Lisa Alvarez-Cohen

N-Nitrosodimethylamine (NDMA) as a Drinking Water Contaminant: A Review

N-Nitrosodimethylamine (NDMA) is a member of a family of extremely potent carcinogens, the N-nitrosamines. Until recently, concerns about NDMA mainly focused on the presence of NDMA in food, consumer products, and polluted air. However, current concern focuses on NDMA as a drinking water contaminant resulting from reactions occurring during chlorination or via direct industrial contamination. Because of the relatively high concentrations of NDMA formed during wastewater chlorination, the intentional and unintentional reuse of municipal wastewater is a particularly important area of concern. Although ultraviolet (UV) treatment can effectively remove NDMA, there is considerable interest in the development of less expensive alternative treatment technologies. These alternative technologies include approaches for removing organic nitrogen-containing NDMA precursors prior to chlorination and the use of sunlight photolysis, and in situ bioremediation to remove NDMA and its precursors.

Kinetics of Aerobic Cometabolism of Chlorinated Solvents

The objectives of this paper are to review the wide range of kinetic models that have been introduced to describe the cometabolic oxidation of chlorinated solvents, to compare modeling approaches and associated experimental data, and to discuss knowledge gaps in the general topic of cometabolism kinetics. To begin, a brief description of the mechanism of oxygenase enzyme metabolism and its qualitative effects on cometabolic degradation kinetics is given. Next, a variety of kinetic expressions that have been used to describe cometabolism, ranging from adaptations of simple metabolic relationships to the development of complex equations that account for intracellular concentrations of key reaction species, are presented. A large number of kinetic coefficients published for a variety of oxygenase populations degrading a broad range of chlorinated solvents are categorized and compared. The discussion section of the paper contains an exploration of knowledge gaps that exist in our understanding of the kinetics of aerobic chlorinated solvent cometabolism. Specific topics covered include: • the use of half saturation constants (Ksc and Ksg) as estimates for inhibition constants (Kisc and Kisg) in saturation modeling expressions, • the specific nature of chlorinated solvent induced product toxicity and the capability for cells to recover from toxic effects, and • methods for incorporating reducing energy limitations into cometabolism models. Finally, the applicability of the broad range of kinetic modeling approaches to scale-up and field applications for in situ bioremediation of chlorinated solvents is discussed.

Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill

The Deepwater Horizon (DWH) oil spill in the spring of 2010 resulted in an input of ∼4.1 million barrels of oil to the Gulf of Mexico; >22% of this oil is unaccounted for, with unknown environmental consequences. Here we investigated the impact of oil deposition on microbial communities in surface sediments collected at 64 sites by targeted sequencing of 16S rRNA genes, shotgun metagenomic sequencing of 14 of these samples and mineralization experiments using 14C-labeled model substrates. The 16S rRNA gene data indicated that the most heavily oil-impacted sediments were enriched in an uncultured Gammaproteobacterium and a Colwellia species, both of which were highly similar to sequences in the DWH deep-sea hydrocarbon plume. The primary drivers in structuring the microbial community were nitrogen and hydrocarbons. Annotation of unassembled metagenomic data revealed the most abundant hydrocarbon degradation pathway encoded genes involved in degrading aliphatic and simple aromatics via butane monooxygenase. The activity of key hydrocarbon degradation pathways by sediment microbes was confirmed by determining the mineralization of 14C-labeled model substrates in the following order: propylene glycol, dodecane, toluene and phenanthrene. Further, analysis of metagenomic sequence data revealed an increase in abundance of genes involved in denitrification pathways in samples that exceeded the Environmental Protection Agency (EPA)’s benchmarks for polycyclic aromatic hydrocarbons (PAHs) compared with those that did not. Importantly, these data demonstrate that the indigenous sediment microbiota contributed an important ecosystem service for remediation of oil in the Gulf. However, PAHs were more recalcitrant to degradation, and their persistence could have deleterious impacts on the sediment ecosystem.

