Management of biofilm by an innovative layer-structured membrane for membrane biofilm reactor (MBfR) to efficient methane oxidation coupled to denitrification (AME-D).

Aerobic methane oxidation coupled with denitrification (AME-D) has garnered significant attention as a promising technology for nitrogen removal from water. Effective biofilm management on the membrane surface is essential to enhance the efficiency of nitrate removal in AME-D systems. In this study, we introduce a novel and scalable layer-structured membrane (LSM) developed using a meticulously designed polyurethane sponge. The application of the LSM in advanced biofilm management for AME-D resulted in a substantial enhancement of denitrification performance. Our experimental results demonstrated remarkable improvements in nitrate-removal flux (92.8 mmol-N m-2 d-1) and methane-oxidation rate (325.6 mmol m-2 d-1) when using an LSM in a membrane biofilm reactor (L-MBfR) compared with a conventional membrane reactor (C-MBfR). The l-MBfR exhibited 12.4-, 6.8- and 3.4-fold increases in nitrate-removal rate, biomass-retention capacity, and methane-oxidation rate, respectively, relative to the control C-MBfR. Notably, the l-MBfR demonstrated a 3.5-fold higher abundance of denitrifying bacteria, including Xanthomonadaceae, Rhodocyclaceae, and Methylophilaceae. In addition, the denitrification-related enzyme activity was twice as high in the l-MBfR than in the C-MBfR. These findings underscore the LSM's ability to create anoxic/anaerobic microenvironments conducive to biofilm formation and denitrification. Furthermore, the LSM exhibited a unique advantage in shaping microbial community structures and facilitating cross-feeding interactions between denitrifying bacteria and aerobic methanotrophs. The results of this study hold great promise for advancing the application of MBfRs in achieving efficient and reliable nitrate removal through the AME-D pathway, facilitated by effective biofilm management.

Intracellular electron competition in response to the oxygen pressure of the aerobic denitrification process in an O2-based membrane biofilm reactor (MBfR) for nitrate removal.

This study quantitatively investigated the effect of dissolved oxygen (DO) concentration on aerobic denitrification, and showed the mechanism of aerobic denitrification from the perspective of electron competition by cultivating Pseudomonas stutzeri T13, a typical aerobic denitrifier, in an oxygen-based membrane biofilm reactor (O2-based MBfR). The experiments showed that when the O2 pressure increased from 2 to 10 psig , the average effluent DO concentration during steady-state phases increased from 0.02 to 4.23 mg/L, and the corresponding mean NO3--N removal efficiency slightly decreased from 97.2 % to 90.9 %. Compared to the maximum theoretical flux of O2 in various phases, the actual O2 transfer flux increased from a limited status (2.07 e- eq m-2 d-1 at 2 psig) to an excessive status (5.58 e- eq m-2 d-1 at 10 psig). The increase of DO inhibited the electron availability for aerobic denitrification, which decreased from 23.97 % to 11.46 %, accompanying the increased electron availability for aerobic respiration from 15.87 % to 28.36 %. Unlike the napA and norB genes, the expression of the nirS and nosZ genes was significantly affected by DO, with the highest relative fold-changes of 6.5 and 6.13 at 4 psig O2, respectively. The results contribute to clarifying the mechanism of aerobic denitrification from the quantitative perspective of electron distribution and the qualitative perspective of gene expression, which benefits the control and practical application of aerobic denitrification for wastewater treatment.

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.

Simultaneous enhancement of nitrate removal flux and methane utilization efficiency in MBfR for aerobic methane oxidation coupled to denitrification by using an innovative scalable double-layer membrane.

