Nitrous oxide (N2O) emissions at wastewater treatment plants result from a complex interplay between environmental, operational, and microbial factors. Effective mitigation requires a comprehensive understanding of these interactions. We investigated process variables linked to N2O emissions and microbial community dynamics over 14 months in independent, parallel activated sludge lines at a full-scale treatment plant with ammonia-based aeration control and temperature-based solids retention time (SRT), focusing on a six-month period when N2O increased from 1% to 3% of nitrogen removed. During the seasonal increase, N2O correlated positively with wastewater conductivity, airflow in the swing zones, and negatively with temperature. The same variables were most strongly associated with microbial community compositions. Denitrifiers and aerobic heterotrophs correlated positively and ammonia oxidizers negatively with N2O emissions during the seasonal increase. Nitrifier abundances decreased with temperature, yet nitrification efficiency remained high and nitrite did not accumulate, while N2O accumulated during aeration. Ammonia oxidizers, linked to changes in temperature and salinity, were a likely source of increased N2O emissions. Species increasing with N2O included potentially incomplete denitrifiers, suggesting a contribution from incomplete denitrification. A longer SRT, tested in one line, supported efficient nitrification and denitrification with low net N2O production. Overall, plant-specific microbial and process data can provide additional information for the selection of mitigation strategies by indicating whether ammonia oxidizers or denitrifiers dominate net N2O production.

Biotransformation is a significant determinant of the fate of organic micropollutants (OMPs) in natural and engineered environments. Here we propose a genome-resolved metatranscriptomics approach for the identification of candidate OMP-transforming microorganisms based on positive relations between biotransformation rate constants and the activity of metagenome-assembled genomes (MAGs). To demonstrate the approach, we used five nitrifier-rich batch cultures, first validating with ammonia (a macropollutant) before applying it to atenolol (an OMP). As expected, the biotransformation rate constant of ammonia was correlated with the activity of an ammonia-oxidizing bacterium, namely Nitrosomonas europaea; it was not correlated with the activity of other bacteria, including several ammonia oxidizers. Additionally, the biotransformation rate constant of ammonia was correlated with the transcript relative abundance of the ammonia monooxygenase (AMO) expressed by N. europaea but not with the transcript abundance of AMO at the community level. The biotransformation rate constant of atenolol was correlated with the activity of four MAGs representing three heterotrophic genera: Terrimonas, Flavobacterium, and Zeimonas. It was not correlated with the total transcript relative abundance of any member of a comprehensive set of amidohydrolases, which are predicted to transform this drug. By contrast, it was correlated with the expression of the amidohydrolase asparagine synthase (AsnB) identified in the Terrimonas and Flavobacterium MAGs. In summary, we present a novel association-based method for investigating biotransformation processes robust to variability in enzyme reaction kinetics with implications for OMP control.

Wastewater produced during oil and gas extraction processes is one of the largest residual water streams. Also known as produced water (PW), it has a complex composition and is typically toxic to aquatic ecosystems. This study explores the use of membrane aerated biofilm reactors (MABR) for biological removal of dissolved organic compounds and associated toxicity. Three different oxygen partial pressures (0.2, 0.6 and 1 bar), the last two mimicking subsea pressures at 20 and 40 m below the sea level, respectively, were tested at three different hydraulic retention times (HRT of 24, 12 and 6 h). Reactors were fed both with onshore and offshore PW. Removal rates for organic carbon increased at decreasing HRT. Highest oxygen partial pressure led to best performance at 6 h HRT (30.1 +/- 3.0 g-COD m(-2) d(-1)) when treating onshore PW and at all HRT when treating offshore PW, with highest COD removal rate still at 6 h HRT (9.4 +/- 1 g-COD m(-2) d(-1)). Removal efficiencies ranged from 78 % when treating onshore PW at 24 h HRT to 36-49 % when treating offshore PW at 6 h HRT. All treatments led to a reduction in toxicity (>92 % reduction for offshore PW), with 0.2 bar oxygen partial pressure showing worst performance, while 0.6 and 1 bar did not show significant differences. The biofilms were dominated by hydrocarbonoclastic, responsible of hydrocarbons biodegradation, and sulfur cycling bacteria. Overall, MABR were effective treating PW and operating at higher oxygen partial pressures yield to better performance, especially at high volumetric loading rates.