Influence of Vitamin B12 and Cocultures on the Growth of Dehalococcoides Isolates in Defined Medium

ABSTRACT Bacteria belonging to the genus Dehalococcoides play a key role in the complete detoxification of chloroethenes as these organisms are the only microbes known to be capable of dechlorination beyond dichloroethenes to vinyl chloride (VC) and ethene. However, Dehalococcoides strains usually grow slowly with a doubling time of 1 to 2 days and have complex nutritional requirements. Here we describe the growth of Dehalococcoides ethenogenes 195 in a defined mineral salts medium, improved growth of strain 195 when the medium was amended with high concentrations of vitamin B12, and a strategy for maintaining Dehalococcoides strains on lactate by growing them in consortia. Although strain 195 could grow in defined medium spiked with ∼0.5 mM trichloroethene (TCE) and 0.001 mg/liter vitamin B12, the TCE dechlorination and cellular growth rates doubled when the vitamin B12 concentration was increased 25-fold to 0.025 mg/liter. In addition, the final ratios of ethene to VC increased when the higher vitamin concentration was used, which reflected the key role that cobalamin plays in dechlorination reactions. No further improvement in dechlorination or growth was observed when the vitamin B12 concentration was increased to more than 0.025 mg/liter. In defined consortia containing strain 195 along with Desulfovibrio desulfuricans and/or Acetobacterium woodii and containing lactate as the electron donor, tetrachloroethene (∼0.4 mM) was completely dechlorinated to VC and ethene and there was concomitant growth of Dehalococcoides cells. In the cultures that also contained D. desulfuricans and/or A. woodii, strain 195 cells grew to densities that were 1.5 times greater than the densities obtained when the isolate was grown alone. The ratio of ethene to VC was highest in the presence of A. woodii, an organism that generates cobalamin de novo during metabolism. These findings demonstrate that the growth of D. ethenogenes strain 195 in defined medium can be optimized by providing high concentrations of vitamin B12 and that this strain can be grown to higher densities in cocultures with fermenters that convert lactate to generate the required hydrogen and acetate and that may enhance the availability of vitamin B12.

Bacterial Community Structure in Geographically Distributed Biological Wastewater Treatment Reactors

Current knowledge of the microbial communities within biological wastewater treatment reactors is incomplete due to limitations of traditional culture-based techniques and despite the emergence of recently applied molecular techniques. Here we demonstrate the application of high-density microarrays targeting universal 16S rRNA genes to evaluate microbial community composition in five biological wastewater treatment reactors in China and the United States. Results suggest a surprisingly consistent composition of microbial community structure among all five reactors. All investigated communities contained a core of bacterial phyla (53-82% of 2119 taxa identified) with almost identical compositions (as determined by colinearity analysis). These core species were distributed widely in terms of abundance but their proportions were virtually the same in all samples. Proteobacteria was the largest phylum and Firmicutes, Actinobacteria, Bacteroidetes were the subdominant phyla. The diversity among the samples can be attributed solely to a group of operational taxonomic units (OTUs) that were detected only in specific samples. Typically, these organisms ranked somewhat lower in terms of abundance but a few were present is much higher proportions.

Temperature effects and substrate interactions during the aerobic biotransformation of BTEX mixtures by toluene-enriched consortia and Rhodococcus rhodochrous