Endevours on the enhancement of nitrate removal efficiency during methane oxidation coupled with denitrification (AME-D) has always overlooked the role of membrane employed. It would be highly beneficial to enrich the biomass content and to manage biofilm on the membrane, in the utilization of methane and denitrification. In this study, an innovative and scalable double-layer membrane (DLM) was designed and prepared for a membrane biofilm reactor (MBfR), to simultaneously enhance nitrate removal flux and methane utilization efficiency during aerobic methane oxidation coupled with the denitrification (AME-D) process. The DLM allowed quick bacterial attachment and biomass accumulation for biofilm growth, which would be then self-regulated for well distribution of functional microbes on/within the DLM. Upon a high biofilm density of over 70 g-VSS m-2 achieved on the DLM, the methane utilization efficiency of the MBfR was enhanced significantly to over 1.3 times than the control MBfR with conventional polypropylene membrane. The MBfR employed DLM also demonstrated the maximum nitrate removal flux of 740 mg-NO3--N m-2 d-1 that was approximately 1.64 times of that in control MBfR at continuous-mode operation. This DLM indeed favored the enrichment of Type II aerobic methanotrophs of Methylocystaceae, and methanol-utilization denitrifiers of Rhodocyclaceae that preferentially utilize methanol as the cross-feeding intermediates to promote the methane utilization efficiency, and thus to enhance the nitrate removal flux. These results raised from new designed DLM confirmed the importance of membrane surface properties on the effectiveness of MBfR, and offered great potential to address challenging problems of MBfRs during engineering application.

Activated sludge filterability improvement by nitrifying bacteria abundance regulation in an adsorption membrane bioreactor (Ad-MBR).

Autotrophic nitrifying bacteria have its intrinsic properties including low EPS production, dense colonial structure and slow-growth rate, favoring the sludge filterability improvement. An adsorption-MBR (Ad-MBR) was developed to enrich nitrifier abundance in the MBR chamber by inlet C/N regulation, and its possible positive effect on sludge filterability and underlying mechanisms were investigated. By DNA extraction, PCR amplification and Illumina high-throughput pyrosequencing, the abundance of nitrifying bacteria was accurately quantified. More than 8.29% nitrifier abundance was achieved in Ad-MBR sludge, which was above three times of that in conventional MBR. Regulated C/N ratio and thereafter nitrifier abundance enrichment improved sludge filterability by altering sludge mixture and its supernatant properties, reflected by a good sludge settleability, a low supernatant viscosity and turbidity, a low supernatant organic substances concentration, and a small amount of strong hydrophobic fractional components, thus to profoundly improve sludge filterability and decelerate membrane fouling.

A comparative study of the bacterial community in denitrifying and traditional enhanced biological phosphorus removal processes.

Denitrifying phosphorus removal is an attractive wastewater treatment process due to its reduced carbon source demand and sludge minimization potential. Two lab-scale sequencing batch reactors (SBRs) were operated in alternating anaerobic-anoxic (A-A) or anaerobic-oxic (A-O) conditions to achieve denitrifying enhanced biological phosphate removal (EBPR) and traditional EBPR. No significant differences were observed in phosphorus removal efficiencies between A-A SBR and A-O SBR, with phosphorus removal rates being 87.9% and 89.0% respectively. The community structures in denitrifying and traditional EBPR processes were evaluated by high-throughput sequencing of the PCR-amplified partial 16S rRNA genes from each sludge. The results obtained showed that the bacterial community was more diverse in A-O sludge than in A-A sludge. Taxonomy and ß-diversity analyses indicated that a significant shift occurred in the dominant microbial community in A-A sludge compared with the seed sludge during the whole acclimation phase, while a slight fluctuation was observed in the abundance of the major taxonomies in A-O sludge. One Dechloromonas-related OTU outside the 4 known Candidatus "Accumulibacter" clades was detected as the main OTU in A-A sludge at the stationary operation, while Candidatus "Accumulibacter" dominated in A-O sludge.

Degradation efficiency and mechanism of azo dye RR2 by a novel ozone aerated internal micro-electrolysis filter.

