Effect of EPS production on the performance of membrane-based biofilm reactors.

This study explored the effect of extracellular polymeric substance (EPS) production on the performance of membrane-based biofilm reactors. Changing EPS production was induced by eliminating one of the main EPS polysaccharides, i.e., Pel. The studies were carried out using a pure culture of either Pseudomonas aeruginosa or an isogenic P. aeruginosa mutant that was unable to produce the Pel polysaccharide. The biofilm cell density for both strains was compared to confirm the Pel deletion mutant decreased overall EPS production in a bioreactor system. When the Pel-deficient mutant was grown as a biofilm, its cell density, i.e., ratio of cells/(cells + EPS), was 74 % higher than the wild type, showing EPS production was reduced by eliminating pel production. The growth kinetics were determined for both strains. The Pel-deficient mutant had a maximum specific growth rate (µ^) that was 14% higher than the wild type. Next, the effects of EPS reduction on reactor performance were assessed for a membrane aerated biofilm reactor (MABR) and a membrane bioreactor (MBR). For the MABR, the organic removal with the Pel-deficient mutant was around 8% higher than for the wild type. For the MBR, the time to reach the fouling threshold was 65 % greater for the Pel-deficient mutant than for the wild type. These results suggest that amount of EPS production can have significant effects on bacterial growth kinetics and bacterial cell density, which in turn can affect the performance of the membrane-based biofilm reactors. In both cases, lower EPS production correlated with more efficient treatment processes.

Effect of nitrous oxide (N2O) on the structure and function of nitrogen-oxide reducing microbial communities.

Nitrous oxide (N2O) is a potent greenhouse gas that can be produced by nitrifying and denitrifying bacteria. Yet the effects of N2O on microbial communities is not well understood. We used batch tests to explore the effects of N2O on mixed denitrifying communities. Batch tests were carried out with acetate as the electron donor and with the following electron acceptors: nitrate (NO3-), nitrite (NO2-), N2O, NO3- + N2O, and NO2- + N2O. Activated sludge from a municipal wastewater treatment plant was used as the inoculum. The bacteria grew readily with N2O as the sole acceptor. When N2O was provided along with NO3- or NO2-, it was used concurrently and resulted in higher growth rates than the same acceptors without added N2O. The microbial communities resulting from N2O addition were significantly different at the genus level from those with just NO3- or NO2-. Tests with N2O as the sole added acceptor revealed a reduced diversity. Analysis of inferred gene content using PICRUSt2 indicated a greater abundance of genera with a complete denitrification pathway when growing on N2O or NO2-, relative to all other tests. This suggests that specific N2O reduction rates are high, and that N2O alone selects for a low-diversity, fully denitrifying community. When N2O is present with NO2- or NO3-, the microbial communities were more diverse and did not select exclusively for full denitrifiers. N2O alone appears to select for a "generalist" community with full denitrification pathways and lower diversity. In terms of denitrification genes, the combination of acceptors with N2O appeared to increase the number of microbes carrying nirK, while fully denitrifying bacteria appear more likely to carry nirS. Lastly, all the taxa in NO2- and N2O samples were predicted to harbor nosZ. This suggests the potential for reduced N2O emissions in denitrifying systems.

Effects of eukaryotic predation on nitrifying MABR biofilms.

This research explored the effects of eukaryotic predation on nitrifying membrane-aerated biofilm reactor (MABR) biofilms. Past research on heterotrophic MABR biofilms showed that predation could create internal voids that promoted sloughing. However, the no past research addressed the effects of predation on nitrifying MABRs, even though nitrification is the most common MABR application. Nitrifying biofilms are typically denser, and ammonia oxidizing bacteria (AOB) form large, dense clusters within the biofilm. This could affect their susceptibility to predation. Nitrifying biofilms were grown in flat-sheet MABRs. Images of the biofilm were captured using optical coherence tomography (OCT). For detachment tests, an increased shear flow (Re≅140) was used, and a shear rheometer was used to measure the biofilm mechanical properties. The nitrifying community was analyzed with fluorescence in situ hybridization (FISH) and quantitative PCR (qPCR). Predation increased internal void ratios from 54 ±â€¯5% to 69 ±â€¯6%. Biofilms were weakened by predation, with a storage modulus (G') and loss modulus (G'') of 242 ±â€¯135 and 1,649 ±â€¯853 Pa with predation and 3,644 ±â€¯1,857 and 23,334 ±â€¯11,481 Pa for the control with suppressed predation. Predation increased the relative biofilm detachment from 4 ±â€¯5 to 18 ±â€¯12%, decreased the amount of biomass, i.e., the average biofilm thickness, from 502 ±â€¯150 to 266 ±â€¯54 µm, and decreased the nitrification flux from 1.00 to 0.61 g NH4+-N/m2day. Also, predation decreased the abundance of nitrite oxidizing bacteria (NOB) relative to AOB, consistent with the observed nitritation. These results show that predation can significantly impact the structural stability, bacterial community and removal rates of nitrifying MABR biofilms. Lumping the effects of predation into the detachment or decay coefficients of biofilm models may not accurately reflect the behavior of nitrifying MABR biofilms.

