Comparison of Estrogenic, Spectroscopic, and Toxicological Analyses of Pilot-Scale Water, Wastewaters, and Processed Wastewaters at Select Military Installations.

Reuse of water requires the removal of contaminants to ensure human health. We report the relative estrogenic activity (REA) of reuse treatment design scenarios for water, wastewaters, and processed wastewaters before and after pilot-scale treatment systems tested at select military facilities. The comparative relationships between REA, several composite toxicological endpoints, and spectroscopic indicators were evaluated for different reuse treatment trains. Four treatment processes including conventional and advanced treatments reduced the estrogenicity by at least 33%. Biologically based methods reduced estrogenicity to below detection levels. Conventional treatment scenarios led to significantly less reduction of adverse biological endpoints compared to the advanced treatment scenarios. Incorporating the anaerobic membrane bioreactor reduced more endpoints with higher reduction percentages compared to the sequencing batch reactor design. Membrane technology and advanced oxidation generated reductions across all biological endpoints, from 65% (genotoxicity) to 100% (estrogenicity). The design scenarios featuring a low-cutoff mechanical screen filter, intermittent activated carbon biofilter, and membrane filtration achieved the highest percent reduction and produced water with the lowest negative biological endpoints. Spectroscopic indicators demonstrated case-specific relationships with estrogenicity and toxicity. Estrogenicity consistently correlated with cytotoxicity and thiol reactivity, indicating the potential for preliminary estrogenicity screening using thiol reactivity.

Water toxicity evaluations: Comparing genetically modified bioluminescent bacteria and CHO cells as biomonitoring tools.

The use of water for drinking and agriculture requires knowledge of its toxicity. In this study, we compared the use of genetically modified bioluminescent (GMB) bacteria whose luminescence increases in the presence of toxicants and Chinese Hamster Ovary (CHO) cells for the characterization of the toxicity of water samples collected from a lake and streams, hydroponic and aquaponic farms, and a wastewater treatment plant. GMB bacteria were used to probe genotoxicity, cytotoxicity and reactive oxygen species-induced effects in the whole water samples. Unlike GMB bacteria, the use of CHO cells requires XAD resin-based pre-concentration of toxic material present in water samples for the subsequent cytotoxicity assay. In addition to the examination of the toxicity of the water from the different sources, the GMB bacteria were also used to test the XAD extracts diluted to the concentrations causing 50% growth inhibition of the CHO cells. The two biomonitoring tools provided different results when they were used to test the above-mentioned diluted XAD extracts. A pre-concentration procedure based on adsorption by XAD resins with subsequent elution was not sufficient to represent the material responsible for the toxicity of the whole water samples toward the GMB bacteria. Therefore, the use of XAD resin extracts may lead to major underestimates of the toxicity of water samples. Although the toxicity findings obtained using the GMB bacteria and CHO cells may not correlate with each another, the GMB bacteria assay did provide a mechanism-specific biomonitoring tool to probe the toxicity of water samples without a need for the pre-concentration step.

Innovative ex-situ biological biogas upgrading using immobilized biomethanation bioreactor (IBBR).

Biogas, which typically consists of about 50-70% of methane gas, is produced by anaerobic digestion of organic waste and wastewater. Biogas is considered an important energy resource with much potential; however, its application is low due to its low quality. In this regard, upgrading it to natural gas quality (above 90% methane) will broaden its application. In this research, a novel ex-situ immobilized biomethanation bioreactor (IBBR) was developed for biologically upgrading biogas by reducing CO2 to CH4 using hydrogen gas as an electron donor. The developed process is based on immobilized microorganisms within a polymeric matrix enabling the application of high recirculation to increase the hydrogen bioavailability. This generates an increase in the consumption rate of hydrogen and the production rate of methane. This process was successfully demonstrated at laboratory-scale system, where the developed process led to a production of 80-89% methane with consumption of more than 93% of the fed hydrogen. However, a lower methane content was achieved in the bench-scale system, likely as a result of lower hydrogen consumption (63-90%). To conclude, the IBBRs show promising results with a potential for simple and effective biogas upgrading.

Influence of Anaerobic Mesophilic and Thermophilic Digestion on Cytotoxicity of Swine Wastewaters.

Recycling wastewater from animal production for fertilizers using anaerobic digestion (AD) is a common method to recover the nutrients in the digestate. However, the digestate toxicity is not well understood because AD is mainly designed for chemical oxygen demand reduction. This study determined the toxicity during AD and the controlling factors with the goal to improve digestate safety during farmer handling to reuse the nutrients. Thermophilic and mesophilic AD of two swine wastewater sources were studied. Mammalian cell cytotoxicity revealed that the effluent after thermophilic digestion was at least 69% more toxic than the mesophilic effluent, owing to higher ammonia and total organic carbon in the former. Ammonia accounted for >55% total cytotoxicity, and the organics of the thermophilic digestate were twice more toxic than those in the mesophilic digestate. Despite less toxicity contribution than the ammonia, the organics did demonstrate significant adverse effects on the thiol-mediated cellular protection mechanism. For swine wastewater nutrient recovery, converting ammonia to less toxic nitrogen forms could lower the toxic hazard of the AD digestate. With much less ammonia, the organics would be the remaining decisive factor for toxicity, which is favorably reduced using thermophilic AD over mesophilic. If the ammonia is not reduced, mesophilic AD would generate a less toxic digestate.

