Distribution of Nitrobacter and Nitrospira Communities in an Aerobic Activated Sludge Bioreactor and their Contributions to Nitrite Oxidation

ABSTRACT An analysis of nitrite-oxidizing bacteria in the activated sludge process of a full-scale partially nitrifying wastewater treatment plant revealed Nitrospira and Nitrobacter averaged 10 13 cells·L -1 and 10 12 cells·L -1 , respectively. Correlation coefficients linking shifts in NOB community to operational or environmental variables illustrated Nitrospira were negatively correlated to nitrite (r = -0.45, P<0.01), while Nitrobacter showed no significant relationship to nitrite (P=0.1017). Nitrospira was negative correlation to DO (r = -0.46, P<0.01) and positively correlated to temperature (r = 0.59, P<0.0001). However, Nitrobacter was positively correlated to DO (r = 0.38, P<0.01) and HRT (R = 0.33, P<0.05), as well as negatively correlated to temperature (r = -0.49, P<0.001) suggesting niche adaptations within the NOB community. The positive association between Nitrobacter and DO supports a selective advantage over Nitrospira in completely nitrifying plants. Given the operational schematic at this WWTP, Nitrospira contributed more to nitrification than Nitrobacter in this WWTP

Development of a quantitative PCR method to differentiate between viable and nonviable bacteria in environmental water samples

Ethidium monoazide bromide (EMA) treatment of pure culture and environmental waters at low concentrations (1.0–7.5 µg/ml) indicated effective enumeration of viable and viable but nonculturable Escherichia coli in pure cultures, creek waters, and secondary activated sludge effluent samples by quantitative polymerase chain reaction (qPCR) amplification of the uidA and fliC gene targets at turbidity values <10 NTU. However, EMA treatment was not effective in primary clarifier and secondary trickling filter effluents where turbidities were ≥10 NTU. In viable pure cultures, rapidly dividing and senescent cells were most affected by increasing EMA concentrations. Amplification of heat-killed pure bacterial cultures decreased 4 to 6 logs depending on EMA concentration and culture age. The greatest difference was observed in 5-h cultures using 7.5 μg/ml EMA. Turbidity (≥100 NTU) in environmental samples inhibited EMA effectiveness on viability discrimination. Enumeration of E. coli in certain wastewaters using EMA-qPCR was similar to culture suggesting that EMA treatment could be incorporated into qPCR assays for the quantification of viable bacteria increasing assay time no more than 30 min. Our results indicate that EMA can be used in routine qPCR assays, but optimum conditions for exposure must be identified for each sample type due to sample matrix effects such as turbidity

Identification of Biomarker Genes To Predict Biodegradation of 1,4-Dioxane

Bacterial multicomponent monooxygenase gene targets in Pseudonocardia dioxanivorans CB1190 were evaluated for their use as biomarkers to identify the potential for 1,4-dioxane biodegradation in pure cultures and environmental samples. Our studies using laboratory pure cultures and industrial activated sludge samples suggest that the presence of genes associated with dioxane monooxygenase, propane monooxygenase, alcohol dehydrogenase, and aldehyde dehydrogenase are promising indicators of 1,4-dioxane biotransformation; however, gene abundance was insufficient to predict actual biodegradation. A time course gene expression analysis of dioxane and propane monooxygenases in Pseudonocardia dioxanivorans CB1190 and mixed communities in wastewater samples revealed important associations with the rates of 1,4-dioxane removal. In addition, transcripts of alcohol dehydrogenase and aldehyde dehydrogenase genes were upregulated during biodegradation, although only the aldehyde dehydrogenase was significantly correlated with 1,4-dioxane concentrations. Expression of the propane monooxygenase demonstrated a time-dependent relationship with 1,4-dioxane biodegradation in P. dioxanivorans CB1190, with increased expression occurring after over 50% of the 1,4-dioxane had been removed. While the fraction of P. dioxanivorans CB1190-like bacteria among the total bacterial population significantly increased with decrease in 1,4-dioxane concentrations in wastewater treatment samples undergoing active biodegradation, the abundance and expression of monooxygenase-based biomarkers were better predictors of 1,4-dioxane degradation than taxonomic 16S rRNA genes. This study illustrates that specific bacterial monooxygenase and dehydrogenase gene targets together can serve as effective biomarkers for 1,4-dioxane biodegradation in the environment

Synergistic Treatment of Mixed 1,4-Dioxane and Chlorinated Solvent Contaminations by Coupling Electrochemical Oxidation with Aerobic Biodegradation

Biodegradation of the persistent groundwater contaminant 1,4-dioxane is often hindered by the absence of dissolved oxygen and the co-occurrence of inhibiting chlorinated solvents. Using flow-through electrolytic reactors equipped with Ti/IrO₂–Ta₂O₅ mesh electrodes, we show that combining electrochemical oxidation with aerobic biodegradation produces an overadditive treatment effect for degrading 1,4-dioxane. In reactors bioaugmented by <i>Pseudonocardia dioxanivorans</i> CB1190 with 3.0 V applied, 1,4-dioxane was oxidized 2.5 times faster than in bioaugmented control reactors without an applied potential, and 12 times faster than by abiotic electrolysis only. Quantitative polymerase chain reaction analyses of CB1190 abundance, oxidation–reduction potential, and dissolved oxygen measurements indicated that microbial growth was promoted by anodic oxygen-generating reactions. At a higher potential of 8.0 V, however, the cell abundance near the anode was diminished, likely due to unfavorable pH and/or redox conditions. When coupled to electrolysis, biodegradation of 1,4-dioxane was sustained even in the presence of the common co-contaminant trichloroethene in the influent. Our findings demonstrate that combining electrolytic treatment with aerobic biodegradation may be a promising synergistic approach for the treatment of mixed contaminants