Effect of predation on the mechanical properties and detachment of MABR biofilms.

The membrane-aerated biofilm reactor (MABR) is an emerging wastewater treatment technology that uses O2-supplying membranes as a biofilm support. Because O2 is supplied from the biofilm base instead of the bulk liquid, MABR biofilms have distinct microbial community structures and behavior. Past research showed that protozoan predation in MABR biofilms can greatly increase biofilm porosity, producing a void layer at the base of the biofilm. We hypothesized that this void layer could weaken the biofilm and promote sloughing, and investigated this with heterotrophic MABR biofilms. A rheometer was used to measure biofilm mechanical strength, and MABR flow cells were used to explore detachment. MABRs supplied with cycloheximide, a protozoan inhibitor, were used as controls. Predation increased the internal void ratio from 6 ± 7% to 50 ± 16%. The storage modulus was 1,780 ± 1,180 Pa with predation condition, compared to 9,800 ± 4,290 Pa for the control. Similarly, the loss modulus was 1,580 ± 729 Pa with predation and 363 ± 189 Pa for the control. When subjected to an increased flow, the biofilm loss was 44 ± 24% for the flow cell with predation, while only 7 ± 9% for the control. This research shows that predation can have an important impact on biofilm porosity in MABRs, reducing the mechanical strength and increasing detachment. Understanding this phenomenon can help develop more effective biofilm control strategies in MABRs.

Predation creates unique void layer in membrane-aerated biofilms.

The membrane-aerated biofilm reactor (MABR) is a novel wastewater treatment technology based on oxygen-supplying membranes. The counter diffusion of oxygen and electron donors in MABRs leads to unique behavior, and we hypothesized it also could impact predation. We used optical coherence tomography (OCT), microsensor analyses, and mathematical modeling to investigate predation in membrane-aerated biofilms (MABs). When protozoa were excluded from the inoculum, the MAB's OCT-observable void fraction was around 5%. When protozoa were included, the void fraction grew to nearly 50%, with large, continuous voids at the base of the biofilm. Real-time OCT imaging showed highly motile protozoa in the voids. MABs with protozoa and a high bulk COD (270 mg/L) only had 4% void fraction. DNA sequencing revealed a high relative abundance of amoeba in both high and low-COD MABs. Flagellates were only abundant in the low-COD MAB. Modeling also suggested a relationship between substrate concentrations, diffusion mode (co- or counter-diffusion), and biofilm void fraction. Results suggest that amoeba proliferate in the biofilm interior, especially in the aerobic zones. Voids form once COD limitation at the base of MABs allows predation rates to exceed microbial growth rates. Once formed, the voids provide a niche for motile protozoa, which expand the voids into a large, continuous gap. This increases the potential for biofilm sloughing, and may have detrimental effects on slow-growing, aerobic microorganisms such as nitrifying bacteria.

Periodic venting of MABR lumen allows high removal rates and high gas-transfer efficiencies.

The membrane-aerated biofilm reactor (MABR) is a novel treatment technology that employs gas-supplying membranes to deliver oxygen directly to a biofilm growing on the membrane surface. When operated with closed-end membranes, the MABR provides 100-percent oxygen transfer efficiencies (OTE), resulting in significant energy savings. However, closed-end MABRs are more sensitive to back-diffusion of inert gases, such as nitrogen. Back-diffusion reduces the average oxygen transfer rates (OTR), consequently decreasing the average contaminant removal fluxes (J). We hypothesized that venting the membrane lumen periodically would increase the OTR and J. Using an experimental flow cell and mathematical modeling, we showed that back-diffusion gas profiles developed over relatively long timescales. Thus, very short ventings could re-establish uniform gas profiles for relatively long time periods. Using modeling, we systematically explored the effect of the venting interval (time between ventings). At moderate venting intervals, opening the membrane for 20 s every 30 min, the venting significantly increased the average OTR and J without substantially impacting the OTEs. When the interval was short enough, in this case shorter than 20 min, the OTR was actually higher than for continuous open-end operation. Our results show that periodic venting is a promising strategy to combine the advantages of open-end and closed end operation, maximizing both the OTR and OTE.

Comparing peracetic acid and hypochlorite for disinfection of combined sewer overflows: Effects of suspended-solids and pH.

