Methane-based Membrane Biofilm Reactor Research Articles

Microbial Stratification Affects Conversions of Nitrogen and Methane in Biofilms Coupling Anammox and n-DAMO Processes.

A methane-based membrane biofilm reactor (MBfR) has a suitable configuration to incorporate anammox and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) processes because of its high gas-transfer efficiency and efficient biomass retention. In this study, the spatial distribution of microorganisms along with the biofilm depth in methane-based MBfRs was experimentally revealed, showing the dominance of anammox bacteria, n-DAMO bacteria, and n-DAMO archaea in the outer layer, middle layer, and inner layer of biofilms, respectively. The long-term and short-term experimental investigations in conjunction with mathematical modeling collectively revealed that microorganisms living in the outer layer of biofilms tend to use substrates from wastewater, while microorganisms inhabiting the inner layer of biofilms tend to use substrates originating from biofilm substratum. Specifically, anammox bacteria dominating the biofilm surface preferentially removed the nitrite provided from wastewater, while n-DAMO bacteria mostly utilized the nitrite generated from n-DAMO archaea as these two methane-related populations spatially clustered together inside the biofilm. Likewise, the methane supplied from the membrane was mostly consumed by n-DAMO archaea, while the dissolved methane in wastewater would be primarily utilized by n-DAMO bacteria. This study offers novel insights into the impacts of microbial stratification in biofilm systems, not only expanding the fundamental understanding of biofilms and microbial interactions therein but also providing a rationale for the potential applications of methane-based MBfRs in sewage treatment.

Model-based analyses of chromate, selenate and sulfate reduction in a methane-based membrane biofilm reactor.

Selenate (SeO42-) and sulfate (SO42-) are frequently present together with chromate (CrO42-) in certain industrial wastewaters. SeO42- and CrO42- are required to be reduced while SO42- reduction should be minimized to avoid the production of toxic sulfide. In this study, a modified biofilm model was employed to investigate the interactions between CrO42-, SeO42- and SO42- bioreduction in a methane (CH4)-based membrane biofilm reactor (MBfR). The model was calibrated using steady-state experimental data of two reported CH4-based MBfRs reducing these oxyanions. The modeling results suggested that the majority of methanotrophs (>80%) were located in the outer layer of the biofilm, while the oxyanions-reducing bacteria preferred to grow close to the membrane. The introduction of SeO42- or SO42- enriched selenate/sulfate-reducing bacteria (SeRB/SRB) but decreased the abundance of chromate-reducing bacteria (CRB). A biofilm thickness of >300μm, an HRT of higher than 4h and an influent dissolved oxygen concentration of 0.3mg /L were favorable for simultaneous high-level CrO42- and SeO42- removal. A two-stage MBfR system with optimized operational conditions showed promise in retaining high-purity (>98%) selenium nanoparticles when treating both CrO42- and SeO42- impacted wastewaters. Moreover, the model indicated that efficient CrO42- removal (>90%) along with minor SO42- reduction (<10%) could be realized via maintaining appropriate biofilm thickness (200-250μm) and influent dissolved oxygen (0.7-0.8mg /L) in a single MBfR. These findings offer insights for the design and operation of CH4-based technology for remediating CrO42- contaminated industrial wastewaters.

Copper stimulation on methane-supported perchlorate reduction in a membrane biofilm reactor

The present study demonstrated that the perchlorate reduction rate in a methane-based membrane biofilm reactor was significantly enhanced from 14.4 to 25.6 mg-Cl/L/d by increasing copper concentration in the feeding medium from 1 to 10 μM, indicating a stimulatory effect of copper on the methane-supported perchlorate reduction process. Batch tests further confirmed that the increased copper concentration enhanced both methane oxidation and perchlorate reduction rates, which was supported by an increasing trend of functional genes (pmoA for methanotrophs and pcrA for specific perchlorate reducers) abundances through quantitative polymerase chain reaction (qPCR). Both 16S rRNA gene sequencing and functional genes (pmoA and pcrA) sequencing jointly revealed that the biofilm supplied with a higher copper concentration exhibited a more diverse microbial community. The methane-supported perchlorate reduction was accomplished through a synergistic association of methanotrophs (Methylocystis, Methylomonas, and Methylocystaceae) and perchlorate reducers (Dechloromonas, Azospira, Magnetospirillum, and Denitratisoma). Acetate may function as the key syntrophic linkage between methanotrophs and perchlorate reducers. It was proposed that the increased copper concentration improved the activity of particulate methane monooxygenase (pMMO) for methane oxidation or promoted the biosynthesis of intracellular carbon storage compounds polyhydroxybutyrate (PHB) in methanotrophs for generating more acetate available for perchlorate reduction.