Differential Sensitivity of Wetland-Derived Nitrogen Cycling Microorganisms to Copper Nanoparticles

Metallic nanoparticles (NPs), the most abundant nanomaterials in consumer and industrial products, are the most probable class to enter the environment. In this study, wetland-derived microcosms were incubated with copper nanoparticles (Cu-NP) and ionic CuCl2 to investigate acute (10 days) and chronic (100 days) exposure towards nitrogen cycling microorganisms. The microbial ecology of wetlands play a crucial role in balancing nitrogen in pristine environments as well as in areas impacted by high nutrient loads (e.g., at wastewater effluent discharges). Gene abundance and expression changes were monitored using the GeoChip 5.0 high throughput functional gene microarray and metatranscriptomic shotgun sequencing (RNA-seq), respectively. After 10 days, the Cu-NP impacted microbial communities experienced structural shifts within microorganisms associated with dissimilatory nitrogen reduction accompanied by lower nitrate removal as compared to the unexposed controls. By day 100, these differences were largely resolved and nitrate removal was similar to the unexposed control. Furthermore, the Cu-NP exposed microcosms tolerated copper and were more resilient and adaptive than the unexposed controls based on the abundance and expression of other functions, including electron transfer, metal homeostasis, and stress response. These findings suggest sudden influxes of Cu-NPs into wetland systems may impair nitrogen removal initially, but long-term microbial shifts and functional redundancy would promote the net flux of total nitrogen out of the wetlands

Adsorption of Contaminants of Emerging Concern (CECs) with Varying Hydrophobicity on Macro- and Microplastic Polyvinyl Chloride, Polyethylene, and Polystyrene: Kinetics and Potential Mechanisms

Microplastic particles are of concern to aquatic environments because their size enables them to be easily ingested by animals and they may become vectors of potentially harmful chemicals. This study focused on understanding the impact of plastic size and plastic types on adsorption and adsorption kinetics of commonly found contaminants of emerging concern (CECs). We exposed macro- and micro-sized polyethylene (PE), polystyrene (PS), and polyvinyl chloride (PVC) to six CECs: diclofenac (DCF), atenolol (ATN), ibuprofen (IBU), 4-acetamidophenol (ACE), bisphenol A (BPA), and 2-mercaptobenzothiazole (MBT). Our results showed that the pseudo-first order model described the adsorption kinetics better than the pseudo-second order model. The rate of adsorption ACE onto macro-PS was the fastest rate of adsorption for all CECs and microplastics evaluated. Generally, the mass fraction of CECs sorbed at equilibrium did not depend on the size of the plastic and chemical hydrophobicity. With a relatively low Kow among the CECs studied here, ACE had the most mass fraction sorbed onto all the plastics in this study. DCF was also consistently sorbed onto all the plastics. The mechanism van der Waals interaction may have dominated in all the adsorptions in this study, but &pi;-&pi; interaction could also be a major mechanism in the adsorption of DCF, IBP, and ACE. Fast adsorption of ATN, IBP, and ACE may occur during wastewater treatment, but slow adsorption may still continue in the wastewater effluent. Our study highlights an ecotoxicological concern for plastics being a vector of commonly found CECs that are not highly hydrophobic

Identification of Biomarker Genes To Predict Biodegradation of 1,4-Dioxane

Bacterial multicomponent monooxygenase gene targets in Pseudonocardia dioxanivorans CB1190 were evaluated for their use as biomarkers to identify the potential for 1,4-dioxane biodegradation in pure cultures and environmental samples. Our studies using laboratory pure cultures and industrial activated sludge samples suggest that the presence of genes associated with dioxane monooxygenase, propane monooxygenase, alcohol dehydrogenase, and aldehyde dehydrogenase are promising indicators of 1,4-dioxane biotransformation; however, gene abundance was insufficient to predict actual biodegradation. A time course gene expression analysis of dioxane and propane monooxygenases in Pseudonocardia dioxanivorans CB1190 and mixed communities in wastewater samples revealed important associations with the rates of 1,4-dioxane removal. In addition, transcripts of alcohol dehydrogenase and aldehyde dehydrogenase genes were upregulated during biodegradation, although only the aldehyde dehydrogenase was significantly correlated with 1,4-dioxane concentrations. Expression of the propane monooxygenase demonstrated a time-dependent relationship with 1,4-dioxane biodegradation in P. dioxanivorans CB1190, with increased expression occurring after over 50% of the 1,4-dioxane had been removed. While the fraction of P. dioxanivorans CB1190-like bacteria among the total bacterial population significantly increased with decrease in 1,4-dioxane concentrations in wastewater treatment samples undergoing active biodegradation, the abundance and expression of monooxygenase-based biomarkers were better predictors of 1,4-dioxane degradation than taxonomic 16S rRNA genes. This study illustrates that specific bacterial monooxygenase and dehydrogenase gene targets together can serve as effective biomarkers for 1,4-dioxane biodegradation in the environment