Co-Removal of Perfluorooctanoic Acid and Nitrate from Water by Coupling Pd Catalysis with Enzymatic Biotransformation.

PFAS (poly- and per-fluorinated alkyl substances) represent a large family of recalcitrant organic compounds that are widely used and pose serious threats to human and ecosystem health. Here, palladium (Pd0)-catalyzed defluorination and microbiological mineralization were combined in a denitrifying H2-based membrane biofilm reactor to remove co-occurring perfluorooctanoic acid (PFOA) and nitrate. The combined process, i.e., Pd-biofilm, enabled continuous removal of ∼4 mmol/L nitrate and ∼1 mg/L PFOA, with 81% defluorination of PFOA. Metagenome analysis identified bacteria likely responsible for biodegradation of partially defluorinated PFOA: Dechloromonas sp. CZR5, Kaistella koreensis, Ochrobacterum anthropic, and Azospira sp. I13. High-performance liquid chromatography-quadrupole time-of-flight mass spectrometry and metagenome analyses revealed that the presence of nitrate promoted microbiological oxidation of partially defluorinated PFOA. Taken together, the results point to PFOA-oxidation pathways that began with PFOA adsorption to Pd0, which enabled catalytic generation of partially or fully defluorinated fatty acids and stepwise oxidation and defluorination by the bacteria. This study documents how combining catalysis and microbiological transformation enables the simultaneous removal of PFOA and nitrate.

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.

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.

Para-Chlorophenol (4-CP) Removal by a Palladium-Coated Biofilm: Coupling Catalytic Dechlorination and Microbial Mineralization via Denitrification.

Rapid dechlorination and full mineralization of para-chlorophenol (4-CP), a toxic contaminant, are unfulfilled goals in water treatment. Means to achieve both goals stem from the novel concept of coupling catalysis by palladium nanoparticles (PdNPs) with biodegradation in a biofilm. Here, we demonstrate that a synergistic version of the hydrogen (H2)-based membrane biofilm reactor (MBfR) enabled simultaneous removals of 4-CP and cocontaminating nitrate. In situ generation of PdNPs within the MBfR biofilm led to rapid 4-CP reductive dechlorination, with >90% selectivity to more bioavailable cyclohexanone. Then, the biofilm mineralized the cyclohexanone by utilizing it as a supplementary electron donor to accelerate nitrate reduction. Long-term operation of the Pd-MBfR enriched the microbial community in cyclohexanone degraders within Clostridium, Chryseobacterium, and Brachymonas. In addition, the PdNP played an important role in accelerating nitrite reduction; while NO3- reduction to NO2- was entirely accomplished by bacteria, NO2- reduction to N2 was catalyzed by PdNPs and bacterial reductases. This study documents a promising option for efficient and complete remediation of halogenated organics and nitrate by the combined action of PdNP and bacterial catalysis.

Three-dimension oxygen gradient induced low energy input for grey water treatment in an oxygen-based membrane biofilm reactor.

Grey water (GW) containing high levels of linear alkylbenzene sulfonates (LAS) can be a threat to human health and organisms in the environment if not treated properly. Although aerobic treatment could achieve high organics removal efficiency, conventional aeration can lead to serious foaming and energy waste. Here, we systematically evaluated an oxygen based membrane biofilm reactor (O2-MBfR) for its capacity to simultaneously remove organics and nitrogen from GW. The dissolved oxygen (DO) concentration inside the reactor was maintained at 0.4 mg/L by gradually controlling the lumen air pressure. Results showed that the O2-MBfR achieved high removal efficiency of total chemical oxygen demand (TCOD), total linear alkylbenzene sulfonates (LAS) and total nitrogen (TN) of 89.7%, 99.1% and 78.1%, respectively, with a hydraulic retention time (HRT) of 7.5 h. Lower HRT (7.0 h) led to the accumulation of LAS in the biofilm, which caused cell lysis and damaged the O2-MBfR system, leading to a discernible and continuous decline of the reactor performance. The O2-MBfR design completely eliminated foaming formation and the three-dimension oxygen gradient design led to low air pressure inside the membrane fiber, which enabled the high removal efficiency for both organics and nitrogen with low energy input and GW treatment cost, providing the fundamental knowledge for practical application of O2-MBfR in wastewater treatment.