A newly designed ozone aerated internal micro-electrolysis filter (OIEF) was developed to investigate its degradation efficiencies and correlated reaction mechanisms of RR2 dye. Complete decolorization and 82% TOC removal efficiency were stably achieved in OIEF process. Based on the comprehensive experimental results, an empirical equation was proposed to illustrate the effects of initial dye concentration and ozone dosage rate on color removal. The results indicated that OIEF process could be operated at wide pH range without significant treatment efficiencies change, while the optimum pH for RR2 dye degradation was 9.0. There were 15, 8 and 6 kinds of identified intermediates during ozonation, IE and OIEF treatment processes, respectively. Less identified intermediates and their lower concentrations in OIEF may attribute to its rather excellent mineralization performance. It was found that ozonation, Fe(2+)/Fe(3+) catalyzed ozonation, the redox reactions of electro-reduction and electro-oxidation are the most important mechanisms in OIEF process. The catalytic effect of Fe(2+)/Fe(3+) would induce mutual conversion between dissolved Fe(2+) and Fe(3+), and then decrease the dissolution rate of ZVI. The excellent treatment performance proved that the OIEF process is one promising technology applied for reactive azo dyes and other refractory wastewater treatment.

Aerobic methane oxidation coupled to denitrification in a membrane biofilm reactor: treatment performance and the effect of oxygen ventilation.

Aerobic methane-oxidation coupled to denitrification (AME-D) process was successfully achieved in a membrane biofilm reactor (MBfR). PVDF membrane was employed to supply the methane and oxygen for biofilm, which was coexistence of methanotrophs and denitrifier. With a feeding NO3(-)-N of 30 mg/L, up to 97% nitrate could be removed stably. The oxygen ventilation modes impacted the denitrification performance remarkably, resulting in different nitrate removal efficiencies and biofilm microorganism distribution. The biofilm sludge showed a high resistance to the DO inhibition, mainly due to the co-existing methanotroph being capable of utilizing oxygen perferentially within biofilm, and create an anoxic micro-environment. The denitrification of both nitrate and nitrite by biofilm sludge conformed to the Monod equation, and the maximum specific nitrate utilization rate (k) ranged from 1.55 to 1.78 NO3(-)-N/g VSS-d. The research findings should be significant to understand the considerable potential of MBfR as a bioprocess for denitrification.

Enhanced denitrification with external carbon sources in a biological anoxic filter.

Three parallel biological anoxic filters (BaFs) were operated to investigate the denitrification kinetics of methanol, brewery wastewater and bakery wastewater. The experiment was conducted within the temperature range of 15-20 °C, with an influent nitrate and carbon dosage of 30 mg/L and 150 mg COD/L (COD: chemical oxygen demand). The denitrification efficiencies of brewery wastewater, bakery wastewater and methanol were 84, 66 and 74%, specific denitrification rates were 1.44, 1.11 and 1.24 kg NO(3)-N/m(3) d, and total nitrogen (TN) removal rates were 74, 62 and 66%, respectively. The volatile attached solid (VAS) tests reveal that methanol has the minimum net biomass yield, so it needs the least carbon to nitrogen (expressed in COD to nitrate, C/N) ratio for complete denitrification. While the brewery wastewater and bakery wastewater need higher C/N ratio to remove all nitrate nitrogen, and they both may need pretreatment to remove phosphate when used as external carbon sources.

Granulation of nitrifying bacteria in a sequencing batch reactor for biological stabilisation of source-separated urine.

Biological stabilisation of human urine highly depends on the abundance and activities of nitrifying bacteria. However, it is quite difficult to enrich nitrifiers as bio-aggregation by self-immobilized biomass. In this study, granulation of nitrifying bacteria involving inoculation strategy was developed. Two sequencing batch reactors, the one inoculated with nitrifying bacteria and the other inoculated with aerobic granules, were operated in laboratory side by side with a feeding of urine solution. Aerobic nitrifying granules (ANG), with compact morphological structure and good nitrifying activity, were achieved in the reactor inoculated with aerobic granules. Enrichment of nitrifying bacteria favors the nutrient uptake, and hence, to obtain a high ammonia oxidation efficiency. Nonetheless, nitrite accumulation gradually dominated in reactor, partly attributes to a high concentration of free nitrous acid and free ammonia in bulk. The matured ANG had a rather stable microbial profile, as that a number of activated bacteria occupied the surface of granule. It was also found that ANG were much more impermeable than aerobic granules and activated sludge, which was demonstrated as smaller porosities, and therewith an excellent settleability. The results herein reveal that granulation of nitrifying bacteria could enrich the biomass to implement stabilisation of urine in biological way.