Toxicological Comparison of Water, Wastewaters, and Processed Wastewaters.

Drinking water utilities will increasingly rely on alternative water sources in the future, including wastewater reuse. Safety must be assured in the application of advanced oxidation processes (AOPs) and supporting treatments for wastewater effluent reuse. This study developed toxicological profiles for source and tap waters, wastewaters, and treated effluents by different processes from four military installation locations. The objective of this study was to evaluate the toxicity of extracted organics from diverse source waters and after reuse treatments. The toxicity analyses included thiol reactivity, mammalian cell cytotoxicity, and genotoxicity. Differences in toxicity between source or tap waters and effluents from wastewater treatment processes supported AOP treatment to reduce risks of potable reuse. An anoxic and aerobic activated sludge process followed by sand filtration controlled toxicity to levels similar to a municipal drinking water. An anaerobic membrane bioreactor process exceeded the toxicity levels of a typical drinking water. Two AOP processes (ultraviolet (UV) + reverse osmosis (RO) + chlorination (NaOCl) or RO + UV-H2O2 + NaOCl) significantly reduced toxicity. The integration of the wastewater systems with ultrafiltration, AOP, and RO was effective to reduce the toxicity to levels comparable to, or better than, tap water samples.

The impact of disinfection Ct values on cytotoxicity of agricultural wastewaters: Ozonation vs. chlorination.

Toxicity arising from toxic disinfection byproducts is an unintended result of disinfection during water reclamation. To ensure safe water reclamation treatment, it is important to develop a disinfection strategy with minimal formation of overall toxicity in the reclaimed water. The cumulative disinfectant concentration over time (Ct) is a useful concept for pathogen control during reuse water disinfection. We evaluated the toxicity impact of Ct values and different methods to achieve identical Ct values by ozonation or chlorination of wastewaters from four agricultural sources on mammalian cells. N-acetylcysteine (NAC) reactivity of the wastewater organic extracts was determined to reveal their impact on the thiol-specific biological detoxification mechanism. The results demonstrated that for two sources and for both ozonation and chlorination, higher Ct values enhanced cytotoxicity. The ozonated waters were at least 10% less toxic and as much as 22.4 times less toxic than either the non-disinfected controls or the chlorinated waters. Chlorination consistently induced higher cytotoxicity than ozonation by between 2.2 and 22.4 fold, respectively, and induced similar or higher cytotoxicity than the non-disinfected controls, by at most 4.4 fold. Given the same Ct values, the combination of high disinfectant concentration and short contact time produced finished wastewaters with higher toxicity, than the combination of low disinfectant concentration and long contact time. NAC thiol reactivity was positively and significantly correlated with mammalian cell cytotoxicity, and agreed with 80% of the cytotoxicity rank order. This suggests that the induction of cytotoxicity involved reactions with agents that acted as thiol pool quenchers. The overall results indicate that the cytotoxicity of wastewaters may increase when higher Ct values are applied to inactivate recalcitrant pathogens. To counteract the potential increase in cytotoxicity at high Ct values, for both ozonation and chlorination, lower disinfectant dose and longer contact time may be adopted.

Abundance and diversity of anammox bacteria in a mainstream municipal wastewater treatment plant.

Among the factors that obstruct the application of anammox-based technology for nitrogen removal from mainstream municipal wastewater is the water's high organic loads. We hypothesized that some anammox species can adapt and grow in mainstream wastewater in which a minimal temperature of 13-15 °C is maintained. Using the AMX368F and AMX820R PCR-primers, anammox bacteria were detected in influent wastewater (COD/N ratio > 13) and in the anaerobic, anoxic, and aerobic chambers of a full-scale municipal wastewater treatment plant, reaching 107 copies/g VSS of the16S rRNA gene. Furthermore, anammox activity was demonstrated by 15N-isotopic tracing. The DNA sequences of clones randomly selected from a clone library were mainly clustered with Candidatus Brocadia flugida in addition to Ca. Brocadia sinica, Ca. Jettenia asiatica, and Ca. Anammoxoglobus propionicus. However, Ca. Brocadia was the only genus detected by high-throughput next-generation sequencing and denaturing gradient gel electrophoresis. The nitrite producers, ammonia-oxidizing archaea and bacteria, were both detected in the influent wastewater and the other chambers, while the nitrite consumers, Nitrospira nitrite oxidizers and the nirS-type denitrifiers, dominated all chambers. The results indicate the occurrence and potential activity of anammox bacteria in mainstream wastewater under certain conditions (proper temperature). The dominance of Brocadia flugida and Anammoxoglobus propionicus suggests a role for volatile fatty acids in selecting the anammox community in wastewater.

Spectroscopic Indicators for Cytotoxicity of Chlorinated and Ozonated Effluents from Wastewater Stabilization Ponds and Activated Sludge.