Peracetic acid (PAA) is an alternative disinfectant that may be effective for combined sewer overflow (CSO) disinfection, but little is known about the effect of particle size on PAA disinfection efficiency. In this work, PAA and hypochlorite were compared as disinfectants, with a focus on the effect of wastewater particles. Inactivation experiments were conducted on suspended cultures of Escherichia coli and wastewater suspended solids. Tested size fractions included particle diameters <10µm, <100µm, and raw wastewater. Chlorine disinfection efficiency decreased with increasing solids size. However, solids size had little effect on PAA disinfection. The PAA disinfection efficiency decreased at pH values above 7.5. Live/dead staining revealed that PAA disinfection leaves most cells in a viable but non-culturable condition. Fourier transform infrared spectroscopy (FTIR) analyses suggests that PAA and hypochlorite may inactivate E. coli bacteria by similar mechanisms.

Kinetics of nitrous oxide (N2O) formation and reduction by Paracoccus pantotrophus.

Nitrous oxide (N2O) is a powerful greenhouse gas emitted from wastewater treatment, as well as natural systems, as a result of biological nitrification and denitrification. While denitrifying bacteria can be a significant source of N2O, they can also reduce N2O to N2. More information on the kinetics of N2O formation and reduction by denitrifying bacteria is needed to predict and quantify their impact on N2O emissions. In this study, kinetic parameters were determined for Paracoccus pantotrophus, a common denitrifying bacterium. Parameters included the maximum specific reduction rates, [Formula: see text], growth rates, [Formula: see text], and yields, Y, for reduction of NO3- (nitrate) to nitrite (NO2-), NO2- to N2O, and N2O to N2, with acetate as the electron donor. The [Formula: see text] values were 2.9 gN gCOD-1 d-1 for NO3- to NO2-, 1.4 gN gCOD-1 d-1 for NO2- to N2O, and 5.3 gN gCOD-1 d-1 for N2O to N2. The [Formula: see text] values were 2.7, 0.93, and 1.5 d-1, respectively. When N2O and NO3- were added concurrently, the apparent (extant) kinetics, [Formula: see text], assuming reduction to N2, were 6.3 gCOD gCOD-1 d-1, compared to 5.4 gCOD gCOD-1 d-1 for NO3- as the sole added acceptor. The [Formula: see text] was 1.6 d-1, compared to 2.5 d-1 for NO3- alone. These results suggest that NO3- and N2O were reduced concurrently. Based on this research, denitrifying bacteria like P. pantotrophus may serve as a significant sink for N2O. With careful design and operation, treatment plants can use denitrifying bacteria to minimize N2O emissions.

A methodology to assess the effects of biofilm roughness on substrate fluxes using image analysis, substrate profiling, and mathematical modelling.

We present a novel approach, based on image analysis and modelling, to study the impact of morphological variability (roughness) and fluid dynamics on substrate mass fluxes in biofilms. Specifically, we used this method to assess substrate fluxes in counter-diffusional autotrophic biofilms in a hydrogen-based membrane biofilm reactor. The physical structure of the biofilm was determined in situ at the meso-scale using stereomicroscopy. Image analysis was used to characterize the biofilm structure, and substrate profiles were obtained using microsensors. A two-dimensional, continuum biofilm model including microbial reactions, mass transport, and fluid dynamics was developed to compute substrate conversion in irregularly shaped counter-diffusional biofilms. Experimental biofilm structures were reproduced in the model and simulated under the prevailing substrate and hydrodynamic conditions for flow velocities varied over three orders of magnitude. Model calculations were consistent with experimental results and showed enhanced conversion rates with increased roughness at higher flow velocities. Also, modelling showed that conversion rates in counter-diffusional biofilms were typically higher than in co-diffusional biofilms. This study highlights the potential to use a simple image acquisition approach coupled to a theoretical model, to evaluate biofilm overall substrate utilization related to biofilm morphological heterogeneity.

Energy-efficient wastewater treatment via the air-based, hybrid membrane biofilm reactor (hybrid MfBR).

We used modeling to predict the energy and cost savings associated with the air-based, hybrid membrane-biofilm reactor (hybrid MfBR). This process is obtained by replacing fine-bubble diffusers in conventional activated sludge with air-supplying, hollow-fiber membrane modules. Evaluated processes included removal of chemical oxygen demand (COD), combined COD and total nitrogen (TN) removal, and hybrid growth (biofilm and suspended). Target concentrations of COD and TN were based on high-stringency water reuse scenarios. Results showed reductions in power requirements as high as 86%. The decrease mainly resulted from the dramatically lower air flows for the MBfR, resulting from its higher oxygen-transfer efficiencies. When the MBfR was used for COD and TN removal, savings up to US$200/1,000 m(3) of treated water were predicted. Cost savings were highly sensitive to the costs of the membrane modules and electrical power. The costs were also very sensitive to membrane oxidation flux for ammonia, and the membrane life. These results suggest the hybrid MBfR may provide significant savings in energy and costs. Further research on the identified key parameters can help confirm these modeling predictions and facilitate scale-up.

