Fate and toxic effects of environmental stressors: environmental control.

The potential for toxicants to harm organisms in the environment is influenced by the physicochemistry of the substances and their environmental behaviors and transformation within ecosystems. This special issue is composed of 20 papers that report on studies which have investigated the fate and toxicity of various toxicants including engineered nanoparticles, pharmaceuticals and personal care products, antibiotics, pathogens, heavy metals, and agricultural nutrients. The environmental transformations of these substances and how these processes affect their toxicity are emphasized. This paper highlights the important findings and perspectives of the selected papers in this special edition, with an aim of providing insights into full-scale evaluation on the toxicity of various contaminants that exist in ecosystems. General suggestions are provided for the future directions of toxicological research.

An overview on the marine neurotoxin, saxitoxin: genetics, molecular targets, methods of detection and ecological functions.

Marine neurotoxins are natural products produced by phytoplankton and select species of invertebrates and fish. These compounds interact with voltage-gated sodium, potassium and calcium channels and modulate the flux of these ions into various cell types. This review provides a summary of marine neurotoxins, including their structures, molecular targets and pharmacologies. Saxitoxin and its derivatives, collectively referred to as paralytic shellfish toxins (PSTs), are unique among neurotoxins in that they are found in both marine and freshwater environments by organisms inhabiting two kingdoms of life. Prokaryotic cyanobacteria are responsible for PST production in freshwater systems, while eukaryotic dinoflagellates are the main producers in marine waters. Bioaccumulation by filter-feeding bivalves and fish and subsequent transfer through the food web results in the potentially fatal human illnesses, paralytic shellfish poisoning and saxitoxin pufferfish poisoning. These illnesses are a result of saxitoxin's ability to bind to the voltage-gated sodium channel, blocking the passage of nerve impulses and leading to death via respiratory paralysis. Recent advances in saxitoxin research are discussed, including the molecular biology of toxin synthesis, new protein targets, association with metal-binding motifs and methods of detection. The eco-evolutionary role(s) PSTs may serve for phytoplankton species that produce them are also discussed.

Genetically modified whole-cell bioreporters for environmental assessment.

Living whole-cell bioreporters serve as environmental biosentinels that survey their ecosystems for harmful pollutants and chemical toxicants, and in the process act as human and other higher animal proxies to pre-alert for unfavorable, damaging, or toxic conditions. Endowed with bioluminescent, fluorescent, or colorimetric signaling elements, bioreporters can provide a fast, easily measured link to chemical contaminant presence, bioavailability, and toxicity relative to a living system. Though well tested in the confines of the laboratory, real-world applications of bioreporters are limited. In this review, we will consider bioreporter technologies that have evolved from the laboratory towards true environmental applications, and discuss their merits as well as crucial advancements that still require adoption for more widespread utilization. Although the vast majority of environmental monitoring strategies rely upon bioreporters constructed from bacteria, we will also examine environmental biosensing through the use of less conventional eukaryotic-based bioreporters, whose chemical signaling capacity facilitates a more human-relevant link to toxicity and health-related consequences.

Horizontal transfer of PAH catabolism genes in Mycobacterium: evidence from comparative genomics and isolated pyrene-degrading bacteria.

Biodegradation of high molecular weight polycyclic aromatic hydrocarbons (PAHs), such as pyrene and benzo[a]pyrene, has only been observed in a few genera, namely fast-growing Mycobacterium and Rhodococcus. In M. vanbaalenii PYR-1, multiple aromatic ring hydroxylating dioxygenase (ARHDOs) genes including pyrene dioxygenases nidAB and nidA3B3 are localized in one genomic region. Here we examine the homologous genomic regions in four other PAH-degrading Mycobacterium (strains JLS, KMS, and MCS, and M. gilvum PYR-GCK), presenting evidence for past horizontal gene transfer events. Seven distinct types of ARHDO genes are present in all five genomes, and display conserved syntenic architecture with respect to gene order, orientation, and association with other genes. Duplications and putative integrase and transposase genes suggest past gene shuffling. To corroborate these observations, pyrene-degrading strains were isolated from two PAH-contaminated sediments: Chattanooga Creek (Tennessee) and Lake Erie (western basin). Some were related to fast-growing Mycobacterium spp. and carried both nidA and nidA3 genes. Other isolates belonged to Microbacteriaceae and Intrasporangiaceae presenting the first evidence of pyrene degradation in these families. These isolates had nidA (and some, nidA3) genes that were homologous to Mycobacterial ARHDO genes, suggesting that horizontal gene transfer events have occurred.

