Modeling versatile and dynamic anaerobic metabolism for PAOs/GAOs competition using agent-based model and verification via single cell Raman Micro-spectroscopy.

Side-stream enhanced biological phosphorus removal process (S2EBPR) has been demonstrated to improve performance stability and offers a suite of advantages compared to conventional EBPR design. Design and optimization of S2EBPR require modification of the current EBPR models that were not able to fully reflect the metabolic functions of and competition between the polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) under extended anaerobic conditions as in the S2EBPR conditions. In this study, we proposed and validated an improved model (iEBPR) for simulating PAO and GAO competition that incorporated heterogeneity and versatility in PAO sequential polymer usage, staged maintenance-decay, and glycolysis-TCA pathway shifts. The iEBPR model was first calibrated against bulk batch testing experiment data and proved to perform better than the previous EBPR model for predicting the soluble orthoP, ammonia, biomass glycogen, and PHA temporal profiles in a starvation batch testing under prolonged anaerobic conditions. We further validated the model with another independent set of anaerobic testing data that included high-resolution single-cell and specific population level intracellular polymer measurements acquired with single-cell Raman micro-spectroscopy technique. The model accurately predicted the temporal changes in the intracellular polymers at cellular and population levels within PAOs and GAOs, and further confirmed the proposed mechanism of sequential polymer utilization, and polymer availability-dependent and staged maintenance-decay in PAOs. These results indicate that under extended anaerobic phases as in S2EBPR, the PAOs may gain competitive advantages over GAOs due to the possession of multiple intracellular polymers and the adaptive switching of the anaerobic metabolic pathways that consequently lead to the later and slower decay in PAOs than GAOs. The iEBPR model can be applied to facilitate and optimize the design and operations of S2EBPR for more reliable nutrient removal and recovery from wastewater.

Oligotyping and metagenomics reveal distinct Candidatus Accumulibacter communities in side-stream versus conventional full-scale enhanced biological phosphorus removal (EBPR) systems.

Candidatus Accumulibacter phosphatis (CAP) and its clade-level micro-diversity has been associated with and implicated in functional differences in phosphorus removal performance in enhanced biological phosphorus removal (EBPR) systems. Side-stream EBPR (S2EBPR) is an emerging process that has been shown to present a suite of advantages over the conventional EBPR design, however, large knowledge gaps remain in terms of its underlying ecological mechanisms. Here, we compared and revealed the higher-resolution differences in microbial ecology of CAP between a full-scale side-stream EBPR configuration and a conventional A2O EBPR process that were operated in parallel and with the same influent feed. Even though the relative abundance of CAP, revealed by 16S rRNA gene amplicon sequencing, was similar in both treatment trains, a clade-level analysis, using combined 16S rRNA-gene based amplicon sequencing and oligotyping analysis and metagenomics analysis, revealed the distinct CAP microdiversity between the S2EBPR and A2O configurations that likely attributed to the improved performance in S2EBPR in comparison to conventional EBPR. Furthermore, genome-resolved metagenomics enabled extraction of three metagenome-assembled genomes (MAGs) belonging to CAP clades IIB (RCAB4-2), IIC (RC14) and II (RC18), from full-scale EBPR sludge for the first time, including a distinct Ca. Accumulibacter clade that is dominant and associated only with the S2EBPR configuration. The results also revealed the temporally increasing predominance of RC14, which belonged to Clade IIC, during the implementation of the S2EBPR configuration. Finally, we also show the existence of previously uncharacterized diversity of clades of CAP, namely the clades IIB and as yet unidentified clade of type II, in full-scale EBPR communities, highlighting the unknown diversity of CAP communities in full-scale EBPR systems.

Intracellular polyphosphate length characterization in polyphosphate accumulating microorganisms (PAOs): Implications in PAO phenotypic diversity and enhanced biological phosphorus removal performance.

