Assessing membrane aerated biofilm reactor configurations in mainstream anammox applications.

Partial nitritation anammox (PNA) membrane aerated biofilm reactors (MABRs) have the potential to be employed in mainstream wastewater treatment and can drastically decrease the energy and carbon requirements for nitrogen removal. Previous PNA MABR studies have looked at 1-stage systems, but no study has holistically compared the performance of different MABR configurations. In this study, a PNA MABR was mechanistically modelled to determine the impact of the reactor configuration (1-stage, hybrid, or 2-stage system) on the location of the preferred niche for anammox bacteria and the overall nitrogen removal performance. Results from this study show that the 2-stage configuration, which used an MABR with a thin biofilm for nitritation and a moving bed biofilm reactor for anammox, had a 20% larger nitrogen removal rate than the 1-stage or hybrid configurations. This suggests that an MABR should focus on maximizing nitrite production with anammox implemented in a second-stage biofilm reactor to achieve the most cost-effective nitrogen removal. However, the optimal configuration will likely be facility specific, as each facility differs in operating costs, construction costs, footprint, and effluent limits. Additional experimentation is required to confirm these results, but this work narrows the number of viable configurations that need to be tested. The results of this study will inform researchers and engineers how to best implement PNA MABRs in mainstream nitrogen removal at larger scales.

Multi-year diagnosis of unpredictable fouling occurrences in a full-scale membrane bioreactor.

The membrane bioreactor (MBR) at the Traverse City Regional Wastewater Treatment Plant has experienced sudden and unpredictable periods of substantial permeability decline since 2011. Early observations detected irregularly-shaped Gram-positive bacteria that correlated with plant upsets. Use of biomolecular techniques, such as DNA sequencing of laboratory isolates and the mixed liquor microbial community, and fluorescent in situ hybridization, identified the dispersed organisms as members of the genus Staphylococcus. However, Staphylococcus species were consistently present during normal operation and therefore were more likely to be an indicator of the upset, not the cause. The results suggest that these microorganisms are responding to specific influent wastewater constituents. We chemically analysed seven mixed liquor samples from periods of permeability decline in 2017 and 2018, and four samples from a period of normal operation. During upset conditions, the total carbohydrate content exceeded that of normal operation by 40%. Additionally, mixed liquor calcium concentrations were 65% above normal during the upset in 2017. It is hypothesized and supported through multivariate statistical analysis and estimation of specific resistance to filtration values that a calcium-intermediated polymer bridging mechanism with extracellular polymeric substance constituents is a major contributor to fouling and permeability disruptions in the Traverse City MBR.

Elucidating the relative roles of ammonia oxidizing and heterotrophic bacteria during the biotransformation of 17α-Ethinylestradiol and Trimethoprim.

The biological fate of 17α-ethinylestradiol (EE2; 500 ng/L to 1 mg/L) and trimethoprim (TMP; 1 µg/L to 1 mg/L) was evaluated with flow through reactors containing an ammonia oxidizing bacterial (AOB) culture, two enriched heterotrophic cultures devoid of nitrifier activity, and nitrifying activated sludge (NAS) cultures. AOBs biotransformed EE2 but not TMP, whereas heterotrophs mineralized EE2, biotransformed TMP, and mineralized EE2-derived metabolites generated by AOBs. Kinetic bioassays showed that AOBs biotransformed EE2 five times faster than heterotrophs. The basal expression of heterotrophic dioxygenase enzymes was sufficient to achieve the high degree of transformation observed at EE2 and TMP concentrations ≤ 1 mg/L, and enhanced enzyme expression was not necessary. The importance of AOBs in removing EE2 and TMP was evaluated further by performing NAS experiments at lower feed concentrations (500-1000 ng/L). EE2 removal slowed markedly after AOBs were inhibited, while TMP removal was not affected by AOB inhibition. Two key EE2 metabolites formed by AOB and heterotrophic laboratory-scale chemostats were also found in independent laboratory-scale mixed culture bioreactors; one of these, sulfo-EE2, was largely resistant to further biodegradation. AOBs and heterotrophs may cooperatively enhance the reliability of treatment systems where efficient removal of EE2 is desired.

Effect of diet on fecal and urinary estrogenic activity.