Enhancing mainstream nitrogen removal by employing nitrate/nitrite-dependent anaerobic methane oxidation processes.

Due to serious eutrophication in water bodies, nitrogen removal has become a critical stage for wastewater treatment plants (WWTPs) over past decades. Conventional biological nitrogen removal processes are based on nitrification and denitrification (N/DN), and are suffering from several major drawbacks, including substantial aeration consumption, high fugitive greenhouse gas emissions, a requirement for external carbon sources, excessive sludge production and low energy recovery efficiency, and thus unable to satisfy the escalating public needs. Recently, the discovery of anaerobic ammonium oxidation (anammox) bacteria has promoted an update of conventional N/DN-based processes to autotrophic nitrogen removal. However, the application of anammox to treat domestic wastewater has been hindered mainly by unsatisfactory effluent quality with nitrogen removal efficiency below 80%. The discovery of nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) during the last decade has provided new opportunities to remove this barrier and to achieve a robust system with high-level nitrogen removal from municipal wastewater, by utilizing methane as an alternative carbon source. In the present review, opportunities and challenges for nitrate/nitrite-dependent anaerobic methane oxidation are discussed. Particularly, the prospective technologies driven by the cooperation of anammox and n-DAMO microorganisms are put forward based on previous experimental and modeling studies. Finally, a novel WWTP system acting as an energy exporter is delineated.

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.

Nitrous oxide emissions from biofilm processes for wastewater treatment.

This paper discusses the microbial basis and the latest research on nitrous oxide (N2O) emissions from biofilms processes for wastewater treatment. Conditions that generally promote N2O formation in biofilms include (1) low DO values, or spatial DO transitions from high to low within the biofilm; (2) DO fluctuations within biofilm due to varying bulk DO concentrations or varying substrate concentrations; (3) conditions with high reaction rates, which lead to greater formation of intermediates, e.g., hydroxylamine (NH2OH) and nitrite (NO2-), that promote N2O formation; and (4) electron donor limitation for denitrification. Formation of N2O directly results from the activities of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and heterotrophic denitrifying bacteria. More research is needed on the roles of AOA, comammox, and specialized denitrifying microorganisms. In nitrifying biofilms, higher bulk ammonia (NH3) concentrations, higher nitrite (NO2-) concentrations, lower dissolved oxygen (DO), and greater biofilm thicknesses result in higher N2O emissions. In denitrifying biofilms, N2O accumulates at low levels as an intermediate and at higher levels at the oxic/anoxic transition regions of the biofilms and where COD becomes limiting. N2O formed in the outer regions can be consumed in the inner regions if COD penetrates sufficiently. In membrane-aerated biofilms, where nitrification takes place in the inner, aerobic biofilm region, the exterior anoxic biofilm can serve as a N2O sink. Reactors that include variable aeration or air scouring, such as denitrifying filters, trickling filters, or rotating biological contactors (RBCs), can form peaks of N2O emissions during or following a scouring or aeration event. N2O emissions from biofilm processes depend on the microbial composition, biofilm thickness, substrate concentrations and variability, and reactor type and operation. Given the complexity and difficulty in quantifying many of these factors, it may be difficult to accurately predict emissions for full-scale treatment plants. However, a better understanding of the mechanisms and the impacts of process configurations can help minimize N2O emission from biofilm processes for wastewater treatment.

Assessing microbial competition in a hydrogen-based membrane biofilm reactor (MBfR) using multidimensional modeling.