Polyphosphate (polyP) accumulating organisms (PAOs) are the key agent to perform enhanced biological phosphorus removal (EBPR) activity, and intracellular polyP plays a key role in this process. Potential associations between EBPR performance and the polyP structure have been suggested, but are yet to be extensively investigated, mainly due to the lack of established methods for polyP characterization in the EBPR system. In this study, we explored and demonstrated that single-cell Raman spectroscopy (SCRS) can be employed for characterizing intracellular polyPs of PAOs in complex environmental samples such as EBPR systems. The results, for the first time, revealed distinct distribution patterns of polyP length (as Raman peak position) in PAOs in lab-scale EBPR reactors that were dominated with different PAO types, as well as among different full-scale EBPR systems with varying configurations. Furthermore, SCRS revealed distinctive polyP composition/features among PAO phenotypic sub-groups, which are likely associated with phylogenetic and/or phenotypic diversity in EBPR communities, highlighting the possible resolving power of SCRS at the microdiversity level. To validate the observed polyP length variations via SCRS, we also performed and compared bulk polyP length characteristics in EBPR biomass using conventional polyacrylamide gel electrophoresis (PAGE) and solution 31P nuclear magnetic resonance (31P-NMR) methods. The results are consistent with the SCRS findings and confirmed the variations in the polyP lengths among different EBPR systems. Compared to conventional methods, SCRS exhibited advantages as compared to conventional methods, including the ability to characterize in situ the intracellular polyPs at subcellular resolution in a label-free and non-destructive way, and the capability to capture subtle and detailed biochemical fingerprints of cells for phenotypic classification. SCRS also has recognized limitations in comparison with 31P-NMR and PAGE, such as the inability to quantitatively detect the average polyP chain length and its distribution. The results provided initial evidence for the potential of SCRS-enabled polyP characterization as an alternative and complementary microbial community phenotyping method to facilitate the phenotype-function (performance) relationship deduction in EBPR systems.

Impact of oxidation processes on the composition and biodegradability of soluble organic nutrients in wastewater effluents.

The characteristics and bioavailability of wastewater derived organic nutrients and their susceptibility to removal technologies have implications in nutrient loading to aquatic environments and their contributions to eutrophication. Therefore, a better understanding of treatability of effluent organic nutrients is of interest for water resource recovery facilities (WRRFs) and regulators. Oxidation processes (OPs) can reduce concentrations of soluble organic nutrients and convert them into more biodegradable forms. In this study, three WRRF effluents were treated with low-pressure ultraviolet (UV) irradiation, hydrogen peroxide (H2 O2 ), and combined UV/H2 O2 . Untreated and treated effluents were subjected to nitrogen and phosphorus speciation analyses and soluble organic nitrogen (SON) biodegradability assays. The OP treatments did not change SON concentrations significantly. For two WRRFs, OP treatments decreased soluble organic phosphorus (SOP) and seemed to convert it into soluble acid hydrolyzable phosphorus (SAHP), indicating possible increases in phosphorus bioavailability. Fingerprinting and quantification of dissolved organic matter (DOM) using fluorescence spectroscopy with parallel factor analysis revealed changes in DOM pool composition in response to OPs treatments, which suggests likely organic nutrients composition changes. Based on biodegradability assessments, OP treatments likely changed the composition and biodegradability of effluent SON compounds. Combined UV/H2 O2 treatment seemed more effective than other OPs at oxidizing some of the organic nutrients. PRACTITIONER POINTS: Treatment of secondary or tertiary effluent with UV, H2 O2 , or UV/ H2 O2 was generally not effective at mineralizing SON and SOP, when applied at the doses used in this study. Treatment resulted in observable changes in DOM compositions, likely including SON and SOP compounds. Combined UV/H2 O2 treatment was more effective than UV or H2 O2 alone at oxidizing some DOM compounds. The BSON (bioavailable SON) assay indicated that the composition of the SON pool in the effluents was likely changed by the OP treatments. This was supported by fluorescence spectroscopy analysis.