Inhibition of copper uptake in yeast reveals the copper transporter Ctr1p as a potential molecular target of saxitoxin.

Saxitoxin is a secondary metabolite produced by several species of dinoflagellates and cyanobacteria which targets voltage-gated sodium and potassium channels in higher vertebrates. However, its molecular target in planktonic aquatic community members that co-occur with the toxin producers remains unknown. Previous microarray analysis with yeast identified copper and iron-homeostasis genes as being differentially regulated in response to saxitoxin. This study sought to identify the molecular target in microbial cells by comparing the transcriptional profiles of key copper and iron homeostasis genes (CTR1, FRE1, FET3, CUP1, CRS5) in cells exposed to saxitoxin, excess copper, excess iron, an extracellular Cu(I) chelator, or an intracellular Cu(I) chelator. Protein expression and localization of Ctr1p (copper transporter), Fet3p (multicopper oxidase involved in high-affinity iron uptake), and Aft1p (iron regulator) were also compared among treatments. Combined transcript and protein profiles suggested saxitoxin inhibited copper uptake. This hypothesis was confirmed by intracellular Cu(I) imaging with a selective fluorescent probe for labile copper. On the basis of the combined molecular and physiological results, a model is presented in which the copper transporter Ctr1p serves as a molecular target of saxitoxin and these observations are couched in the context of the eco-evolutionary role this toxin may serve for species that produce it.

Implications of fecal bacteria input from latrine-polluted ponds for wells in sandy aquifers.

Ponds receiving latrine effluents may serve as sources of fecal contamination to shallow aquifers tapped by millions of tube-wells in Bangladesh. To test this hypothesis, transects of monitoring wells radiating away from four ponds were installed in a shallow sandy aquifer underlying a densely populated village and monitored for 14 months. Two of the ponds extended to medium sand. Another pond was sited within silty sand and the last in silt. The fecal indicator bacterium E. coli was rarely detected along the transects during the dry season and was only detected near the ponds extending to medium sand up to 7 m away during the monsoon. A log-linear decline in E. coli and Bacteroidales concentrations with distance along the transects in the early monsoon indicates that ponds excavated in medium sand were the likely source of contamination. Spatial removal rates ranged from 0.5 to 1.3 log(10)/m. After the ponds were artificially filled with groundwater to simulate the impact of a rain storm, E. coli levels increased near a pond recently excavated in medium sand, but no others. These observations show that adjacent sediment grain-size and how recently a pond was excavated influence the how much fecal contamination ponds receiving latrine effluents contribute to neighboring groundwater.

Pathogen detection using engineered bacteriophages.

Bacteriophages, or phages, are bacterial viruses that can infect a broad or narrow range of host organisms. Knowing the host range of a phage allows it to be exploited in targeting various pathogens. Applying phages for the identification of microorganisms related to food and waterborne pathogens and pathogens of clinical significance to humans and animals has a long history, and there has to some extent been a recent revival in these applications as phages have become more extensively integrated into novel detection, identification, and monitoring technologies. Biotechnological and genetic engineering strategies applied to phages are responsible for some of these new methods, but even natural unmodified phages are widely applicable when paired with appropriate innovative detector platforms. This review highlights the use of phages as pathogen detector interfaces to provide the reader with an up-to-date inventory of phage-based biodetection strategies.

Unsealed tubewells lead to increased fecal contamination of drinking water.