The membrane biofilm reactor (MBfR) is a novel technology that safely delivers hydrogen to the base of a denitrifying biofilm via gas-supplying membranes. While hydrogen is an effective electron donor for denitrifying bacteria (DNB), it also supports sulfate-reducing bacteria (SRB) and methanogens (MET), which consume hydrogen and create undesirable by-products. SRB and MET are only competitive for hydrogen when local nitrate concentrations are low, therefore SRB and MET primarily grow near the base of the biofilm. In an MBfR, hydrogen concentrations are greatest at the base of the biofilm, making SRB and MET more likely to proliferate in an MBfR system than a conventional biofilm reactor. Modeling results showed that because of this, control of the hydrogen concentration via the intramembrane pressure was a key tool for limiting SRB and MET development. Another means is biofilm management, which supported both sloughing and erosive detachment. For the conditions simulated, maintaining thinner biofilms promoted higher denitrification fluxes and limited the presence of SRB and MET. The 2-d modeling showed that periodic biofilm sloughing helped control slow-growing SRB and MET. Moreover, the rough (non-flat) membrane assembly in the 2-d model provided a special niche for SRB and MET that was not represented in the 1-d model. This study compared 1-d and 2-d biofilm model applicability for simulating competition in counter-diffusional biofilms. Although more computationally expensive, the 2-d model captured important mechanisms unseen in the 1-d model.

Impact of Nitrate on the Removal of Pollutants from Water in Reducing Gas-Based Membrane Biofilm Reactors: A Review.

The membrane biofilm reactor (MBfR) is a novel wastewater treatment technology, garnering attention due to its high gas utilization rate and effective pollutant removal capability. This paper outlines the working mechanism, advantages, and disadvantages of MBfR, and the denitrification pathways, assessing the efficacy of MBfR in removing oxidized pollutants (sulfate (SO4-), perchlorate (ClO4-)), heavy metal ions (chromates (Cr(VI)), selenates (Se(VI))), and organic pollutants (tetracycline (TC), p-chloronitrobenzene (p-CNB)), and delves into the role of related microorganisms. Specifically, through the addition of nitrates (NO3-), this paper analyzes its impact on the removal efficiency of other pollutants and explores the changes in microbial communities. The results of the study show that NO3- inhibits the removal of other pollutants (oxidizing pollutants, heavy metal ions and organic pollutants), etc., in the simultaneous removal of multiple pollutants by MBfR.

Microbial interactions facilitating efficient methane driven denitrification via in-situ utilization of short chain fatty acids.

High nitrate pollution in agriculture and industry poses a challenge to emerging methane oxidation coupled denitrification. In this study, an efficient nitrate removal efficiency of 100 % was achieved at an influent loading rate of 400 mg-N/L·d, accompanied by the production of short chain fatty acids (SCFAs) with a maximum value of 80.9 mg/L. Batch tests confirmed that methane was initially converted to acetate, which then served as a carbon source for denitrification. Microbial community characterization revealed the dominance of heterotrophic denitrifiers, including Simplicispira (22.8 %), Stappia (4.9 %), and the high­nitrogen-tolerant heterotrophic denitrifier Diaphorobacter (19.0 %), at the nitrate removal rate of 400 mg-N/L·d. Notably, the low abundance of methanotrophs ranging from 0.24 % to 3.75 % across all operational stages does not fully align with the abundance of pmoA genes, suggesting the presence of other functional microorganisms capable of methane oxidation and SCFAs production. These findings could facilitate highly efficient denitrification driven by methane and contributed to the development of denitrification using methane as an electron donor.

Elemental sulfur biorecovery from phosphogypsum using oxygen-membrane biofilm reactor: Bioreactor parameters optimization, metagenomic analysis and metabolic prediction of the biofilm activity.

This work investigated elemental sulfur (S0) biorecovery from Phosphogypsum (PG) using sulfur-oxidizing bacteria in an O2-based membrane biofilm reactor (MBfR). The system was first optimized using synthetic sulfide medium (SSM) as influent, then switched to biogenic sulfide medium (BSM) generated by biological reduction of PG alkaline leachate. The results using SSM had high sulfide-oxidation efficiency (98 %), sulfide to S0 conversion (∼90 %), and S0 production rate up to 2.7 g S0/(m2.d), when the O2/S ratio was ∼0.5 g O2/g S. With the BSM influent, the system maintained high sulfide-to-S0 conversion rate (97 %), and S0-production rate of 1.6 g S0/(m2.d). Metagenomic analysis revealed that Thauera was the dominant genus in SSM and BSM biofilms. Furthermore, influent composition affected the bacterial community structure and abundances of functional microbial sulfur genes, modifying the sulfur-transformation pathways in the biofilms. Overall, this work shows promise for O2-MBfR usage in S0 biorecovery from PG-leachate and other sulfidogenic effluents.