The United States Environmental Protection Agency has identified estrogens from animal feeding operations as a major environmental concern, but few data are available to quantify the excretion of estrogenic compounds by dairy cattle. The objectives of this study were to quantify variation in estrogenic activity in feces and urine due to increased dietary inclusion of phytoestrogens. Ten Holstein heifers were assigned to 2 groups balanced for age and days pregnant; groups were randomly assigned to treatment sequence in a 2-period crossover design. Dietary treatments consisted of grass hay or red clover hay, and necessary supplements. Total collection allowed for sampling of feed refusals, feces, and urine during the last 4 d of each period. Feces and urine samples were pooled by heifer and period, and base extracts were analyzed for estrogenic activity (estrogen equivalents) using the yeast estrogen screen bioassay. Feces and urine samples collected from 5 heifers were extracted and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify excretion of 7 phytoestrogenic compounds. Excretion of 17-beta estradiol equivalents in urine was higher and tended to be higher in feces for heifers fed red clover hay (84.4 and 120.2 mg/d for feces and urine, respectively) compared with those fed grass hay (57.4 and 35.6 mg/d). Analysis by LC-MS/MS indicated greater fecal excretion of equol, genistein, daidzein, coumestrol, and formononetin by heifers fed red clover hay (1634, 29.9, 96.3, 27.8, and 163 mg/d, respectively) than heifers fed grass hay (340, 3.0, 46.2, 8.8, and 18.3 mg/d, respectively). Diet had no effect on fecal biochanin A or 2-carbethoxy-5, 7-dihydroxy-4'-methoxyisoflavone. Four phytoestrogens were detected in urine (2-carbethoxy-5, 7-dihydroxy-4'-methoxyisoflavone, daidzein, equol, and formononetin) and their excretion was not affected by diet. Identifying sources of variation in estrogenic activity of manure will aid in the development of practices to reduce environmental estrogen accumulation.

The use of qualitative system dynamics to identify sustainability characteristics of decentralized wastewater management alternatives.

In order to pursue more sustainable alternatives in wastewater management, it is vital that we understand how a given infrastructure alternative will impact the various aspects of sustainability. A set of qualitative tools (force field diagrams and causal loop diagrams (CLDs)) for the assessment of wastewater management alternatives is proposed and demonstrated in the context of a decentralized wastewater infrastructure upgrade. The objective for the application of these tools is to improve decision makers' understanding of how a given alternative will impact the economic, environmental/ecological, social, and functional aspects of sustainability. In the proposed method, each aspect of sustainability is treated as a stock, and its movement (up or down) can be inferred using both qualitative and quantitative data. By incorporating these tools into a participatory planning process, project-specific CLDs can be developed and loops of interest can be identified to help elucidate stakeholder values. The ultimate goal of this methodology is to facilitate the pursuit of sustainability in wastewater management by allowing decision makers to address specific sustainability challenges without creating new ones.

Characterization of metabolites formed during the biotransformation of 17alpha-ethinylestradiol by Nitrosomonas europaea in batch and continuous flow bioreactors.

The biotransformation of 17alpha-ethinylestradiol (EE2) by an ammonia oxidizing bacteria, Nitrosomonas europaea, grown in batch (ammonia-rich) and continuous flow (chemostat, ammonia-limited) reactors was investigated. Both C-14 labeled EE2 (10 gammag/L) and unlabeled EE2 (1 mg/L) were used to facilitate metabolite identification under environmentally relevant physiological conditions. Whole cell ammonia monooxygenase (AMO) activity was not inhibited at the EE2 concentrations used in this study. Characterization of the primary metabolite formed during batch cultivation by liquid chromatography/ion-trap mass spectrometry (LC-ITMS) and nuclear magnetic resonance (NMR) spectroscopy showed modification at the ethinyl group and addition of a carboxyl group. This metabolite (M386) (revealed by m/z 385 in negative mode electrospray LC/ MS) was not formed in the abiotic control. In contrast, biotransformation of EE2 under continuous flow conditions showed formation of a monohydroxylated EE2 (revealed by m/z 311), but not M386. Furthermore, nitrated EE2 derivatives were formed in both batch and continuous flow cultures, as a result of abiotic transformation of EE2 in the presence of high concentrations of nitrite in the bioreactors. Results from this study underscore the importance of physiological state and growth conditions as critical variables that can dictate the metabolic pathway for EE2 biodegradation and the nature of byproducts formed.

Chemical inhibition of nitrification in activated sludge.