Isolation of selenate from selenite, carbonate, phosphate, and arsenate solutions for δ18O-selenate determination.

Selenium and oxygen isotope systematics can be useful tools for tracing sources and fate of Se oxyanions in water. In order to measure δ18O values of selenate, SeO4 2- must first be sequestered from water by precipitation as BaSeO4(s). However, other dissolved oxyanions insoluble with Ba2+ require removal. Dissolved selenate was separated from dissolved selenite, carbonate, phosphate, and arsenate by addition of Ce3+ cations that quantitatively removed these oxyanions by precipitation as insoluble Ce2(SeO3)3(s), Ce2(CO3)3(s), CePO4(s), and CeAsO4(s), respectively. δ18O-selenate (-8.19 ± 0.17 ‰) did not change after four replicates of selenite removal by Ce2(SeO3)3(s) precipitation and Ce3+ removal by cation exchange (-8.20 ± 0.14, -8.32 ± 0.09, -8.17 ± 0.13, and -8.29 ± 0.13 ‰). δ18O-selenate values (-10.86 ± 0.45 ‰) were preserved also when selenate was pre-concentrated on anion exchange resin, quantitatively retrieved by elution, and processed with Ce3+ to remove interfering oxyanions (-10.77 ± 0.07 ‰). The extraction and purification steps developed here successfully isolated dissolved selenate from interfering oxyanions while preserving δ18O-selenate values. This method should be useful for characterizing δ18O-selenate when present with the co-occurring oxyanions above in laboratory experiments and field sites with high Se concentrations, although further research is required for methods to eliminate any co-occurring sulphate.

Survey of full-scale sidestream enhanced biological phosphorus removal (S2EBPR) systems and comparison with conventional EBPRs in North America: Process stability, kinetics, and microbial populations.

Sidestream EBPR (S2EBPR) is an emerging alternative process to address common challenges in EBPR related to weak wastewater influent and may improve EBPR process stability. A systematic evaluation and comparison of the process performance and microbial community structure was conducted between conventional and S2EBPR facilities in North America. The statistical analysis suggested higher performance stability in S2EBPR than conventional EBPR, although possible bias associated with other plant-specific factors might have affected the comparison. Variations in stoichiometric values related to EBPR activity and discrepancies between the observed values and current model predictions suggested a varying degree of metabolic versatility of PAOs in S2EBPR systems that warrant further investigation. Microbial community analysis using various techniques suggested comparable known candidate PAO relative abundances in S2EBPR and conventional EBPR systems, whereas the relative abundance of known candidate GAOs seemed to be consistently lower in S2EBPR facilities than conventional EBPR facilities. 16S rRNA gene sequencing analysis revealed differences in the community phylogenetic fingerprints between S2EBPR and conventional facilities and indicated statistically higher microbial diversity index values in S2EBPR facilities than those in conventional EBPRs. PRACTITIONER POINTS: Sidestream EBPR (S2EBPR) can be implemented with varying and flexible configurations, and they offer advantages over conventional configurations for addressing the common challenges in EBPR related to weak wastewater influent and may improve EBPR process stability. Survey of S2EBPR plants in North America suggested statistically more stable phosphorus removal performance in S2EBPR plants than conventional EBPRs, although possible bias might affect the comparison due to other plant-specific factors. The EBPR kinetics and stoichiometry of the S2EBPR facilities seemed to vary and are associated with metabolic versatility of PAOs in S2EBPR systems that warrant further investigation. The abundance of known candidate PAOs in S2EBPR plants was similar to those in conventional EBPRs, and the abundance of known candidate GAOs was generally lower in S2EBPR than conventional EBPR facilities. Further finer-resolution analysis of PAOs and GAOs, as well as identification of other unknown PAOs and GAOs, is needed. Microbial diversity is higher in S2EBPR facilities compared with conventional ones, implying that S2EBPR microbial communities could show better resilience to perturbations due to potential functional redundancy.

