution of acetate for methanol, the lasting starvation following saturation of electron donor was another pivotal selection pressure that favored the growth of denitratating bacteria, which was supported by the distinctive physiological strategy involving the higher growth rate combining with larger poly-hydroxybutyrate (PHB) accumulation at sufficient electron donor situation and then manage the stress of electron donor starvation by consumpiton of the PHB. High-throughput 16S rRNA gene sequencing analysis indicated that non-methylotrophic Halomonas campisalis (48.1 %) and Halomonas campaniensis (30.4 %) dominated in the denitratating community. Moreover the denitratation was driven by the nitrate inhibiting the nirS transcription in the Halomonas species.

Complete perchlorate reduction using methane as the sole electron donor and carbon source.

Using a CH4-based membrane biofilm reactor (MBfR), we studied perchlorate (ClO4(-)) reduction by a biofilm performing anaerobic methane oxidation coupled to denitrification (ANMO-D). We focused on the effects of nitrate (NO3(-)) and nitrite (NO2(-)) surface loadings on ClO4(-) reduction and on the biofilm community's mechanism for ClO4(-) reduction. The ANMO-D biofilm reduced up to 5 mg/L of ClO4(-) to a nondetectable level using CH4 as the only electron donor and carbon source when CH4 delivery was not limiting; NO3(-) was completely reduced as well when its surface loading was ≤ 0.32 g N/m(2)-d. When CH4 delivery was limiting, NO3(-) inhibited ClO4(-) reduction by competing for the scarce electron donor. NO2(-) inhibited ClO4(-) reduction when its surface loading was ≥ 0.10 g N/m(2)-d, probably because of cellular toxicity. Although Archaea were present through all stages, Bacteria dominated the ClO4(-)-reducing ANMO-D biofilm, and gene copies of the particulate methane mono-oxygenase (pMMO) correlated to the increase of respiratory gene copies. These pieces of evidence support that ClO4(-) reduction by the MBfR biofilm involved chlorite (ClO2(-)) dismutation to generate the O2 needed as a cosubstrate for the mono-oxygenation of CH4.

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.

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.

Interactions between perchlorate and nitrate reductions in the biofilm of a hydrogen-based membrane biofilm reactor.

We studied the microbial functional and structural interactions between nitrate (NO(3)(-)) and perchlorate (ClO(4)(-)) reductions in the hydrogen (H(2))-based membrane biofilm reactor (MBfR). When H(2) was not limiting, ClO(4)(-) and NO(3)(-) reductions were complete, and the MBfR's biofilm was composed mainly of bacteria from the ε- and ß-proteobacteria classes, with autotrophic genera Sulfuricurvum, Hydrogenophaga, and Dechloromonas dominating the biofilm. Based on functional-gene and pyrosequencing assays, Dechloromonas played the most important role in ClO(4)(-) reduction, while Sulfuricurvum and Hydrogenophaga were responsible for NO(3)(-) reduction. When H(2) delivery was insufficient to completely reduce both electron acceptors, NO(3)(-) reduction out-competed ClO(4)(-) reduction for electrons from H(2), and mixotrophs become important in the MBfR biofilm. ß-Proteobacteria became the dominant class, and Azonexus replaced Sulfuricurvum as a main genus. The changes suggest that facultative, NO(3)(-)-reducing bacteria had advantages over strict autotrophs when H(2) was limiting, because organic microbial products became important electron donors when H(2) was severely limiting.

Methane oxidation coupled to perchlorate reduction in a membrane biofilm batch reactor.

A specially designed CH4-based membrane biofilm batch reactor (MBBR) was applied to investigate anaerobic methane oxidation coupled to perchlorate reduction (AnMO-PR). The 0.21 mM ClO4- added in the first stage of operation was completely reduced in 28 days, 0.40 mM ClO4- was reduced within 23 days in stage 2, and 0.56 mM of ClO4- was reduced within 30 days in stage 3. Although some chlorate (ClO3-) accumulated, the recovery of Cl- was over 92%. Illumina sequencing of the 16S rRNA gene documented that the bacterial community was mainly composed by perchlorate-reducing bacteria (PRB), methanotrophic bacteria, and archaea. Real-time quantitative PCR showed the archaeal 16S rRNA and mcrA genes increased as more ClO4- was reduced, and the predominant archaea belonged to Methanosarcina mazei, which is related to ANME-3, an archaeon able to perform reverse methanogenesis. Several pieces of evidence support that ClO4- reduction by the MBBR biofilm occurred via a synergism between Methanosarcina and PRB: Methanosarcina oxidized methane through reverse methanogesis and provided electron donor for PRB to reduce ClO4-. Because methanotrophs were present, we cannot rule out that they also were involved in AnMO-PR if they received O2 generated by disproportionation of ClO2- from the PRB.