Bangladesh is underlain by shallow aquifers in which millions of drinking water wells are emplaced without annular seals. Fecal contamination has been widely detected in private tubewells. To evaluate the impact of well construction on microbial water quality 35 private tubewells (11 with intact cement platforms, 19 without) and 17 monitoring wells (11 with the annulus sealed with cement, six unsealed) were monitored for culturable Escherichia coli over 18 months. Additionally, two 'snapshot' sampling events were performed on a subset of wells during late-dry and early-wet seasons, wherein the fecal indicator bacteria (FIB) E. coli, Bacteroidales and the pathogenicity genes eltA (enterotoxigenic E. coli; ETEC), ipaH (Shigella) and 40/41 hexon (adenovirus) were detected using quantitative polymerase chain reaction (qPCR). No difference in E. coli detection frequency was found between tubewells with and without platforms. Unsealed private wells, however, contained culturable E. coli more frequently and higher concentrations of FIB than sealed monitoring wells (p < 0.05), suggestive of rapid downward flow along unsealed annuli. As a group the pathogens ETEC, Shigella and adenovirus were detected more frequently (10/22) during the wet season than the dry season (2/20). This suggests proper sealing of private tubewell annuli may lead to substantial improvements in microbial drinking water quality.

Detection of dichloromethane with a bioluminescent (lux) bacterial bioreporter.

The focus of this research effort was to develop an autonomous, inducible, lux-based bioluminescent bioreporter for the real-time detection of dichloromethane. Dichloromethane (DCM), also known as methylene chloride, is a volatile organic compound and one of the most commonly used halogenated solvents in the U.S., with applications ranging from grease and paint stripping to aerosol propellants and pharmaceutical tablet coatings. Predictably, it is released into the environment where it contaminates air and water resources. Due to its classification as a probable human carcinogen, hepatic toxin, and central nervous system effector, DCM must be carefully monitored and controlled. Methods for DCM detection usually rely on analytical techniques such as solid-phase microextraction (SPME) and capillary gas chromatography or photoacoustic environmental monitors, all of which require trained personnel and/or expensive equipment. To complement conventional monitoring practices, we have created a bioreporter for the self-directed detection of DCM by taking advantage of the evolutionary adaptation of bacteria to recognize and metabolize chemical agents. This bioreporter, Methylobacterium extorquens DCM( lux ), was engineered to contain a bioluminescent luxCDABE gene cassette derived from Photorhabdus luminescens fused downstream to the dcm dehalogenase operon, which causes the organism to generate visible light when exposed to DCM. We have demonstrated detection limits down to 1.0 ppm under vapor phase exposures and 0.1 ppm under liquid phase exposures with response times of 2.3 and 1.3 h, respectively, and with specificity towards DCM under relevant industrial environmental monitoring conditions.

Pseudomonas fluorescens HK44: lessons learned from a model whole-cell bioreporter with a broad application history.

Initially described in 1990, Pseudomonas fluorescens HK44 served as the first whole-cell bioreporter genetically endowed with a bioluminescent (luxCDABE) phenotype directly linked to a catabolic (naphthalene degradative) pathway. HK44 was the first genetically engineered microorganism to be released in the field to monitor bioremediation potential. Subsequent to that release, strain HK44 had been introduced into other solids (soils, sands), liquid (water, wastewater), and volatile environments. In these matrices, it has functioned as one of the best characterized chemically-responsive environmental bioreporters and as a model organism for understanding bacterial colonization and transport, cell immobilization strategies, and the kinetics of cellular bioluminescent emission. This review summarizes the characteristics of P. fluorescens HK44 and the extensive range of its applications with special focus on the monitoring of bioremediation processes and biosensing of environmental pollution.

Domestic plant food loss and waste in the United States: Environmental footprints and mitigation strategies.