Pharmaceuticals and other organic micropollutants (OMPs) present in wastewater effluents are of growing concern, as they threaten environmental and human health. Conventional biological treatments lead to limited removal of OMPs. Methanotrophic bacteria can degrade a variety of OMPs. By employing a novel bubble-free hybrid membrane biofilm bioreactor (hMBfR), we grew methanotrophic bacteria at three CH4 loading rates. Biomass productivity and CH4 loading showed a linear correlation, with a maximum productivity of 372 mgVSS center dot L-1 center dot d 1, with corresponding biomass concentration of 1117.6 +/- 56.4 mg-VSS center dot L-1. Furthermore, the biodegradation of sulfamethoxazole and 1H-benzotriazole positively correlated with CH4 oxidation rates, with highest biodegradation kinetic constants of 3.58 L center dot g(-1)center dot d(-1) and 5.42 L center dot g(-1)center dot d(-1), respectively. Additionally, the hMBfR recovered nutrients as microbial proteins, with an average content 39% DW. The biofilm community was dominated by Methylomonas, while the bulk was dominated by aerobic heterotrophic bacteria. The hMBfR removed OMPs, allowing for safer water reuse while valorising CH4 and nutrients.

Membrane-aerated biofilm reactors (MABRs) are an emerging technology for nutrient removal; however, a trade-off remains between their removal rate and oxygen transfer efficiency. This study compares nitrifying flow-through MABRs operated under continuous and intermittent aeration modes at mainstream wastewater ammonia levels. The intermittently-aerated MABRs maintained maximal nitrification rates, including under conditions allowing the oxygen partial pressure on the gas side of the membrane to considerably drop during the no-aeration period. Nitrous oxide emissions of all reactors were comparable and amounted to approximately 20 % of the converted ammonia. Intermittent aeration increased the transformation rate constant of atenolol, yet did not affect the removal of sulfamethoxazole. Seven additional trace organic chemicals were not biodegraded by any of the reactors. The ammonia-oxidizing bacteria in the intermittently-aerated MABRs were dominated by Nitrosospira, previously shown to be abundant at low oxygen concentrations and provide reactor stability under changing conditions. Our findings indicate that intermittently-aerated flow-through MABRs can achieve high nitrification rates and oxygen transfer efficiencies, highlighting the possible implications of air supply discontinuity on nitrous oxide emissions and trace organic chemical biotransformation.

A possible solution for the standoff detection of buried landmines is based on the use of microbial bioreporters, genetically engineered to emit a remotely detectable optical signal in response to trace amounts of explosives' signature chemicals, mostly 2,4-dinitrotoluene (DNT). Previously developed DNT sensor strains were based on the fusion of a DNT-inducible gene promoter to a reporting element, either a fluorescent protein gene or a bacterial bioluminescence gene cassette. In the present study, a different approach was used: the DNT-inducible promoter activates, in Escherichia coli, the quorum-sensing luxI and luxR genes of Aliivibrio fischeri. N-Acyl homoserine lactone (AHL), synthesized by LuxI, combines with LuxR and activates the bioluminescence reporter genes. The resulting bioreporter displayed a dose-dependent luminescent signal in the presence of DNT. Performance of the sensor strain was further enhanced by manipulation of the sensing element (combining the E. coli DNT-inducible azoR and yqjF gene promoters), by replacing the luminescence gene cassette of Photorhabdus luminescens luxCDABE with A. fischeri luxCDABEG, and by introducing two mutations, eutE and ygdD, into the host strain. DNT detection sensitivity of the final bioreporter was over 340-fold higher than the original construct.