Dissolved oxygen has no inhibition on methane oxidation coupled to selenate reduction in a membrane biofilm reactor.

Methane oxidation coupled to selenate reduction has been suggested as a promising technology to bio-remediate selenium contaminated environments. However, the effect of dissolved oxygen (DO) on this process remained unclear. Here, we investigate the feasibility of selenate removal at two distinct DO concentrations. A membrane biofilm reactor (MBfR) was initially fed with ∼5 mg Se/L and then lowered to ∼1 mg Se/L of selenate, under anoxic condition containing ∼0.2 mg/L of influent DO. Selenate removal reached approximately 90% without selenite accumulation after one-month operation. Then 6-7 mg/L of DO was introduced and showed no apparent effect on selenate reduction in the subsequent operation. Electron microscopy suggested elevated oxygen exposure did not affect microbial shapes. 16S rDNA sequencing showed the aerobic methanotroph Methylocystis increased, while possible selenate reducers, Ignavibacterium and Bradyrhizobium, maintained stable after oxygen boost. Gene analysis indicated that nitrate/nitrite reductases positively correlated with selenate removal flux and were not remarkably affected by oxygen addition. Reversely, enzymes related with aerobic methane oxidation were obviously improved. This study provides a potential technology for selenate removal from oxygenated environments in a methane-based MBfR.

Formation of nanoscale Te0 and its effect on TeO32- reduction in CH4-based membrane biofilm reactor.

Formation and recovery of elemental tellurium (Te0) from wastewaters are required by increasing demands and scarce resources. Membrane biofilm reactor (MBfR) using gaseous electron donor has been reported as a low-cost and benign technique to reduce and recover metal (loids). In this study, we demonstrate the feasibility of nanoscale Te0 formation by tellurite (TeO32-) reduction in a CH4-based MBfR. Biogenic Te0 intensively attached on cell surface, within diameters ranging from 10 nm to 30 nm and the hexagonal nanostructure. Along with the Te0 formation, the TeO32- reduction was inhibited. After flushing, biofilm resumed the TeO32- reduction ability, suggesting that the formed nanoscale Te0 might inhibit the reduction by hindering substrate transfer of TeO32- to microbes. The 16S rRNA gene amplicon sequencing revealed that Thermomonas and Hyphomicrobium were possibly responsible for TeO32- reduction since they increased consecutively along with the experiment operation. The PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) analysis showed that the sulfite reductases were positively correlated with the TeO32- flux, indicating they were potential enzymes involved in reduction process. This study confirms the capability of CH4-based MBfR in tellurium reduction and formation, and provides more techniques for resources recovery and recycles.

Binding of Sudan II and IV to lecithin liposomes and E. coli membranes: insights into the toxicity of hydrophobic azo dyes.

BACKGROUND: Sudan red compounds are hydrophobic azo dyes, still used as food additives in some countries. However, they have been shown to be unsafe, causing tumors in the liver and urinary bladder in rats. They have been classified as category 3 human carcinogens by the International Agency for Research on Cancer. A number of hypotheses that could explain the mechanism of carcinogenesis have been proposed for dyes similar to the Sudan red compounds. Traditionally, investigations of the membrane toxicity of organic substances have focused on hydrocarbons, e.g. polycyclic aromatic hydrocarbons (PAHs), and DDT. In contrast to hydrocarbons, Sudan red compounds contain azo and hydroxy groups, which can form hydrogen bonds with the polar head groups of membrane phospholipids. Thus, entry may be impeded. They could have different toxicities from other lipophilic hydrocarbons. The available data show that because these compounds are lipophilic, interactions with hydrophobic parts of the cell are important for their toxicity. Lipophilic compounds accumulate in the membrane, causing expansion of the membrane surface area, inhibition of primary ion pumps and increased proton permeability. RESULTS: This work investigated the interactions of the amphiphilic compounds Sudan II and IV with lecithin liposomes and live Escherichia coli (E. coli). Sudan II and IV binding to lecithin liposomes and live E. coli corresponds to the Langmuir adsorption isotherm. In the Sudan red compounds--lecithin liposome solutions, the binding ratio of Sudan II to lecithin is 1/31 and that of Sudan IV to 1/314. The binding constant of the Sudan II-lecithin complex is 1.75 x 104 and that of the Sudan IV-lecithin complex 2.92 x 105. Besides, the influences of pH, electrolyte and temperature were investigated and analyzed quantitatively. In the Sudan red compounds--E.coli mixture, the binding ratios of Sudan II and Sudan IV to E.coli membrane phospholipid are 1/29 and 1/114. The binding constants of the Sudan II--and Sudan IV- E.coli membrane phospholipid complexes are 1.86 x 104 and 6.02 x 104. Over 60% of Sudan II and 75% of Sudan IV penetrated into E.coli, in which 90% of them remained in the E.coli membrane. CONCLUSION: Experiments of Sudan II and IV binding to lecithin liposomes and live E. coli indicates that amphiphilic compounds may be sequestered in the lecithin liposomes and membrane phospholipid bilayer according to the Langmuir adsorption law. Penetration into the cytosol was impeded and inhibited for Sudan red compounds. It is possible for such compounds themselves (excluding their metabolites and by-products)not result directly in terminal toxicity. Therefore, membrane toxicity could be manifested as membrane blocking and membrane expansion. The method established here may be useful for evaluating the interaction of toxins with membranes.