Impact of solid residence time (SRT) on functionally relevant microbial populations and performance in full-scale enhanced biological phosphorus removal (EBPR) systems.

Investigations of the impact of solid residence time (SRT) on microbial ecology and performance of enhanced biological phosphorus removal (EBPR) process in full-scale systems have been scarce due to the challenges in isolating and examining the SRT from other complex plant-specific factors. This study performed a comprehensive evaluation of the influence of SRT on polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) dynamics and on P removal performance at Clark County Water Reclamation District Facility in Las Vegas, USA. Five parallel treatment trains with separated clarifiers were operated with five different SRTs ranging from 6 to 40 days. Microbial community analysis using multiple molecular and Raman techniques suggested that the relative abundances and diversity of PAOs and GAOs in EBPR systems are highly affected by the SRT. The resultant EBPR system stability and performance can be potentially controlled and optimized by manipulating the system SRT, and shorter SRT (<10 days) seems to be preferred. PRACTITIONER POINTS: Phosphorus removal performance and kinetics are highly affected by the operational solid residence time (SRT), with lower and more stable effluent P level achieved at SRT < 10 days. Excessive long SRTs above that needed for nitrification may harm EBPR performance; additionally, excessive long SRT may favor GAOs to dominate over PAOs and thus further reducing efficient use of rbCOD for EBPR. Microbial population abundance and diversity, especially those functionally relevant PAOs and GAOs, can impact the P removal performances, and they are highly dependent on the operational solid residence time. EBPR performance can be potentially controlled and optimized by manipulating the system SRT, and shorter SRT (≤10 days) seems to be preferred at the influent rbCOD/P ratio and environmental conditions as in the plant studied.

Side-stream enhanced biological phosphorus removal (S2EBPR) process improves system performance - A full-scale comparative study.

To address the common challenges in enhanced biological phosphorus removal (EBPR) related to stability and unfavorable influent carbon to phosphorus ratio, a side-stream EBPR (S2EBPR) process that involves a side-stream anaerobic biological sludge hydrolysis and fermentation reactor was proposed as an emerging alternative. In this study, a full-scale pilot testing was performed with side-by-side operation of a conventional anaerobic-anoxic-aerobic (A2O) process versus a S2EBPR process. A comparison of the performance, activity and microbial community between the two configurations was performed. The results demonstrated that, with the same influent wastewater characteristics, S2EBPR configuration showed improved P removal performance and stability than the conventional A2O configuration, especially when the mixers in the side-stream anaerobic reactor were operated intermittently. Mass balance analysis illustrated that both denitrification and EBPR were enhanced in S2EBPR configuration, where return activated sludge was diverted into the anaerobic zone to promote fermentation and enrichment of polyphosphate accumulating organisms (PAOs), and the influent was bypassed to the anoxic zone for enhancing denitrification. A relatively higher PAO activity and total PAO abundance were observed in S2EBPR than in A2O configuration, accompanied by a higher degree of dependence on glycolysis pathway than tricarboxylic acid cycle. No significant difference in the relative abundances of putative PAOs, including Ca. Accumulibacter and Tetrasphaera, were observed between the two configurations. However, higher microbial community diversity indices were observed in S2EBPR configuration than in conventional one. In addition, consistently lower relative abundance of known glycogen accumulating organisms (GAOs) was observed in S2EBPR system. Extended anaerobic retention time and conditions that generate continuous and more complex volatile fatty acids in the side-stream anaerobic reactor of S2EBPR process likely give more competitive advantage for PAOs over GAOs. PAOs exhibited sustained EBPR activity and delayed decay under extended anaerobic condition, likely due to their versatile metabolic pathways depending on the availability and utilization of multiple intracellular polymers. This study provided new insights into the effects of implementing side-stream EBPR configuration on microbial populations, EBPR activity profiles and resulted system performance.