Detection of buried landmines is a dangerous and complicated task that consumes large financial resources and poses significant risks to the personnel involved. A potential alternative to conventional detection methodologies is the use of microbial bioreporters, capable of emitting an optical signal upon exposure to explosives, thus revealing to a remote detector the location of buried explosive devices. We have previously reported the design, construction, and optimization of an Escherichia coli -based bioreporter for the detection of 2,4,6-trinitrotoluene (TNT) and its accompanying impurity 2,4-dinitrotoluene (DNT). Here we describe the further enhancement of this bioreporter by the directed evolution of YhaJ, the transcriptional activator of the yqjF gene promoter, the sensing element of the bioreporter’s molecular circuit. This process resulted in a 37-fold reduction of the detection threshold, as well as significant enhancements to signal intensity and response time, rendering this sensor strain more suitable for detecting the minute concentrations of DNT in the soil above buried landmines. The capability of this enhanced bioreporter to detect DNT buried in sand is demonstrated.

Landmines and explosive remnants of war pose a global humanitarian problem and cause numerous casualties long after the conflict has ended. The current approaches for locating landmines, such as metal detection, which require one’s physical presence at the minefield, involve high risk to personnel; these methods are also costly, time-consuming, and have a high rate of false-positive results. There is no currently viable technology for the remote detection of buried explosive devices. A possible solution to this may be the use of genetically engineered microorganisms, molecularly “tailored” to emit an optical signal in the presence of trace explosives escaping for the landmine and accumulating in the soil above it. This optical signal, imaged from a remote location, can then be used to generate a physical map of the mine’s location. A few years ago, we have described the remote detection of buried landmines using alginate-encapsulated fluorescent microbial (Escherichia coli) bioreporters spread over the tested minefield. Since then, we have modified the system to one based on bioluminescent (rather than fluorescent) bacteria and have employed several synthetic biology approaches to significantly enhance their major performance parameters: higher signal intensity, faster response time, and lower detection threshold of the target explosives. These molecular approaches and their effect on sensor performance will be described.

Landmines and other explosive remnants of war pose a global humanitarian problem that claims numerous casualties long after the conflict has ended. As there are no acceptable methodologies for the remote discovery of such devices, current detection practices still require the risky presence of personnel in the minefield. We have recently described bacterial sensor strains capable of reporting the existence of 2,4-dinitrotoluene (DNT) vapors in the soil above 2,4,6-trinitrotoluene (TNT)-based landmines, by generating a bioluminescent or a fluorescent signal. This may allow the identification of landmine location by remote imaging of an area over which the bacteria have been spread. In the study reported herein, we have improved the DNT-detection capabilities of these sensor strains by combining two DNT-responsive Escherichia coli gene promoters, yqjF and azoR, and subjecting them to three cycles of random mutagenesis by error-prone PCR, combined with segmentation and rearrangement (“DNA shuffling”). The activity of selected modified promoters was evaluated with the Aliivibrio fischeri and Photobacterium leiognathi luxCDABEG gene cassettes as the bioluminescent reporters, exhibiting a ten-fold background reduction that has led to a three-fold decrease in detection threshold. Signal intensity was further enhanced by modifying the ribosomal binding site of the yqjF gene promoter. The superior DNT detection capabilities on a solid matrix by the improved sensor strain were demonstrated. • Performance of microbial sensor strains for buried explosives was molecularly enhanced. • Manipulations included random mutagenesis, “DNA shuffling,” and RBS reprogramming. • The re-engineered constructs exhibited superior detection of trace explosives.

We describe a biosensing module in which live bacteria, genetically "tailored" to respond to the presence of a specific target material, constitute the core sensing element, reporting their response by bioluminescence. The module is constructed of two channels: an 'induced' channel that measures the bioluminescent light emitted by bacteria exposed to the inspected area, and a 'reference' channel that measures in parallel the bioluminescent light emitted spontaneously by bacteria of the same batch. This enables to overcome signal variations generated by different batches of bacteria, and due to varying environmental operating conditions. A special low-noise optoelectronic circuit was constructed to detect the bioluminescence emitted by the bacteria in both channels. The bacteria are encapsulated in polymer beads that also contain nutrients and water, enabling long-term maintenance-free operation. The beads are packaged in special cassettes at the bottom of the module, so that the induced channel cassette is in direct contact with the ground underneath the module, whereas the reference channel cassette is isolated from the ground. The module contains, in addition, a digital signal processing unit, and a wireless communication unit. The module is designed to operate outdoors as an autonomous network element designed for large scale in-situ deployment. The module described herein was developed for the detection of buried landmines, by sensing the presence of 2,4-dinitrotoluene (DNT) vapors released by the mine, accumulating in the ground above it. Detection of DNT in the sub-ppm range is demonstrated.