The United States (U.S.) aims to reduce half of food loss and waste (FLW) by 2030. To achieve this goal, the public, academic, and political attentions on FLW have been increasing, and a series of actions have been implemented. However, the actions lack consideration on the categorical priority of FLW mitigation in relation to environmental footprints. In this article, we compare the FLW of three main plant food categories (i.e., grains, vegetables, and fruits) and their water and carbon footprints during 1970-2017. The vegetable FLW doubled during the period, reaching 3.39 × 1010 kg in 2017, which was 5- and 2-fold higher than the FLW of grains and fruits, respectively. The FLW of vegetables, grains, and fruits contributed 29%, 47%, and 24% to the total blue water wasted through FLW. The total carbon dioxide emissions generated by plant FLW were contributed by vegetables with 50%, grains with 31%, and fruits with 19%. Canonical correspondence analysis indicates that vegetable FLW had a higher positive correlation with urbanization, household incomes, gross domestic product, and high-income population than grain FLW, whereas fruit FLW was not influenced by these socioeconomic factors. Therefore, we suggest that the FLW mitigation should be prioritized on vegetables. Specific strategies include local food sourcing, shortening food miles, building food belts, and developing controlled-environment agriculture. Our data-based comparisons provide valuable insights into food policy improvement for achieving the 2030 reduction goal of the U.S., but the insights could be improved by considering the influences of foods imported from other nations.

Draft genome sequence of the polycyclic aromatic hydrocarbon-degrading, genetically engineered bioluminescent bioreporter Pseudomonas fluorescens HK44.

Pseudomonas fluorescens strain HK44 (DSM 6700) is a genetically engineered lux-based bioluminescent bioreporter. Here we report the draft genome sequence of strain HK44. Annotation of ∼6.1 Mb of sequence indicates that 30% of the traits are unique and distributed over five genomic islands, a prophage, and two plasmids.

Global gene expression profiling in larval zebrafish exposed to microcystin-LR and microcystis reveals endocrine disrupting effects of Cyanobacteria.

Microcystis blooms occur worldwide and threaten aquatic ecosystems and human health. Sublethal effects on early developmental stages of fish are largely unknown, and research has mainly focused on microcystin toxins (such as MC-LR) rather than Microcystis cells. We exposed (96 h) zebrafish larvae to purified MC-LR (0-1000 µg/L) or lyophilized Microcystis aeruginosa containing 4.5 µg/L MC-LR and evaluated changes in global gene expression (Affymetrix GeneChip zebrafish genome arrays). Significant changes in gene expression (≥ 1.7-fold change, p < 0.0001) were determined with Rosetta Resolver 7.0, and ontology analysis was conducted with the DAVID bioinformatics tool. The number of differentially expressed genes relative to control increased with MC-LR concentration and included genes related to known mechanisms of action for MC-LR in mammals and older life stages of fish, as well as genes unique to larval zebrafish. Up-regulation of vitellogenin genes (vtg) (19.2-fold to >100-fold on arrays; 619.3-fold confirmed by quantitative PCR) was observed in Microcystis-exposed larvae but not in larvae exposed to MC-LR. Up-regulation of vtg indicates exposure to estrogenic substance(s) and suggests that Microcystis may be a natural source of environmental estrogens. Concerns about effects of Microcystis blooms may extend beyond those associated with the microcystin toxin.

In vivo bioluminescent imaging (BLI): noninvasive visualization and interrogation of biological processes in living animals.

In vivo bioluminescent imaging (BLI) is increasingly being utilized as a method for modern biological research. This process, which involves the noninvasive interrogation of living animals using light emitted from luciferase-expressing bioreporter cells, has been applied to study a wide range of biomolecular functions such as gene function, drug discovery and development, cellular trafficking, protein-protein interactions, and especially tumorigenesis, cancer treatment, and disease progression. This article will review the various bioreporter/biosensor integrations of BLI and discuss how BLI is being applied towards a new visual understanding of biological processes within the living organism.

Transcription levels (amoA mRNA-based) and population dominance (amoA gene-based) of ammonia-oxidizing bacteria.