Bioreduction of chromate in a syngas-based membrane biofilm reactor.

This study leveraged synthesis gas (syngas), a renewable resource attainable through the gasification of biowaste, to achieve efficient chromate removal from water. To enhance syngas transfer efficiency, a membrane biofilm reactor (MBfR) was employed. Long-term reactor operation showed a stable and high-level chromate removal efficiency > 95%, yielding harmless Cr(III) precipitates, as visualised by scanning electron microscopy and energy dispersive X-ray analysis. Corresponding to the short hydraulic retention time of 0.25 days, a high chromate removal rate of 80 µmol/L/d was attained. In addition to chromate reduction, in situ production of volatile fatty acids (VFAs) by gas fermentation was observed. Three sets of in situ batch tests and two groups of ex situ batch tests jointly unravelled the mechanisms, showing that biological chromate reduction was primarily driven by VFAs produced from in situ syngas fermentation, whereas hydrogen originally present in the syngas played a minor role. 16 S rRNA gene amplicon sequencing has confirmed the enrichment of syngas-fermenting bacteria (such as Sporomusa), who performed in situ gas fermentation leading to the synthesis of VFAs, and organics-utilising bacteria (such as Aquitalea), who utilised VFAs to drive chromate reduction. These findings, combined with batch assays, elucidate the pathways orchestrating synergistic interactions between fermentative microbial cohorts and chromate-reducing microorganisms. The findings facilitate the development of cost-effective strategies for groundwater and drinking water remediation and present an alternative application scenario for syngas.

Biological bromate reduction coupled with in situ gas fermentation in H2/CO2-based membrane biofilm reactor.

Bromate, a carcinogenic contaminant generated in water disinfection, presents a pressing environmental concern. While biological bromate reduction is an effective remediation approach, its implementation often necessitates the addition of organics, incurring high operational costs. This study demonstrated the efficient biological bromate reduction using H2/CO2 mixture as the feedstock. A membrane biofilm reactor (MBfR) was used for the efficient delivery of gases. Long-term reactor operation showed a high-level bromate removal efficiency of above 95 %, yielding harmless bromide as the final product. Corresponding to the short hydraulic retention time of 0.25 d, a high bromate removal rate of 4 mg Br/L/d was achieved. During the long-term operation, in situ production of volatile fatty acids (VFAs) by gas fermentation was observed, which can be regulated by controlling the gas flow. Three sets of in situ batch tests and two groups of ex situ batch tests jointly unravelled the mechanisms underpinning the efficient bromate removal, showing that the microbial bromate reduction was primarily driven by the VFAs produced from in situ gas fermentation. Microbial community analysis showed an increased abundance of Bacteroidota group from 4.0 % to 18.5 %, which is capable of performing syngas fermentation, and the presence of heterotrophic denitrifiers (e.g., Thauera and Brachymonas), which are known to perform bromate reduction. Together these results for the first time demonstrated the feasibility of using H2/CO2 mixture for bromate removal coupled with in situ VFAs production. The findings can facilitate the development of cost-effective strategies for groundwater and drinking water remediation.

Impact of hydrogen sulfide on anammox and nitrate/nitrite-dependent anaerobic methane oxidation coupled technologies.

The coupling between anammox and nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) has been considered a sustainable technology for nitrogen removal from sidestream wastewater and can be implemented in both membrane biofilm reactor (MBfR) and granular bioreactor. However, the potential influence of the accompanying hydrogen sulfide (H2S) in the anaerobic digestion (AD)-related methane-containing mixture on anammox/n-DAMO remains unknown. To fill this gap, this work first constructed a model incorporating the C/N/S-related bioprocesses and evaluated/calibrated/validated the model using experimental data. The model was then used to explore the impact of H2S on the MBfR and granular bioreactor designed to perform anammox/n-DAMO at practical levels (i.e., 0∼5% (v/v) and 0∼40 g/S m3, respectively). The simulation results indicated that H2S in inflow gas did not significantly affect the total nitrogen (TN) removal of the MBfR under all operational conditions studied in this work, thus lifting the concern about applying AD-produced biogas to power up anammox/n-DAMO in the MBfR. However, the presence of H2S in the influent would either compromise the treatment performance of the granular bioreactor at a relatively high influent NH4+-N/NO2--N ratio (e.g., >1.0) or lead to increased energy demand associated with TN removal at a relatively low influent NH4+-N/NO2--N ratio (e.g., <0.7). Such a negative effect of the influent H2S could not be attenuated by regulating the hydraulic residence time and should therefore be avoided when applying the granular bioreactor to perform anammox/n-DAMO in practice.