The need for simple and rapid means for evaluating the potential toxic effects of environmental samples has prompted the development of reporter gene assays, based on tester cells (bioreporters) genetically engineered to report on sample toxicity by producing a readily quantifiable signal. Bacteria are especially suitable to serve as bioreporters owing to their fast responses, low cost, convenient preservation, ease of handling, and amenability to genetic manipulations. Various bacterial bioreporters have been introduced for general toxicity and genotoxicity assessment, and the monitoring of endocrine disrupting and dioxin-like compounds has been mostly covered by similarly engineered eukaryotic cells. Some reporter gene assays have been validated, standardized, and accredited, and many others are under constant development. Efforts are aimed at broadening detection spectra, lowering detection thresholds, and combining toxicity identification capabilities with characterization of the toxic effects. Taking advantage of bacterial robustness, attempts are also being made to incorporate bacterial bioreporters into field instrumentation for online continuous monitoring or on-site spot checks. However, key hurdles concerning test validation, cell preservation, and regulatory issues related to the use of genetically modified organisms still remain to be overcome.

We assessed the applicability of multi-strain bacterial bioreporter bioassays to drug screening. To this end, we investigated the reactions of a panel of 15 luminescent recombinant Escherichia coli bacterial bioreporters to a library of 420 pharmaceuticals. The panel included bacterial bioreporters associated with oxidative stress, DNA damage, heat shock, and efflux of excess metals. Eighty nine drugs elicited a response from at least one of the panel members and formed distinctive clusters, some of which contained closely related drugs. In addition, we tested a group of selected nine drugs against a collection of about 2000 different fluorescent transcriptional reporters that covers the great majority of gene promoters in E. coli. The sets of induced genes were in accord with the in vitro toxicity of the tested drugs, as reflected by the response patterns of the 15-member panel, and provided more insights into their toxicity mechanisms. Facilitated by microplates and robotic systems, all assays were conducted in high-throughput. Our results thus suggest that multi-strain assemblages of bacterial bioreporters have the potential for playing a significant role in drug development alongside current in vitro toxicity tests.

A methodology for oxygen profile measurements in the unsaturated zone is developed based on direct push drilling using sampling liners equipped with homemade silicone septa. The oxygen measurement is carried out by puncturing the septum with a commercial retractable optode needle fitted with a fluorescent tip. Metrological characteristics and method validation were carried out under laboratory conditions using different levels of oxygen and various water contents. The relative standard deviations under dry and water saturated soil conditions were less than 0.3% and 5% for 0.5 mg L(-1) of oxygen and less than 2% and 3% for 9 mg L(-1). Field demonstrations in a calcareous-sandstone soil aquifer treatment system with a layered clayey, marl and sandstone lithology of widely different water contents provided down to 30 m deep profiles of the dissolved oxygen level with less than 1.5 m spatial resolution. A single sensor was used for over 50 field measurements, though recalibration was required after approximately 30 measurements due to the deterioration of the fluorescent tip.

An approach for the rapid detection and classification of a broad spectrum of water pollutants, based on a genome-wide reporter bacterial live cell array, is proposed and demonstrated. An array of ca. 2000 Escherichia coli fluorescent transcriptional reporters was exposed to 25 toxic compounds as well as to unpolluted water, and its responses were recorded after 3 h. The 25 toxic compounds represented 5 pollutant classes: genotoxicants, metals, detergents, alcohols, and monoaromatic hydrocarbons. Identifying unique gene expression patterns, a nearest neighbour-based model detected pollutant presence and predicted class attribution with an estimated accuracy of 87%. Sensitivity and positive predictive values varied among classes, being higher for pollutant classes that were defined by mode of action than for those defined by structure only. Sensitivity for unpolluted water was 0.90 and the positive predictive value was 0.79. All pollutant classes induced the transcription of a statistically significant proportion of membrane associated genes; in addition, the sets of genes responsive to genotoxicants, detergents and alcohols were enriched with genes involved in DNA repair, iron utilization and the translation machinery, respectively. Following further development, a methodology of the type described herein may be suitable for integration in water monitoring schemes in conjunction with existing analytical and biological detection techniques.