Roles of Oxygen in Methane-dependent Selenate Reduction in a Membrane Biofilm Reactor: Stimulation or Suppression

Although methane (CH4) has been proven to be able to serve as an electron donor for bio-reducing various oxidized contaminants (e.g., selenate (SeO42−)), little is known regarding the roles of oxygen in methane-based reduction processes. Here, a methane-based membrane biofilm reactor (MBfR) was established for evaluating the effects of oxygen supply rates on selenate reduction performance and microbial communities. The oxygen supply rate played a dual role (stimulatory or suppressive effect) in selenate reduction rates, depending on the presence or absence of dissolved oxygen (DO). Specifically, selenate reduction rate was substantially enhanced when an appropriate oxygen rate (e.g., 12 to 184 mg/L.d in this study) was supplied but with negligible DO. The highest selenate reduction rate (up to 34 mg-Se/L.d) was obtained under an oxygen supply rate of 184 mg/L.d. In contrast, excessive oxygen supply rate (626 mg/L.d) would significantly suppress selenate reduction rate under DO level of 3 mg/L. Accordingly, though the high oxygen supply rate (626 mg/L.d) would promote the expression of pmoA (5.9 × 109 copies g−1), the expression level of narG (a recognized gene to mediate selenate reduction) would be significantly downregulated (6.1 × 109 copies g−1), thus suppressing selenate reduction. In contrast, the expression of narG gene significantly increased to 2.8 × 1010 copies g−1, and the expression of pmoA gene could still maintain at 1.1 × 109 copies g−1 under an oxygen supply rate of 184 mg/L.d. High-throughput sequencing targeting 16S rRNA gene, pmoA, and narG collectively suggested Methylocystis acts as the major aerobic methanotroph, in synergy with Arthrobacter and Variovorax which likely jointly reduce selenate to selenite (SeO32−), and further to elemental selenium (Se0). Methylocystis was predominant in the biofilm regardless of variations of oxygen supply rates, while Arthrobacter and Variovorax were sensitive to oxygen fluctuation. These findings provide insights into the effects of oxygen on methane-dependent selenate reduction and suggest that it is feasible to achieve a higher selenate removal by regulating oxygen supply rates.

Applications of methane-based membrane biofilm reactor for oxidized pollutants removal from wastewater: a short review

Methane-based membrane biofilm reactor (CH4-MBfR) is an emerging wastewater treatment technology based on gas-permeable membranes for oxidized pollutants (e.g., NO3 -) abatement. This paper reviews the operation principle of CH4-MBfR, the involved pathways in the aerobic/anaerobic methane oxidation coupled to denitrification (AMO/ANMO-D) process, as well as the recent key advances of CH4-MBfR for environmental applications.

Efficient nitrate removal from synthetic groundwater via in situ utilization of short-chain fatty acids from methane bioconversion

Abstract Nitrate (NO3−) removal from groundwater is of great importance since it has posed a major risk to human health. Although methane-based denitrification processes have been developed, the practical application of methane as a carbon source for denitrification is constrained by either a long start-up time or a relatively low nitrate removal rate. This study investigated nitrate removal capacity by using a methane-based membrane biofilm reactor (MBfR) feeding with synthetic groundwater. A high nitrate removal rate of 706 mg-N L−1 d−1 was achieved, which is by far the highest rate reported for methane-based denitrification reactors. Short-chain fatty acids (SCFAs) were detected as products of methane bioconversion, with concentrations of acetic and propionic acid up to 168.8 mg L−1 and 50.2 mg L−1, respectively. Long-term operation and batch test results revealed that the SCFAs generated from methane bioconversion were in situ utilized for supporting nitrate removal as carbon and electron sources, thus leading to a practically useful removal rate. The findings suggest that it is promising to apply the methane-based MBfR to remove nitrate from nitrate contaminated groundwater.

Model-based assessment of chromate reduction and nitrate effect in a methane-based membrane biofilm reactor

Chromate contamination can pose a high risk to both the environment and public health. Previous studies have shown that CH4-based membrane biofilm reactor (MBfR) is a promising method for chromate removal. In this study, we developed a multispecies biofilm model to study chromate reduction and its interaction with nitrate reduction in a CH4-based MBfR. The model-simulated results were consistent with the experimental data reported in the literature. The model showed that the presence of nitrate in the influent promoted the growth of heterotrophs, while suppressing methanotrophs and chromate reducers. Moreover, it indicated that a biofilm thickness of 150 μm and an influent dissolved oxygen concentration of 0.5 mg O2/L could improve the reactor performance by increasing the chromate removal efficiency under the simulated conditions.