Conventional aerobic nitrification was adversely affected by single pulse inputs of six different classes of industrially relevant chemical toxins: an electrophilic solvent (1-chloro-2,4-dinitrobenzene, CDNB), a heavy metal (cadmium), a hydrophobic chemical (1-octanol), an uncoupling agent (2,4-dinitrophenol, DNP), alkaline pH, and cyanide in its weak metal complexed form. The concentrations of each chemical source that caused 1 5, 25, and 50% respiratory inhibition of a nitrifying mixed liquor during a short-term assay were used to shock sequencing batch reactors containing nitrifying conventional activated sludge. The reactors were monitored for recovery over a period of 30 days or less. All shock conditions inhibited nitrification, but to different degrees. The nitrate generation rate (NGR) of the shocked reactors recovered overtime to control reactor levels and showed that it was a more sensitive indicator of nitrification inhibition than both initial respirometric tests conducted on unexposed biomass and effluent nitrogen species analyses. CDNB had the most severe impact on nitrification, followed by alkaline pH 11, cadmium, cyanide, octanol, and DNP. Based on effluent data, cadmium and octanol primarily inhibited ammonia-oxidizing bacteria (AOB) while CDNB, pH 11,and cyanide inhibited both AOB and nitrite-oxidizing bacteria (NOB). DNP initially inhibited nitrification but quickly increased the NGR relative to the control and stimulated nitrification after several days in a manner reflective of oxidative uncoupling. The shocked mixed liquor showed trends toward recovery from inhibition for all chemicals tested, but in some cases this reversion was slow. These results contribute to our broader effort to identify relationships between chemical sources and the process effects they induce in activated sludge treatment systems.

Deflocculation effects due to chemical perturbations in sequencing batch reactors.

Toxic shock-induced deflocculation was examined for activated sludge exposed to six different classes of industrially relevant chemical toxins: an electrophilic solvent (1-chloro-2,4-dinitrobenzene, CDNB), a heavy metal (cadmium), a hydrophobic chemical (1-octanol), an uncoupling agent (2,4-dinitrophenol, DNP), alkaline pH, and weakly complexed cyanide. The concentrations required to inhibit respiration by 50% were used to shock sequencing batch reactors (SBRs) containing a nitrifying (10-day solids retention time (SRT)) and a non-nitrifying (2-day SRT) biomass. Effluent total suspended solids (TSS) and soluble potassium were monitored to examine deflocculation caused by a bacterial stress response mechanism called glutathione-gated potassium efflux (GGKE). Reactors were monitored for recovery over a period of 3 SRTs or less. At the concentrations tested, CDNB, cadmium and pH 11 were found to cause significant increases in effluent TSS concentrations and showed elevated levels of potassium. In contrast, octanol, DNP and cyanide did not induce severe deflocculation and showed moderate increases in effluent potassium levels. Recovery of effluent TSS and potassium concentrations to control levels generally did not correlate, supporting the hypothesis that reflocculation requires regrowth of biomass. These results suggest that different chemicals induce deflocculation in SBRs, but deflocculation is not necessarily caused by the GGKE mechanism in all cases.

Evaluating the role of microbial stress response mechanisms in causing biological treatment system upset.

It is known that microbial stress mechanisms play a significant role in short-term microbial adaptation to environmental perturbations, and activation of these mechanisms enhance a cell's chance for surviving the perturbation with minimal damage. Although the target of these mechanisms is protective at the cellular level, the effect may be disruptive at the macroscopic level in engineered bioreactor systems. In this paper, it is proposed that these mechanisms are activated in response to wastewater influent perturbations and may be a significant cause of activated sludge treatment process upset. Selected microbial stress responses are reviewed and hypotheses indicating their potential role in treatment process upset are proposed. A research approach that was previously used to identify the mechanistic cause of deflocculation during perturbation by electrophilic chemicals is summarized, and a protocol for future experiments geared toward establishing source-cause-effect relationships for a range of wastewater upset conditions is put forth. Identifying source-cause-effect relationships will provide a basis for development of new monitoring technologies and operational strategies for systems under the influence of influent chemical perturbations.

Stress protein expression in domestic activated sludge in response to xenobiotic shock loading.