Investigation of the role of biopolymer clusters in MBR membrane fouling using flash freezing and environmental scanning electron microscopy.

The technique that employs flash freezing and environmental scanning electron microscopy (ESEM) was utilised for detailed investigation of the fouling materials in a membrane bioreactor (MBR). The method involves the flash freezing of a wet sample in liquid nitrogen for 10s to preserve its structure for direct ESEM observation with a high image resolution. ESEM images show that the sludge cake formed by simple filtration of the MBR bulk sludge has a highly porous, sponge-like structure with a fairly low resistance. However, the fouling layer attached to the membrane surface contains a thin gel layer under the main body of the sponge-like sludge cake, which is similar to that formed by filtration of a dispersion of biopolymer clusters (BPCs). It is apparent that BPCs tend to accumulate on the membrane surface, and the gel layer is largely responsible for the high filtration resistance of the cake layer on the fouled membranes.

Change in the fouling propensity of sludge in membrane bioreactors (MBR) in relation to the accumulation of biopolymer clusters.

A membrane bioreactor (MBR) and an activated sludge process (ASP) were operated side by side to evaluate the change of sludge supernatant characteristics and the evolution of the sludge fouling propensity. The MBR sludge had a higher organic concentration and more biopolymer clusters (BPC) in the supernatant compared with ASP. BPC increased in both concentration and size in the MBR. The results show that the change in the liquid-phase property had a profound effect on the sludge fouling propensity. MBR operation transformed typical activated sludge to MBR sludge with a higher fouling propensity. Distinct from the ASP, membrane filtration retained soluble microbial products (SMP) within the MBR, and the vast membrane surface provided a unique environment for the transformation of SMP to large size BPC, leading to further sludge deposition on the membrane surface. Thus, membrane filtration is the crucial cause of the inevitable fouling problem in submerged MBRs.

[Aerobic nitrifying granulation fed with source-separated urine in sequencing batch reactor].

Morphological characteristics of aerobic nitrifying granules that were utilized to treat the diluted source-separated urine were investigated in two lab-scale sequencing batch reactors. In the first sequencing batch reactor, which was inoculated with nitrifying bacteria, the COD of effluent was markedly decreased while the nitrification rate was very low. Aerobic nitrifying granules were not appeared in the first bioreactor. In the other SBR system that was inoculated with aerobic granules cultivated in the laboratory, the COD and ammonia in source-separated urine were effectively removed and the removal rate was more than 90%. Under operational condition that influent ammonia volume rate was 0.5 kg/(m3 x d) for 70 days, the aerobic nitrifying granules were stable in the reactor. Aerobic nitrifying granules have well settleability and metabolize activity, the surface of granules was occupied by nitrifying bacillus and cocci bacteria. Contrast to the inoculating aerobic granules, the diameter of aerobic nitrifying granules dramatically decreased to more than 2 mm and the settle velocity were greater than that of aerobic granules which have the same diameter.

Influence of different substrates on the formation and characteristics of aerobic granules in sequencing batch reactors.

The effects of different substrates on the aerobic granulation process were studied using laboratory-scale sequencing batch reactors (SBRs). Four parallel granules sequencing batch reactors (GSBR): R1, R2, R3, and R4 were fed with acetate, glucose, peptone and fecula, respectively. Stable aerobic granules were successfully cultivated in R1, R2, R4, and smaller granules less than 500 microm were formed in R3. Morphology and the physic-chemical characteristics of aerobic granules fed with different carbon substrates were investigated by the four reactors operated under the same pressure. The aerobic granules in the four reactors were observed and found that peptone was the most stable one due to its good settleability even after a sludge age as short as 10 d. A strong correlation was testified between the characteristics of aerobic granules and the properties of carbon substrates. The stability of aerobic granules was affected by extracellular polymer substances (EPS) derived from microorganism growth during feast time fed with different carbon substrates, and the influence of the property of storage substance was greater than that of its quantity. Optimal carbon substrates, which are helpful in the cultivation and retention of well-settling granules and in the enhancement of the overall ability of the aerobic granules reactors, were found.