A population shift of ammonia-oxidizing bacteria (AOB) was described within a bench-scale activated sludge process treating an industrial wastewater in a previous report (Kuo et al. in Environ Eng Sci 23:507-520, 2006). In this investigation, transcriptional levels (amoA mRNA-based) of the three AOB groups (i.e., RI-27, B2-3, and Nitrosomonas nitrosa) identified in the treatment process were determined by quantitative real-time reverse transcription (RT-PCR) assays to circuitously evaluate AOB ammonia-oxidizing activity and to assess the presumed correlation between cellular activity and the dominant (greatest number) AOB population. Results demonstrated that the AOB group with higher amoA mRNA levels dominated the overall AOB population in the wastewater treatment process. Although AOB population dominance did not correlate well with transcripts at a normalized cellular level (amoA mRNA/DNA ratio), overall amoA mRNA levels did reflect the activity of distinct AOB groups under different N-loading conditions. Thus, an additional molecular parameter (amoA mRNA) was successfully utilized to assess timely shifts in AOB population structure that may impact nitrification treatment performance.

Bioenergy sustainability in China: potential and impacts.

The sustainability implications of bioenergy development strategies are large and complex. Unlike conventional agriculture, bioenergy production provides an opportunity to design systems for improving eco-environmental services. Different places have different goals and solutions for bioenergy development, but they all should adhere to the sustainability requirements of the environment, economy, and society. This article serves as a brief overview of China's bioenergy development and as an introduction to this special issue on the impacts of bioenergy development in China. The eleven articles in this special issue present a range of perspectives and scenario analyses on bioenergy production and its impacts as well as potential barriers to its development. Five general themes are covered: status and goals, biomass resources, energy plants, environmental impacts, and economic and social impacts. The potential for bioenergy production in China is huge, particularly in the central north and northwest. China plans to develop a bioenergy capacity of 30GW by 2020. However, realization of this goal will require breakthroughs in bioenergy landscape design, energy plant biotechnology, legislation, incentive policy, and conversion facilities. Our analyses suggest that (1) the linkage between bioenergy, environment, and economy are often circular rather than linear in nature; (2) sustainability is a core concept in bioenergy design and the ultimate goal of bioenergy development; and (3) each bioenergy development scheme must be region-specific and designed to solve local environmental and agricultural problems.

Bayesian estimation and sensitivity analysis of toluene and trichloroethylene biodegradation kinetic parameters.

Parameter estimation is needed for process management, design, and reactor scaling when values from the literature vary tremendously or are unavailable. A Bayesian approach, implemented via Markov chain Monte Carlo (MCMC) simulations using SAS software, was used to estimate the kinetic parameters of toluene and trichloroethylene (TCE) biodegradation by the microorganism Pseudomonas putida F1 in batch cultures. The prediction capabilities of Bayesian estimation were illustrated by comparing predicted and observed data and reported in goodness-of-fit statistics. The sensitivity analysis showed that the parameters obtained using this approach were consistent under the designated toluene and TCE concentration range. Moreover, the impact of TCE on toluene degradation kinetics was numerically exhibited, verifying the fact that TCE was able to stimulate toluene degradation; hence, TCE's presence increased the apparent maximum toluene-specific rate. Various kinetic models were explored at different degrees of complexity. At a low TCE concentration range (e.g., <2 mg L-1 ), a simplified Michaelis-Menten model (i.e., substrate half-saturation parameters approximated the inhibition parameters) was adequate to describe the reaction kinetics. However, at a higher TCE range (e.g., 5 mg L-1 ), a full-scale Michaelis-Menten model was needed to discriminate among the inhibition parameters in the model. The results demonstrated that a Bayesian estimation method is particularly useful for determining complex bioreaction kinetic parameters in the presence of a small volume of experimental data.

Use of gene probes to assess the impact and effectiveness of aerobic in situ bioremediation of TCE.