Effects of salinity on simultaneous reduction of perchlorate and nitrate in a methane-based membrane biofilm reactor.

This study builds upon prior work showing that methane (CH4) could be utilized as the sole electron donor and carbon source in a membrane biofilm reactor (MBfR) for complete perchlorate (ClO4-) and nitrate (NO3-) removal. Here, we further investigated the effects of salinity on the simultaneous removal of the two contaminants in the reactor. By testing ClO4- and NO3- at different salinities, we found that the reactor performance was very sensitive to salinity. While 0.2 % salinity did not significantly affect the hydrogen-based MBfR for ClO4- and NO3- removals, 1 % salinity completely inhibited ClO4- reduction and significantly lowered NO3- reduction in the CH4-based MBfR. In salinity-free conditions, NO3- and ClO4- removal fluxes were 0.171 g N/m2-day and 0.091 g/m2-day, respectively, but NO3- removal fluxes dropped to 0.0085 g N/m2-day and ClO4- reduction was completely inhibited when the medium changed to 1 % salinity. Scanning electron microscopy (SEM) showed that the salinity dramatically changed the microbial morphology, which led to the development of wire-like cell structures. Quantitative real-time PCR (qPCR) indicated that the total number of microorganisms and abundances of functional genes significantly declined in the presence of NaCl. The relative abundances of Methylomonas (methanogens) decreased from 31.3 to 5.9 % and Denitratisoma (denitrifiers) decreased from 10.6 to 4.4 % when 1 % salinity was introduced.

Removal of multiple electron acceptors by pilot-scale, two-stage membrane biofilm reactors.

We studied the performance of a pilot-scale membrane biofilm reactor (MBfR) treating groundwater containing four electron acceptors: nitrate (NO3(-)), perchlorate (ClO4(-)), sulfate (SO4(2-)), and oxygen (O2). The treatment goal was to remove ClO4(-) from ∼200 µg/L to less than 6 µg/L. The pilot system was operated as two MBfRs in series, and the positions of the lead and lag MBfRs were switched regularly. The lead MBfR removed at least 99% of the O2 and 63-88% of NO3(-), depending on loading conditions. The lag MBfR was where most of the ClO4(-) reduction occurred, and the effluent ClO4(-) concentration was driven to as low as 4 µg/L, with most concentrations ≤10 µg/L. However, SO4(2-) reduction occurred in the lag MBfR when its NO3(-) + O2 flux was smaller than ∼0.18 g H2/m(2)-d, and this was accompanied by a lower ClO4(-) flux. We were able to suppress SO4(2-) reduction by lowering the H2 pressure and increasing the NO3(-) + O2 flux. We also monitored the microbial community using the quantitative polymerase chain reaction targeting characteristic reductase genes. Due to regular position switching, the lead and lag MBfRs had similar microbial communities. Denitrifying bacteria dominated the biofilm when the NO3(-) + O2 fluxes were highest, but sulfate-reducing bacteria became more important when SO4(2-) reduction was enhanced in the lag MBfR due to low NO3(-) + O2 flux. The practical two-stage strategy to achieve complete ClO4(-) and NO3(-) reduction while suppressing SO4(2-) reduction involved controlling the NO3(-) + O2 surface loading between 0.18 and 0.34 g H2/m(2)-d and using a low H2 pressure in the lag MBfR.

Comparative transmembrane transports of four typical lipophilic organic chemicals.

Transmembrane transports of four kinds of lipophilic organic chemicals (LOCs) on suspending multilamellar liposomes (SML) and Escherichia coli (E. coli) were investigated, where both anthracene and phenanthrene were accorded to the lipid-water partition law and Sudan I and III to the Langmuir isothermal adsorption. Less than half of phenanthrene is transported into E. coli, where more than 60% are located in the cytoplasm. About 60% of anthracene entered the E. coli where only 10% was released into the cytoplasm. The partition coefficients of phenanthrene and anthracene partitioning from the extracellular liquid into membrane are 502 and 1190L/kg but their inverse partition coefficients are only 0.180 and 0.018kg/L. Over 60% of Sudan I and less than 40% of Sudan III accumulated on E. coli where most of them remained on the membrane. The transmembrane impedance effect (TMIE) is proposed for evaluating the cell-transport of polar LOCs.