Dispersal limitation in phyllosphere communities was measured on the leaf surfaces of salt-excreting Tamarix trees, which offer unique, discrete habitats for microbial assemblages. We employed 16S rRNA gene pyrosequencing to measure bacterial community dissimilarity on leaves of spatially dispersed Tamarix specimens in sites with uniform climatic conditions across the Sonoran Desert in the Southwestern United States. Our analyses revealed diverse bacterial communities with four dominant phyla that exhibited differential effects of environmental and geographic variables. Geographical distance was the most important parameter that affected community composition, particularly that of betaproteobacteria, which displayed a statistically significant, distance-decay relationship.

The ever-growing use of pharmaceutical compounds, including antibacterial substances, poses a substantial pollution load on the environment. Such compounds can compromise water quality, contaminate soils, livestock and crops, enhance resistance of microorganisms to antibiotic substances, and hamper human health. We report the construction of a novel panel of genetically engineered Escherichia coli reporter strains for the detection and classification of antibiotic substances. Each of these strains harbours a plasmid that carries a fusion of a selected gene promoter to bioluminescence (luxCDABE) reporter genes and an alternative tryptophan auxotrophy-based non-antibiotic selection system. The bioreporter panel was tested for sensitivity and responsiveness to diverse antibiotic substances by monitoring bioluminescence as a function of time and of antibiotic concentrations. All of the tested antibiotics were detected by the panel, which displayed different response patterns for each substance. These unique responses were analysed by several algorithms that enabled clustering the compounds according to their functional properties, and allowed the classification of unknown antibiotic substances with a high degree of accuracy and confidence.

Motivated by the advantages endowed by high-throughput analysis, researchers have succeeded in incorporating multiple reporter cells into a single platform; the technology now allows the simultaneous scrutiny of a large collection of sensor strains. We review current aspects in cell array technology with emphasis on microbial sensor arrays. We consider various techniques for patterning live cells on solid surfaces, describe different array-based applications and devices, and highlight recent efforts for live cell storage. We review mathematical approaches for deciphering the data emanating from bioreporter collections, and discuss the future of single cell arrays. Innovative technologies for cell patterning, preservation and interpretation are continuously being developed; when they all mature, cell arrays may become an efficient analytical tool, in a scope resembling that of DNA microarray biochips.

Chip-integrated luminescent recombinant reporter bacteria were combined with fluidics and light detection systems to form a real-time water biomonitor. The biomonitor was exposed to a continuous water flow for up to ten days, in the course of which it was challenged with spikes of both model toxic compounds and toxic environmental samples. All simulated contamination events were reported within 0.5–2.5 h. Furthermore, the response pattern of the reporter bacteria was indicative of the nature of the contaminating chemicals. Efforts were aimed at improving signal quality and at the development of an alarm management software. Following further research, a device of the proposed design could be implemented in monitoring networks as an early warning system against water pollution by toxic chemicals.

An international round‐robin study on the Ames fluctuation test [ISO 11350, 2012], a microplate version of the classic plate‐incorporation method for the detection of mutagenicity in water, wastewater and chemicals was performed by 18 laboratories from seven countries. Such a round‐robin study is a precondition for both the finalization of the ISO standardization process and a possible regulatory implementation in water legislation. The laboratories tested four water samples (spiked/nonspiked) and two chemical mixtures with and without supplementation of a S9‐mix. Validity criteria (acceptable spontaneous and positive control‐induced mutation counts) were fulfilled by 92–100%, depending on the test conditions. A two‐step method for statistical evaluation of the test results is proposed and assessed in terms of specificity and sensitivity. The data were first subjected to powerful analysis of variance (ANOVA) after an arcsine‐square‐root transformation to detect significant differences between the test samples and the negative control (NC). A threshold (TH) value based on a pooled NC was then calculated to exclude false positive test results. Statistically, positive effects observed by the William's test were considered negative, if the mean of all replicates of a sample did not exceed the calculated TH. By making use of this approach, the overall test sensitivity was 100%, and the test specificity ranged from 80 to 100%. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.