Gene probe hybridization was used to determine distribution and expression of co-metabolic genes at a contaminated site as it underwent in situ methanotrophic bioremediation of trichloroethylene (TCE). The bioremediation strategies tested included a series of air, air:methane, and air:methane:nutrient pulses of the test plot using horizontal injection wells. During the test period, the levels of TCE reduced drastically in almost all test samples. Sediment core samples (n=367) taken from 0 m (surface)-43 m depth were probed for gene coding for methanotrophic soluble methane monooxygenase (sMMO) and heterotrophic toluene dioxygenase (TOD), which are known to co-metabolize TCE. The same sediment samples were also probed for genes coding for methanol dehydrogenase (MDH) (catalyzing the oxidation of methanol to formaldehyde) to assess specifically changes in methylotrophic bacterial populations in the site. Gene hybridization results showed that the frequency of detection of sMMO genes were stimulated approximately 250% following 1% methane:air (v/v) injection. Subsequent injection of 4% methane:air (v/v) resulted in an 85% decline probably due to nutrient limitations, since addition of nutrients (gaseous nitrogen and phosphorus) thereafter caused an increase in the frequency of detection of sMMO genes. Detection of TOD genes declined during the process, and eventually they were non-detectable by the final treatment, suggesting that methanotrophs displaced the TOD gene containing heterotrophs. Active transcription of sMMO and TOD was evidenced by hybridization to mRNA. These analyses combined with results showing the concomitant decline in TCE concentrations, increases in chloride concentration and increases in methanotroph viable counts, provide multiple lines of evidence that TCE remediation was caused specifically by methanotrophs. Our results suggest that sMMO genes are responsible for most, if not all, of the observed biodegradation of TCE. This study demonstrates that the use of nucleic acid analytical methods provided a gene specific assessment of the effects of in situ treatment technologies.

Factors influencing the persistence of fecal Bacteroides in stream water.

Laboratory microcosm experiments were used to assess the effects of environmental parameters on the persistence of the Bacteroides 16S rRNA genes derived from equine fecal samples in stream water to investigate the utility of Bacteroides spp. as fecal indicator organisms. Quantitative real-time polymerase chain reaction (qPCR) was used to measure gene concentrations over time with treatments designed to compare filtered vs. unfiltered stream water, fecal aggregate size, initial fecal concentrations, and water temperatures. Comparison of Bacteroides16S rRNA genes/mL in microcosms constructed with unfiltered stream water and filtered stream water indicated that stream water filtration to remove indigenous microorganisms followed by temperature had the largest effects on gene persistence. First-order exponential decay functions were fitted to the data from each microcosm constructed using unfiltered stream water, and the decay constants (k) ranged from 0.0071 h(-1) in the microcosms incubated at 5 degrees C to 0.0336 h(-1) in a set of microcosms incubated at 25 degrees C. Analysis of k calculated from the 10 experimental treatments indicated that k is more highly correlated to temperature than initial Bacteroides 16S rRNA gene starting concentrations. The equation resulting from graphing k (as the dependent variable) vs. temperature (as the independent variable) best fit a peak, Gaussian, 3 parameter function with a maximum decay at 30 degrees C, a r(2) of 0.83 and all parameters were significant (P < 0.0015). Thus this data suggest that factors that reduce biological activity, such as physical removal of stream microorganisms by filtration and low temperature, result in slower Bacteroides 16S rRNA gene decay.

Reporter proteins in whole-cell optical bioreporter detection systems, biosensor integrations, and biosensing applications.

Whole-cell, genetically modified bioreporters are designed to emit detectable signals in response to a target analyte or related group of analytes. When integrated with a transducer capable of measuring those signals, a biosensor results that acts as a self-contained analytical system useful in basic and applied environmental, medical, pharmacological, and agricultural sciences. Historically, these devices have focused on signaling proteins such as green fluorescent protein, aequorin, firefly luciferase, and/or bacterial luciferase. The biochemistry and genetic development of these sensor systems as well as the advantages, challenges, and common applications of each one will be discussed.