Biological reduction and hydrodechlorination of chlorinated nitroaromatic antibiotic chloramphenicol under H2-transfer membrane biofilm reactor.

Chlorinated nitroaromatic antibiotic chloramphenicol (CAP) is a persistent pollutant that is widely present in environments. A H2 transfer membrane biofilm reactor (H2-MBfR) and short-term batch tests were setup to investigate the co-removal of CAP and NO3-. Results showed that the presence of CAP (<10 mg L-1) has no effect on the denitrification process while 100% removal efficiency of CAP can be obtained when nitrate was absent. Nitroaromatic reduction and completely dechlorination were successfully realized when CAP was removed. The CAP transformation product p-aminobenzoic acid (PABA) was detected and batch tests revealed that the hydroxy carboxylation was far faster than nitroaromatic reduction when p-nitrobenzyl alcohol (PNBOH) was conversed to p-aminobenzoic acid (PABA). The path way of CAP degradation was proposed based on the intermediate's analysis. Microbial community analysis indicated that Pleomorphomonadaceae accounts for the dechlorination of CAP.

Simultaneous removal of nitrate and ammonium by hydrogen-based partial denitrification coupled with anammox in a membrane biofilm reactor.

Hydrogen-based membrane biofilm reactors (MBfRs) are effective for nitrogen removal. However, the safety of hydrogen limited the application of MBfR. Here, a hydrogen-based partial denitrification system coupled with anammox (H2-PDA) was constructed in an MBfR for reducing hydrogen demand significantly. The metabolomics and structures of microbial communities were analyzed to determine the phenotypic differences and drivers underlying denitrification, anammox, and H2-PDA. These findings indicated that total nitrogen (TN) removal increased from 57.1% in S1 to 93.7% in S2. During the H2-PDA process, partial denitrification and anammox contributed to TN removal by 93.7% and 6.3%, respectively. Community analysis indicated that the H2-PDA system was dominated by the genus Meiothermus, which is involved in partial denitrification. Collectively, these findings confirmed the feasibility of incorporating the H2-PDA process in a MBfR and form a foundation for the establishment of novel and practical methods for efficient nitrogen removal.

The structure of biodegradable surfactants shaped the microbial community, antimicrobial resistance, and potential for horizontal gene transfer.

While most household surfactants are biodegradable in aerobic conditions, their biodegradability may obscure their environmental risks. The presence of surfactants in a biological treatment process can lead to the proliferation of antimicrobial-resistance genes (ARG) in the biomass. Surfactants can be cationic, anionic, or zwitterionic, and these different classes may have different effects on the proliferation ARG. Cationic hexadecyltrimethyl-ammonium (CTAB), anionic sodium dodecyl sulfate (SDS), and zwitterionic 3-(decyldimethylammonio)-propanesulfonate inner salt (DAPS) were used to represent the three classes of surfactants in domestic household clean-up products. This study focused on the removal of these surfactants by the O2-based Membrane Biofilm Reactor (O2-MBfR) for hotspot scenarios (∼1 mM) and how the three classes of surfactants affected the microbial community's structure and ARG. Given sufficient O2 delivery, the MBfR provided at least 98% surfactant removal. The presence and biodegradation for each surfactant uniquely shaped the biofilms' microbial communities and the presence of ARG. CTAB had by far the strongest impact and the higher ARG abundance. In particular, Pseudomonas and Stenotrophomonas, the two main genera in the biofilm treating CTAB, were highly correlated to the abundance of ARG for efflux pumps and antibiotic inactivation. CTAB also led to more functional genes relevant to the Type-IV secretion system and protection against oxidative stress, which also could encourage horizontal gene transfer. Our findings highlight that the biodegradation of quaternary ammonium surfactants, while beneficial, can pose public health concerns from its ability to promote the proliferation of ARG.