Comparative Life Cycle Assessment of Advanced Wastewater Treatment Processes for Removal of Chemicals of Emerging Concern.

The potential health effects associated with contaminants of emerging concern (CECs) have motivated regulatory initiatives and deployment of energy- and chemical-intensive advanced treatment processes for their removal. This study evaluates life cycle environmental and health impacts associated with advanced CEC removal processes, encompassing both the benefits of improved effluent quality as well as emissions from upstream activities. A total of 64 treatment configurations were designed and modeled for treating typical U.S. medium-strength wastewater, covering three policy-relevant representative levels of carbon and nutrient removal, with and without additional tertiary CEC removal. The USEtox model was used to calculate characterization factors of several CECs with missing values. Stochastic uncertainty analysis considered variability in influent water quality and uncertainty in CEC toxicity and associated characterization factors. Results show that advanced tertiary treatment can simultaneously reduce nutrients and CECs in effluents to specified limits, but these direct water quality benefits were outweighed by even greater increases in indirect impacts for the toxicity-related metrics, even when considering order-of-magnitude uncertainties for CEC characterization factors. Future work should consider water quality aspects not currently captured in life cycle impact assessment, such as endocrine disruption, in order to evaluate the full policy implications of the CEC removal.

Biotransformation of Two Pharmaceuticals by the Ammonia-Oxidizing Archaeon Nitrososphaera gargensis.

The biotransformation of some micropollutants has previously been observed to be positively associated with ammonia oxidation activities and the transcript abundance of the archaeal ammonia monooxygenase gene (amoA) in nitrifying activated sludge. Given the increasing interest in and potential importance of ammonia-oxidizing archaea (AOA), we investigated the capabilities of an AOA pure culture, Nitrososphaera gargensis, to biotransform ten micropollutants belonging to three structurally similar groups (i.e., phenylureas, tertiary amides, and tertiary amines). N. gargensis was able to biotransform two of the tertiary amines, mianserin (MIA) and ranitidine (RAN), exhibiting similar compound specificity as two ammonia-oxidizing bacteria (AOB) strains that were tested for comparison. The same MIA and RAN biotransformation reactions were carried out by both the AOA and AOB strains. The major transformation product (TP) of MIA, α-oxo MIA was likely formed via a two-step oxidation reaction. The first hydroxylation step is typically catalyzed by monooxygenases. Three RAN TP candidates were identified from nontarget analysis. Their tentative structures and possible biotransformation pathways were proposed. The biotransformation of MIA and RAN only occurred when ammonia oxidation was active, suggesting cometabolic transformations. Consistently, a comparative proteomic analysis revealed no significant differential expression of any protein-encoding gene in N. gargensis grown on ammonium with MIA or RAN compared with standard cultivation on ammonium only. Taken together, this study provides first important insights regarding the roles played by AOA in micropollutant biotransformation.

Life-Cycle Assessment of Advanced Nutrient Removal Technologies for Wastewater Treatment.

Advanced nutrient removal processes, while improving the water quality of the receiving water body, can also produce indirect environmental and health impacts associated with increases in usage of energy, chemicals, and other material resources. The present study evaluated three levels of treatment for nutrient removal (N and P) using 27 representative treatment process configurations. Impacts were assessed across multiple environmental and health impacts using life-cycle assessment (LCA) following the Tool for the Reduction and Assessment of Chemical and Other Environmental Impacts (TRACI) impact-assessment method. Results show that advanced technologies that achieve high-level nutrient removal significantly decreased local eutrophication potential, while chemicals and electricity use for these advanced treatments, particularly multistage enhanced tertiary processes and reverse osmosis, simultaneously increased eutrophication indirectly and contributed to other potential environmental and health impacts including human and ecotoxicity, global warming potential, ozone depletion, and acidification. Average eutrophication potential can be reduced by about 70% when Level 2 (TN = 3 mg/L; TP = 0.1 mg/L) treatments are employed instead of Level 1 (TN = 8 mg/L; TP = 1 mg/L), but the implementation of more advanced tertiary processes for Level 3 (TN = 1 mg/L; TP = 0.01 mg/L) treatment may only lead to an additional 15% net reduction in life-cycle eutrophication potential.