Methane-based denitrification kinetics and syntrophy in a membrane biofilm reactor at low methane pressure

A methane-based membrane biofilm reactor (MBfR) was assessed for a tertiary nitrogen removal process in domestic wastewater treatment. To mitigate effluent dissolved methane concentrations, the MBfR was operated with a 20% methane mixing ratio and a low pressure of 0.003 atm. The nitrate concentration was reduced from 20 to 4 mg/L with a low methane concentration of 3.3 mg/L in the effluent at 4 h hydraulic retention time (HRT). An in situ dissolved oxygen sensor showed a concentration of 0.045 mg/L in the MBfR, demonstrating methane oxidation under hypoxic conditions. Both 16S rRNA gene sequencing and metagenomic analysis identified bacteria capable of oxidation of methane coupled to denitrification (Methylocystis), whereas other bacteria were implicated in either methane oxidation (Methylococcus) or nitrate reduction (Escherichia). Reduced genetic potential for nitrate reduction to nitrite at a shorter HRT coincided with a decreased efficiency of denitrification, suggesting rate limitation by this initial step of denitrification. Genes encoding nitrite reduction to dinitrogen were at similar relative abundance under both HRT conditions. Our results provide mechanistic evidence for microbial syntrophy between aerobic methanotrophs and denitrifiers in methane-fed MBfRs operated under varying HRTs, with important implications for novel biological nitrogen removal to dilute wastewater.

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.

Perchlorate bio-reduction in a methane-based membrane biofilm reactor in the presence and absence of oxygen

Perchlorate has been widely detected in various water environments and could cause serious health problems. Methane has been proposed as a promising electron donor to remove perchlorate from contaminated water, yet it is unclear whether and how microbial methane oxidation couples with perchlorate reduction, in particular under anoxic conditions. Here, the feasibility and performance of perchlorate reduction driven by methane in the presence and absence of oxygen were investigated and compared in a lab-scale methane-based membrane biofilm reactor. Long-term operational performance suggested that perchlorate was reduced to chloride, with 4 mg Cl/L/d of perchlorate removal rate under anoxic conditions. Differently, perchlorate removal rate increased to 16 mg Cl/L/d, and volatile fatty acids (VFAs) were produced from methane partial oxidation when a limited oxygen (10 mg/L/d) was externally supplied. Regardless of oxygen conditions, microbial perchlorate reduction driven by methane might be mediated through synergistic interactions by a microbial consortium, but with different key microbial members under both oxygen regimes. Under anoxic conditions, aerobic methanotrophs (likely Methylocystaceae and Methylococcaceae) might micro-aerobically oxidize methane by utilizing internal oxygen from microbial perchlorate reduction, which might be mediated by Rhodocyclaceae. In contrast, under oxygen-limiting conditions, methanogens (e.g., Methanosarcina) and fermenters (e.g., Veillonellaceae) likely jointly converted methane into VFAs, then dissimilatory perchlorate-reducing bacteria (e.g., Rhodocyclaceae) utilized the produced VFAs to reduce perchlorate to chloride. Our findings provide evidence to link methane oxidation with perchlorate reduction under both oxygen regimes, which could be facilitated to design a process to remove perchlorate from groundwater.

Role of Extracellular Polymeric Substances in a Methane Based Membrane Biofilm Reactor Reducing Vanadate

For the first time, we demonstrated vanadate (V(V)) reduction in a membrane biofilm reactor (MBfR) using CH4 as the sole electron donor. The V(V)-reducing capability of the biofilm kept increasing, with complete removal of V(V) achieved when the influent surface loading of V(V) was 363 mg m-2 day-1. Almost all V(V) was reduced to V(IV) precipitates, which is confirmed by a scanning electron microscope coupled to energy dispersive X-ray spectroscopy (SEM-EDS) and X-ray photoelectron spectroscopy (XPS). Microbial community analysis revealed that denitrifiers Methylomonas and Denitratisoma might be the main genera responsible for V(V) reduction. The constant enrichment of Methylophilus suggests that the intermediate (i.e., methanol) from CH4 metabolism might be used as the electron carriers for V(V) bioreduction. Intrusion of V(V) (2-5 mg/L, at the surface loading of 150-378 mg m-2 day-1) into the biofilm stimulated the secretion of extracellular polymeric substances (EPS), but high loading of V(V) (10 mg/L, at the surface loading of 668 mg m-2 day-1) decreased the amount of EPS. Metagenomic prediction analysis established the strong correlation between the secretion of EPS and the microbial metabolism associated with V(V) reduction, tricarboxylic acid cycle (TCA) cycle, methane oxidation, and ATP production, and EPS might relieve the oxidative stress induced by high loading of V(V). Colorimetric determination and a three-dimensional excitation-emission matrix (3D-EEM) showed that tryptophan and humic acid-like substances might play important roles in microbial cell protection and V(V) binding. Fourier transform infrared (FTIR) spectroscopy identified hydroxyl (-OH) and carboxyl (COO-) groups in EPS as the candidate functional groups for binding V(V).

Bromate and Nitrate Bioreduction Coupled with Poly-β-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-β-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.