We investigated chronic mammalian cell cytotoxicity of wastewaters from four sources and their optical spectroscopic properties with or without chlorination or ozonation. Samples from effluents of activated sludge, nitrification tower, facultative waste stabilization pond, and maturation waste stabilization pond were either chlorinated or ozonated. The wastewater samples were analyzed for fluorescence excitation emission matrix, specific fluorescence index (SFI), and specific UV absorbance at 254 nm (SUVA). Before and after disinfection the wastewater samples were quantitatively analyzed for in vitro mammalian cell cytotoxicity. We found that the organic extracts from the ozonated samples induced lower cytotoxicity responses than those from the chlorinated or the nondisinfected samples. To develop correlations between SFI, SUVA, and cytotoxicity, we analyzed 21 independent samples. Significant linear correlations found among these samples suggest that under the tested conditions, cytotoxicity was preferentially influenced by the fluorescence and SUVA of their composite organic agents. These two spectroscopic parameters may be used as indicators for the potential cytotoxicity of nondisinfected, ozonated, or chlorinated municipal wastewaters.

Application of immobilized and granular dried anaerobic biomass for stabilizing and increasing anaerobic bio-systems tolerance for high organic loads and phenol shocks.

This study focuses on the stability and tolerance of continuous-flow bioreactors inoculated with anaerobic methanogens in three different configurations: (R1) dried granular biomass immobilized in PAC-enriched hydrophilic polyurethane foam, (R2) dried granular biomass, and (R3) wet granular biomass. These systems were tested under two different organic loading rates (OLR) of 6.25 and 10.94 (gCOD/(Lreactor∗d)), using a glucose-based synthetic mixture. The effect of an instantaneous shock load of phenol (5g/L for three days), and of phenol inclusion in the feed (0.5g/L) were also tested. At the lower OLR, all reactors performed similarly, however, increasing the OLR lead to a significant biomass washout and failure of R3. Biomass in R1 was more tolerant to phenol shock load than R2, though activity was recovered in both systems after about one month. PAC provided protection and shortened the adaptation time for 0.5g/L phenol that continuously was fed.

The effect of anaerobic biomass drying and exposure to air on their recovery and evolution.

The main goal of this study was to test the effect of various drying methods of granular anaerobic biomass on biomass survival, potential and rate of methane re-production, and structure. This may facilitate the development of drying methods to preserve excess anaerobic biomass in dry form for re-inoculation of existing digesters after process failure or wash out or for the start-up of new digesters. To that end, anaerobic granular biomass was collected from an up-flow anaerobic sludge blanket (UASB) reactor. The biomass was dried using two alternative methods: oven with air circulation at 50 °C for 24 h (DAO), and vacuum rotary evaporator at anaerobic conditions (DAN). For comparison, the control was a biomass with no drying (WET). Biomass samples were tested for specific methanogenic activity using synthetic wastewater. The microbial communities were also tested for viability using the LIVE/DEAD kit, and total biomass was initially quantified by qPCR targeting 16S rRNA genes. In addition, the mcrA functional gene was used s a target for the detection of the most abundant methanogens. Basic bacterial morphology classification was done by VIT(®) gene probe technology using a fluorescence microscope. Dried DAN and DAO biomasses required approximately four operational runs to recover their initial methanogenic activity compared to WET biomass. LIVE/DEAD results showed clear increases in the proportions of the viable biomass of the total bacterial communities over time, especially for the DAN and DAO samples. A comparison of the qPCR results of both DAN and DAO to the WET biomass showed that the methanogenic mcrA gene fraction of the total biomass population of 16S rRNA gene concentrations decreased moderately by about 17.2% in the samples of DAO and by approximately 6.7% in the samples of DAN over all runs after Run1.

Modeling the effect of immobilization of microorganisms on the rate of biodegradation of phenol under inhibitory conditions.

The main objective of this research is to model the effect of biodegradation process of phenol at high initial concentrations using a well known immobilization technique of the biomass. This work focused on testing the effect of activated carbon and clay while considering the diffusive internal mass transfer limitations. Biodegradation of phenol was performed by using enriched microorganisms from a compost of agricultural wastes. The average phenol biodegradation rate (uptake) of free biomass system was 235.3 mg g(-1) h(-1) at initial concentration range of 212-260 mg/L. However, the values for the systems of immobilized biomass in alginate and activated carbon (1 mm), alginate, activated carbon (4 mm), alginate, activated carbon and clay (1 mm) and alginate, activated carbon and clay (4 mm) were 64.9, 27.6, 27.5, and 8 mg g(-1) h(-1) respectively. The effective diffusion factors in different matrix were obtained using an intra-particle diffusion-based mathematical model. Diffusion limitation was observed when the matrix contained clay in addition to activated carbon. The diffusion coefficient was decreased from 1.6 × 10(-8) to 1.2 × 10(-9) cm(2)/s when clay was added to the matrix of 1 mm of alginate and activated carbon. Also, slight differences between the diffusion factors were observed for larger beads. The combination of clay and AC contributes to better mineralization of phenol at high concentrations. This could be attributed to the synergism of both additives.