Universal quantifier derived from AFM analysis links cellular mechanical properties and cell-surface integration forces with microbial deposition and transport behavior.

In this study, we employed AFM analysis combined with mathematical modeling for quantifying cell-surface contact mechanics and magnitude and range of cell-surface interaction forces for seven bacterial strains with a wide range of cell morphology, dimension, and surface characteristics. Comprehensive cell-surface characterization including surface charge, extracellular polymeric substance content, hydrophobicity, and cell-cell aggregation analyses were performed. Flow-through column tests were employed to determine the attachment efficiency and deposition-transport behavior of these bacterial strains. No statistically significant correlation between attachment efficiency and any single-cell surface property was identified. Single-cell characterization by atomic force microscopy (AFM) yielded the mechanical deformation and elastic modulus, penetration resistance to AFM probe penetration by cellular surface substances (CSS), range and magnitude of the repulsive-attractive intersurface forces, and geometry of each strain. We proposed and derived a universal dimensionless modified Tabor's parameter to integrate all these properties that account for their collective behavior. Results showed that the Tabor parameter derived from AFM analysis correlated well with experimentally determined attachment efficiency (α), which therefore is able to link microscale cell-surface properties with macroscale bacterial transport behavior. Results suggested that the AFM tests performed between a single cell and a surface captured the key quantities of the interactions between the cell and the surface that dictate overall cell attachment behavior. Tabor's parameter therefore can be potentially incorporated into the microbial transport model.

Correlation of macroscopic aggregation behavior and microscopic adhesion properties of bacteria strains using a dimensionless Tabor's parameter.

Macroscopic adhesion-aggregation, floc formation, and subsequent transportation of microorganisms in porous media are closely related to the microscopic behavior and properties of individual cells. The classical Tabor's parameter in colloidal science is modified to correlate the macroscopic aggregation and microscopic adhesion properties of microorganisms. Seven bacterial strains relevant to wastewater treatment and bioremediation were characterized in terms of their macroscopic aggregation index (AI) using an optical method, and their microscopic coupled adhesion and deformation properties using atomic force microscopy (AFM). Single cells were indented to measure the range and magnitude of the repulsive-attractive intersurface forces, elastic modulus, thickness and density of the cellular surface substances (CSS). The strong correlation suggests that cost and time effective microscopic AFM characterization is capable of making reliable prediction of macroscopic behavior.

An antimicrobial polycationic sand filter for water disinfection.

A new sand filtration water disinfection technology is developed which relies on the antimicrobial properties of hydrophobic polycations (N-hexylated polyethylenimine) covalently attached to the sand's surface. The efficacy of the filter disinfection process was evaluated both with water spiked with E. coli and with real aqueous effluent from a wastewater treatment plant. For the former, over 7-log reduction in bacterial count was achieved. With real environmental wastewater secondary effluent samples, the E. coli concentration reduction declined to under 2 logs. This reduced inactivation efficiency compared to the model aqueous sample is likely due to the particulate or colloidal matter present that diminishes the contact between the immobilized polycation and the suspended bacteria. Preliminary sand washing methods were tested to assess potential 'regeneration' approaches. Potential advantages of the proposed approach over conventional disinfection in terms of eliminating harmful by-products and reducing energy consumption are discussed.

Process optimization by decoupled control of key microbial populations: distribution of activity and abundance of polyphosphate-accumulating organisms and nitrifying populations in a full-scale IFAS-EBPR plant.