A biofilm model for assessing perchlorate reduction in a methane-based membrane biofilm reactor

Perchlorate (ClO4−) is recognized as an important contaminant in surface water and groundwater, which would pose health risks at very low concentrations. A methane-based membrane biofilm reactor (MBfR) has been successfully demonstrated for perchlorate reduction, which provided an alternative solution for perchlorate remediation with low cost. In this work, a multispecies biofilm model was developed to evaluate perchlorate reduction in the methane-based MBfR under different operational conditions. The model was calibrated and validated using the experimental data from the long-term operation of the MBfR at seven distinct stages. The results suggested that the developed model could satisfactorily describe perchlorate reduction and denitrification performances in the MBfR (R2>0.9). The modeling results provided insight into the microbial community distribution in the biofilm, with aerobic methanotrophs and perchlorate reduction bacteria being mainly located at the membrane side (∼60%) and heterotrophic bacteria being situated near the liquid side (∼50%). The model simulations indicated that over 80% of perchlorate removal efficiency could be achieved through controlling the optimal combinations of methane pressure (PCH4) and perchlorate loading (LClO4) (e.g., applying a PCH4 of 30kPa at a LClO4 of 0.08gCl/m2/d). In addition, the perchlorate reduction would be inhibited by the presence of nitrate and nitrite in the MBfR, which should be appropriately controlled during the future practical application of the promising process.

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 0.171g N/m2-day and 0.091g/m2-day, respectively, but NO3- removal fluxes dropped to 0.0085g 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.

Bioreduction of Chromate in a Methane-Based Membrane Biofilm Reactor.

For the first time, we demonstrate chromate (Cr(VI)) bioreduction using methane (CH4) as the sole electron donor in a membrane biofilm reactor (MBfR). The experiments were divided into five stages lasting a total of 90 days, and each stage achieved a steady state for at least 15 days. Due to continued acclimation of the microbial community, the Cr(VI)-reducing capacity of the biofilm kept increasing. Cr(VI) removal at the end of the 90-day test reached 95% at an influent Cr(VI) concentration of 3 mg Cr/L and a surface loading of 0.37g of Cr m(-2) day(-1). Meiothermus (Deinococci), a potential Cr(VI)-reducing bacterium, was negligible in the inoculum but dominated the MBfR biofilm after Cr(VI) was added to the reactor, while Methylosinus, a type II methanotrophs, represented 11%-21% of the total bacterial DNA in the biofilm. Synergy within a microbial consortia likely was responsible for Cr(VI) reduction based on CH4 oxidation. In the synergy, methanotrophs fermented CH4 to produce metabolic intermediates that were used by the Cr(VI)-reducing bacteria as electron donors. Solid Cr(III) was the main product, accounting for more than 88% of the reduced Cr in most cases. Transmission electron microscope (TEM) and energy dispersive X-ray (EDS) analysis showed that Cr(III) accumulated inside and outside of some bacterial cells, implying that different Cr(VI)-reducing mechanisms were involved.

Evolution of the microbial community of the biofilm in a methane-based membrane biofilm reactor reducing multiple electron acceptors.

Previous work documented complete perchlorate reduction in a membrane biofilm reactor (MBfR) using methane as the sole electron donor and carbon source. This work explores how the biofilm's microbial community evolved as the biofilm stage-wise reduced different combinations of perchlorate, nitrate, and nitrite. The initial inoculum, carrying out anaerobic methane oxidation coupled to denitrification (ANMO-D), was dominated by uncultured Anaerolineaceae and Ferruginibacter sp. The microbial community significantly changed after it was inoculated into the CH4-based MBfR and fed with a medium containing perchlorate and nitrite. Archaea were lost within the first 40days, and the uncultured Anaerolineaceae and Ferruginibacter sp. also had significant losses. Replacing them were anoxic methanotrophs, especially Methylocystis, which accounted for more than 25% of total bacteria. Once the methanotrophs became important, methanol-oxidizing denitrifying bacteria, namely, Methloversatilis and Methylophilus, became important in the biofilm, probably by utilizing organic matter generated by the metabolism of methanotrophs. When methane consumption was equal to the maximum-possible electron-donor supply, Methylomonas, also an anoxic methanotroph, accounted for >10% of total bacteria and remained a major part of the community until the end of the experiments. We propose that aerobic methane oxidation coupled to denitrification and perchlorate reduction (AMO-D and AMO-PR) directly oxidized methane and reduced NO3 (-) to NO2 (-) or N2O under anoxic condition, producing organic matter for methanol-assimilating denitrification and perchlorate reduction (MA-D and MA-PR) to reduce NO3 (-). Simultaneously, bacteria capable of anaerobic methane oxidation coupled to denitrification and perchlorate reduction (ANMO-D and ANMO-PR) used methane as the electron donor to respire NO3 (-) or ClO4 (-) directly. Graphical Abstract ᅟ.