Using the Western blot immunochemical analysis method, the heat shock protein, GroEL, was found to be either induced or repressed in activated sludge microorganisms exposed to a range of xenobiotics. At the EC25 concentration, pentachlorophenol (PCP), cadmium, nickel, 2,4-dichloroaniline, benzoquinone, 2,4-dinitrophenol, and 1,1,1-trichloroethane all rapidly induced measurable GroEL expression, even though the time-dependent response for each of these compounds was somewhat varied. Toluene and hydroquinone resulted in repression of GroEL expression to levels below that measured in the control mixed liquor. For PCP concentrations at or exceeding the EC25, there was a significant and consistent increase in effluent volatile suspended solids from activated sludge sequencing batch reactors relative to unstressed controls. These preliminary results indicate that stress proteins may serve as sensitive and rapid indicators of toxicity which can adversely impact treatment process performance in activated sludge systems.

Creating anoxic and microaerobic conditions in sequencing batch reactors treating volatile BTX compounds.

An experimental strategy is introduced for studying the biodegradation of wastewaters containing volatile contaminants using an alternating anoxic/microaerobic sequencing batch reactor (SBR). Benzene, toluene, and the xylene isomers (BTX) served as model volatile contaminants for this study. The reactor was configured to overcome stripping the volatile BTX compounds into the atmosphere to provide opportunities for BTX biodegradation. Oxygen-free anoxic and microaerobic (< 0.2 mg/L dissolved oxygen) conditions were established using a novel laboratory reactor configuration. ORP was successfully used to monitor different electron acceptor conditions in the SBR. Toluene and m-xylene were amenable to anoxic (denitrifying) metabolism while benzene, o-, and p-xylene were biodegradable under microaerobic conditions. The results demonstrate that establishing microaerobic conditions in full-scale bioreactors may be an appropriate way to encourage the biodegradation of aerobically biodegradable volatile contaminants. Additionally, the laboratory reactor configuration introduced in this paper may be useful in subsequent studies involving microaerobic metabolism.

The immunochemical detection of stress proteins in activated sludge exposed to toxic chemicals.

The heat shock protein, GroEL, was found to be induced in activated sludge cultures exposed to perturbations of chemicals (cadmium, pentachlorophenol, and acetone) or heat stress. In laboratory activated sludge reactors, GroEL was rapidly induced (within minutes) in the presence of 5 mg/l or greater total cadmium. At 5 mg/l cadmium, however, moderate to insignificant changes in activated sludge process performance indicators [effluent suspended solids concentration, chemical oxygen demand (COD) removal, and specific oxygen uptake rate] were observed. As total cadmium concentrations increased above 5 mg/l, there was a significant and consistent increase in effluent volatile suspended solids concentrations from activated sludge sequencing batch reactors relative to unstressed controls. These results indicate that stress proteins may serve as sensitive and rapid indicators of mixed liquor toxicity which can adversely impact treatment process performance, but that GroEL may not be a good candidate protein for this purpose.

Detection of GroEL in activated sludge: a model for detection of system stress.

GroEL is a ubiquitous constitutively synthesized protein that is also stress inducible. Activated sludge, which is a standard biological process used in wastewater treatment systems, is made up of a diverse microbial consortium. The synthesis of GroEL in activated sludge was significantly induced after heat (42 degrees C) shock. The increased level of GroEL expression was shown to be due to de novo protein synthesis. We have demonstrated a method which shows that stress proteins can be detected in activated sludge, and propose their use as specific indicators of system stress.

Microscopic methods for distinguishing among three cell types in TOL plasmid-carrying Pseudomonas putida cultures.

Microscopic methods were developed that enable the sensitive quantification of different cell types that are generated by plasmid instability processes when Pseudomonas putida PaW164 (X+), which carries a TOL plasmid (pWW0-164), is grown in chemostat culture. Cells that have lost the structural TOL genes (X-) or the entire TOL plasmid (X0) can be quantified in a background of 6000 X+ cells using catechol agarose miniplates. X0 cells can be quantified in a background of 3500 X+ or X- cells using carbenicillin agarose miniplates. These methods represent significant improvements in sensitivity over conventional plating methods.

Impact of Growth in Benzoate and m-Toluate Liquid Media on Culturability of Pseudomonas putida on Benzoate and m-Toluate Plates.

Pseudomonas putida grown in continuous culture on benzoate or m-toluate lost the ability to grow on benzoate or m-toluate plates. A similar effect was not seen with a glucose continuous culture. Cells carrying and expressing a TOL plasmid rapidly lost their ability to grow on benzoate solid medium.