Nitrogen Removal From Nitrate-Containing Wastewaters in Hydrogen-Based Membrane Biofilm Reactors via Hydrogen Autotrophic Denitrification: Biofilm Structure, Microbial Community and Optimization Strategies.

The hydrogen-based membrane biofilm reactor (MBfR) has been widely applied in nitrate removal from wastewater, while the erratic fluctuation of treatment efficiency is in consequence of unstable operation parameters. In this study, hydrogen pressure, pH, and biofilm thickness were optimized as the key controlling parameters to operate MBfR. The results of 653.31 µm in biofilm thickness, 0.05 MPa in hydrogen pressure and pH in 7.78 suggesting high-efficiency NO 3 - - N removal and the NO 3 - - N removal flux was 1.15 g·m-2 d-1. 16S rRNA gene analysis revealed that Pseudomonas, Methyloversatilis, Thauera, Nitrospira, and Hydrogenophaga were the five most abundant bacterial genera in MBfRs after optimization. Moreover, significant increases of Pseudomonas relative abundances from 0.36 to 9.77% suggested that optimization could effectively remove nitrogen from MBfRs. Membrane pores and surfaces exhibited varying degrees of calcification during stable operation, as evinced by Ca2+ precipitation adhering to MBfR membrane surfaces based on scanning electron microscopy (SEM), atomic force microscopy (AFM) analyses. Scanning electron microscopy-energy dispersive spectrometer (SEM-EDS) analyses also confirmed that the primary elemental composition of polyvinyl chloride (PVC) membrane surfaces after response surface methodology (RSM) optimization comprised Ca, O, C, P, and Fe. Further, X-ray diffraction (XRD) analyses indicated the formation of Ca5F(PO4)3 geometry during the stable operation phase.

Biofilm stratification in counter-diffused membrane biofilm bioreactors (MBfRs) for aerobic methane oxidation coupled to aerobic/anoxic denitrification: Effect of oxygen pressure.

Aerobic methane oxidation coupled with denitrification (AME-D) executed in membrane biofilm bioreactors (MBfRs) provides a high promise for simultaneously mitigating methane (CH4) emissions and removing nitrate in wastewater. However, systematically experimental investigation on how oxygen partial pressure affects the development and characteristics of counter-diffusional biofilm, as well as its spatial stratification profiles, and the cooperative interaction of the biofilm microbes, is still absent. In this study, we combined Optical Coherence Tomography (OCT) with Confocal Laser Scanning Microscopy (CLSM) to in-situ characterize the development of counter-diffusion biofilm in the MBfR for the first time. It was revealed that oxygen partial pressure onto the MBfR was capable of manipulating biofilm thickness and spatial stratification, and then managing the distribution of functional microbes. With the optimized oxygen partial pressure of 5.5 psig (25% oxygen content), the manipulated counter-diffusional biofilm in the AME-D process obtained the highest denitrification efficiency, due mainly to that this biofilm had the proper dynamic balance between the aerobic-layer and anoxic-layer where suitable O2 gradient and sufficient aerobic methanotrophs were achieved in aerobic-layer to favor methane oxidation, and complete O2 depletion and accessible organic sources were kept to avoid constraining denitrification activity in anoxic-layer. By using metagenome analysis and Fluorescence in situ hybridization (FISH) staining, the spatial distribution of the functional microbes within counter-diffused biofilm was successfully evidenced, and Rhodocyclaceae, one typical aerobic denitrifier, was found to survive and gradually enriched in the aerobic layer and played a key role in denitrification aerobically. This in-situ biofilm visualization and characterization evidenced directly for the first time the cooperative path of denitrification for AME-D in the counter-diffused biofilm, which involved aerobic methanotrophs, heterotrophic aerobic denitrifiers, and heterotrophic anoxic denitrifiers.