Evidence of specialized bromate-reducing bacteria in a hollow fiber membrane biofilm reactor.

Bromate is a carcinogenic disinfection by-product formed from bromide during ozonation or advanced oxidation. We previously observed bromate reduction in a hydrogen-based, denitrifying hollow fiber membrane biofilm reactor (MBfR). In this research, we investigated the potential existence of specialized bromate-reducing bacteria. Using denaturing gradient gel electrophoresis (DGGE), we compared the microbial ecology of two denitrifying MBfRs, one amended with nitrate as the electron acceptor and the other with nitrate plus bromate. The DGGE results showed that bromate exerted a selective pressure for a putative, specialized bromate-reducing bacterium, which developed a strong presence only in the reactor with bromate. To gain further insight into the capabilities of specialized, bromate-reducing bacteria, we explored bromate reduction in a control MBfR without any primary electron acceptors. A grown biofilm in the control MBfR reduced bromate without previous exposure, but the rate of reduction decreased over time, especially after perturbations resulting in biomass loss. The decrease in bromate reduction may have been the result of the toxic effects of bromate. We also used batch tests of the perchlorate-reducing pure culture, Dechloromonas sp. PC1 to test bromate reduction and growth. Bromate was reduced without measurable growth. Based on these results, we speculate bromate's selective pressure for the putative, specialized BRB observed in the DGGE was not growth related, but possibly based on resistance to bromate toxicity.

Performance and microbial ecology of the hybrid membrane biofilm process for concurrent nitrification and denitrification of wastewater.

We report on a novel process for total nitrogen (TN) removal, the hybrid membrane biofilm process (HMBP). The HMBP uses air-supplying hollow-fibre membranes inside an activated sludge tank, with suppressed aeration, to allow concurrent nitrification and denitrification. We hypothesised that a nitrifying biofilm would form on the membranes, and that the low bulk-liquid BOD concentrations would encourage heterotrophic denitrifying bacteria to grow in suspension. A nitrifying biofilm was initially established by supplying an influent ammonia concentration of 20 mgN/L. Subsequently, 120 mg/L acetate was added to the influent as BOD. With a bulk-liquid SRT of only 5 days, nitrification rates were 0.85 gN/m(2) per day and the TN removal reached 75%. The biofilm thickness was approximately 500 lim. We used DGGE to obtain a microbial community fingerprint of suspended and attached growth, and prepared a clone library. The DGGE results, along with the clone library and operating data, suggest that nitrifying bacteria were primarily attached to the membranes, while heterotrophic bacteria were predominant in the bulk liquid. Our results demonstrate that the HMBP is effective for TN removal, achieving high levels of nitrification with a low bulk-liquid SRT and concurrently denitrifying with BOD as the sole electron donor.

Hydrogen-based, hollow-fiber membrane biofilm reactor for reduction of perchlorate and other oxidized contaminants.

Many oxidized pollutants, such as nitrate, perchlorate, bromate, and chlorinated solvents, can be microbially reduced to less toxic or less soluble forms. For drinking water treatment, an electron donor must be added. Hydrogen is an ideal electron donor, as it is non-toxic, inexpensive, and sparsely soluble. We tested a hydrogen-based, hollow-fiber membrane biofilm reactor (MBfR) for reduction of perchlorate, bromate, chlorate, chlorite, chromate, selenate, selenite, and dichloromethane. The influent included 5 mg/L nitrate or 8 mg/L oxygen as a primary electron accepting substrate, plus 1 mg/L of the contaminant. The mixed-culture reactor was operated at a pH of 7 and with a 25 minute hydraulic detention time. High recirculation rates provided completely mixed conditions. The objective was to screen for the reduction of each contaminant. The tests were short-term, without allowing time for the reactor to adapt to the contaminants. Nitrate and oxygen were reduced by over 99 percent for all tests. Removals for the contaminants ranged from a minimum of 29% for chlorate to over 95% for bromate. Results show that the tested contaminants can be removed as secondary substrates in an MBfR, and that the MBfR may be suitable for treating these and other oxidized contaminants in drinking water.

A factor analytic study of branching patient management problems.

This study was undertaken to determine the factor analytic structure of patient management problems (PMPs) and to determine whether such factors are stable for different groups taking the same examination and for the same group over time. Two examinations were administered to a group of medical students, the first during their junior year and the second during their senior year. The second examination was also administered to a second class of students during their junior year. Factor analyses results indicated there are two components to medical problem-solving as measured by PMPs-data gathering and management. Both factors were stable over groups and over time.