This study investigated the abundance and distribution of key functional microbial populations and their activities in a full-scale integrated fixed film activated sludge-enhanced biological phosphorus removal (IFAS-EBPR) process. Polyphosphate accumulating organisms (PAOs) including Accumulibacter and EBPR activities were predominately associated with the mixed liquor (>90%) whereas nitrifying populations and nitrification activity resided mostly (>70%) on the carrier media. Ammonia oxidizer bacteria (AOB) were members of the Nitrosomonas europaea/eutropha/halophila and the Nitrosomonas oligotropha lineages, while nitrite oxidizer bacteria (NOB) belonged to the Nitrospira genus. Addition of the carrier media in the hybrid activated sludge system increased the nitrification capacity and stability; this effect was much greater in the first IFAS stage than in the second one where the residual ammonia concentration becomes limiting. Our results show that IFAS-EBPR systems enable decoupling of solid residence time (SRT) control for nitrifiers and PAOs that require or prefer conflicting SRT values (e.g. >15 days required for nitrifiers and <5 days preferred for PAOs). Allowing the slow-growing nitrifiers to attach to the carrier media and the faster-growing phosphorus (P)-removing organisms (and other heterotrophs, e.g. denitrifiers) to be in the suspended mixed liquor (ML), the EBPR-IFAS system facilitates separate SRT controls and overall optimization for both N and P removal processes.

Mechanistic toxicity assessment of nanomaterials by whole-cell-array stress genes expression analysis.

This study performed mechanistic toxicity assessment of nanosilver (nAg) and nanotitanium dioxide anatase (nTiO2_a) via toxicogenomic approach, employing a whole-cell-array library consisting of 91 recombinated Escherichia coli K12 strains with transcriptional GFP-fusions covering most known stress response genes. The results, for the first time, revealed more detailed transcriptional information on the toxic mechanism of nAg and nTiO2_a, and led to a better understanding of the mode of action (MOA) of metal and metal oxide nanomaterials (NMs). The detailed pathways network established for the oxidative stress system and for the SOS (DNA damage) repair system based on the temporal gene expression profiling data revealed the relationships and sequences of key genes involved in these toxin response systems. Both NMs were found to cause oxidative stress as well as cell membrane and transportation damage. Genotoxicity and DNA damage were also observed, although nTiO2_a induced SOS response via previously identified pathway and nAg seemed to induce DNA repair via a pathway different from SOS. We observed that the NMs at lower concentration tend to induce more chemical-specific toxicity response, while at higher concentrations, more general global stress response dominates. The information-rich real-time gene expression data allowed for identification of potential biomarkers that can be employed for specific toxin detection and biosensor developments. The concentration-dependent gene expression response led to the determination of the No Observed Transcriptional Effect Level (NOTEL) values, which can be potentially applied in the regulatory and risk assessment framework as an alternative toxicity assessment end point.

Implication of using different carbon sources for denitrification in wastewater treatments.

Application of external carbon sources for denitrification becomes necessary for wastewater treatment plants that have to meet very stringent effluent nitrogen limits (e.g., 3 to 5 mgTN/L). In this study, we evaluated and compared three carbon sources--MicroC (Environmental Operating Solutions, Bourne, Massachusetts), methanol, and acetate-in terms of their denitrification rates and kinetics, effect on overall nitrogen removal performance, and microbial community structure of carbon-specific denitrifying enrichments. Denitrification rates and kinetics were determined with both acclimated and non-acclimated biomass, obtained from laboratory-scale sequencing batch reactor systems or full-scale plants. The results demonstrate the feasibility of the use of MicroC for denitrification processes, with maximum denitrification rates (k(dmax)) of 6.4 mgN/gVSSh and an observed yield of 0.36 mgVSS/mgCOD. Comparable maximum nitrate uptake rates were found with methanol, while acetate showed a maximum denitrification rate nearly twice as high as the others. The maximum growth rates measured at 20 degrees C for MicroC and methanol were 3.7 and 1.2 day(-1), respectively. The implications resulting from the differences in the denitrification rates and kinetics of different carbon sources on the full-scale nitrogen removal performance, under various configurations and operational conditions, were assessed using Biowin (EnviroSim Associates, Ltd., Flamborough, Ontario, Canada) simulations for both pre- and post-denitrification systems. Examination of microbial population structures using Automated Ribosomal Intergenic Spacer Analysis (ARISA) throughout the study period showed dynamic temporal changes and distinct microbial community structures of different carbon-specific denitrifying cultures. The ability of a specific carbon-acclimated denitrifying population to instantly use other carbon source also was investigated, and the chemical-structure-associated behavior patterns observed suggested that the complex biochemical pathways/enzymes involved in the denitrification process depended on the carbon sources used.

Prokaryotic real-time gene expression profiling for toxicity assessment.

Examining global effects of toxins on gene expression profiles is proving to be a powerful method for toxicity assessment and for investigating mechanisms of toxicity. This study demonstrated the application of prokaryotic real-time gene expression profiling in Escherichia coli for toxicity assessment of environmental pollutants in water samples, by use of a cell-array library of 93 E. coli K12 strains with transcriptional green fluorescent protein (GFP) fusions covering most known stress response genes. The high-temporal-resolution gene expression data, for the first time, revealed complex and time-dependent transcriptional activities of various stress-associated genes in response to mercury and mitomycin (MMC) exposure and allowed for gene clustering analysis based on temporal response patterns. Compound-specific and distinctive gene expression profiles were obtained for MMC and mercury at different concentrations. MMC (genotoxin) induced not only the SOS response, which regulates DNA damage and repair, but also many other stress genes associated with drug resistance/sensitivity and chemical detoxification. A number of genes belonging to the P-type ATPase family and the MerR family were identified to be related to mercury resistance, among which zntA was found to be up-regulated at an increasing level as the mercury concentration increased. A mechanism-based evaluation of toxins based on real-time gene expression profiles promises, to be an efficient and informative method for toxicity assessment in environmental samples.

Nitrifying community analysis in a single submerged attached-growth bioreactor for treatment of high-ammonia waste stream.

This study investigated the nitrifying community structure in a single-stage submerged attached-growth bioreactor (SAGB) that successfully achieved stable nitrogen removal over nitrite of a high-strength ammonia wastewater. The reactor was operated with intermittent aeration and external carbon addition (methanol). With influent ammonia and total Kjeldahl nitrogen ranging from 537 to 968 mg/L and 643 to 1510 mg/L, respectively, 85% nitrogen removal was obtained, and effluent was dominated by nitrite (NO2-/NOx > 0.95). Nitrifying community analysis using fluorescence in situ hybridization (FISH), with a hierarchical set of probes targeting known ammonia-oxidizing bacteria (AOB) within beta-proteobacteria, showed that the AOB community of the biofilter consists almost entirely of members of the Nitrosomonas europaea/eutropha and the Nitrosococcus mobilis lineages. Image analysis of FISH pictures was used to quantify the identified AOB, and it was estimated that Nitrosomonas europaea/eutropha-like AOB accounted for 4.3% of the total volume of the biofilm, while Nitrosococcus mobilis-like AOB made up 1.2%; these numbers summed up to a total AOB fraction of 5.5% of the total volume on the biofilm. Nitrite-oxidizing bacteria (NOB) were not detectable in the biofilm samples with probes for either Nitrospira sp. or Nitrobacter sp., which indicated that NOB were either absent from the biofilters or present in numbers below the detection limit of FISH (< 0.1% of the total biofilm). Nitrite oxidizers were likely outcompeted from the system because of the free ammonia inhibition and the possibility that the aeration period (from intermittent aeration) was not sufficiently long for the NOB to be released from the competition for oxygen with heterotrophs and AOB. The nitrogen removal via nitrite in a SAGB reactor described in this study is applicable for high-ammonia-strength wastewater treatment, such as centrate or industrial wastes.