A microbial consortium derived from a gasoline-contaminated aquifer was enriched on toluene (T) in a chemostat at 20 degrees C and was found to degrade benzene (B), ethylbenzene (E), and xylenes (X). Studies conducted to determine the optimal temperature for microbial activity revealed that cell growth and toluene degradation were maximized at 35 degrees C. A consortium enriched at 35 degrees C exhibited increased degradation rates of benzene, toluene, ethylbenzene, and xylenes in single-substrate experiments; in BTEX mixtures, enhanced benzene, toluene, and xylene degradation rates were observed, but ethylbenzene degradation rates decreased. Substrate degradation patterns over a range of BTEX concentrations (0 to 80 mg/L) for individual aromatics were found to differ significantly from patterns for aromatics in mixtures. Individually, toluene was degraded fastest, followed by benzene, ethylbenzene, and the xylenes. In BTEX mixtures, degradation followed the order of ethylbenzene, toluene, and benzene, with the xylenes degraded last. A pure culture isolated from the 35 degrees C-enriched consortium was identified as Rhodococcus rhodochrous. This culture was shown to degrade each of the BTEX compounds, individually and in mixtures, following the same degradation patterns as the mixed cultures. Additionally, R. rhodochrous was shown to utilize benzene, toluene, and ethylbenzene as primary carbon and energy sources. Studies conducted with the 35 degrees C-enriched consortium and R. rhodochrous to evaluate potential substrate interactions caused by the concurrent presence of multiple BTEX compounds revealed a range of substrate interaction patterns including no interaction, stimulation, competitive inhibition, noncompetitive inhibition, and cometabolism. In the case of the consortium, benzene and toluene degradation rates were slightly enhanced by the presence of o-xylene, whereas the presence of toluene, benzene, or ethylbenzene had a negative effect on xylene degradation rates. Ethylbenzene was shown to be the most potent inhibitor of BTEX degradation by both the mixed and pure cultures. Attempted quantification of these inhibition effects in the case of the consortium suggested a mixture of competitive and noncompetitive inhibition kinetics. Benzene, toluene, and the xylenes had a negligible effect on the biodegradation of ethylbenzene by both cultures. Cometabolism of o-, m-, and p-xylene was shown to be a positive substrate interaction.

Aerobic MTBE biodegradation: an examination of past studies, current challenges and future research directions.

With the current practice of amending gasoline with up to 15% by volume MTBE, the contamination of groundwater by MTBE has become widespread. As a result, the bioremediation of MTBE-impacted aquifers has become an active area of research. A review of the current literature on the aerobic biodegradation of MTBE reveals that a number of cultures from diverse environments can either partially degrade or completely mineralize MTBE. MTBE is either utilized as a sole carbon and energy source or is degraded cometabolically by cultures grown on alkanes. Reported degradation rates range from 0.3 to 50 mg MTBE/g cells/h while growth rates (0.01–0.05 g MTBE/g cells/d) and cellular yields (0.1–0.2 g cells/g MTBE) are generally low. Studies on the mechanisms of MTBE degradation indicate that a monooxygenase enzyme cleaves the ether bond yielding tert-butyl alcohol (TBA) and formaldehyde as the dominant detectable intermediates. TBA is further degraded to 2-methyl-2-hydroxy-1-propanol, 2-hydroxyisobutyric acid, 2-propanol, acetone, hydroxyacteone and eventually, carbon dioxide. The majority of these intermediates are also common to mammalian MTBE metabolism. Laboratory studies on the degradation of MTBE in the presence of gasoline aromatics reveal that while degradation rates of other gasoline components are generally not inhibited by MTBE, MTBE degradation could be inhibited in the presence of more easily biodegradable compounds. Controlled field studies are clearly needed to elucidate MTBE degradation potential in co-contaminant plumes. Based on the reviewed studies, it is likely that a bioremediation strategy involving direct metabolism, cometabolism, bioaugmentation, or some combination thereof, could be applied as a feasible and cost-effective treatment method for MTBE contamination.

Model for the cometabolic biodegradation of chlorinated organics.

A mechanistic model is developed to describe the kinetics of the cometabolic degradation of chlorinated organics by oxygenase-expressing cultures. The oxidization of chlorinated organics has been shown to cause decreased cell activity due to product toxicity and/or the depletion of reducing energy. In addition, competition between growth substrate and cometabolic substrate for oxygenase enzymes may significantly affect cometabolic degradation rates. Typical saturation kinetic expressions for substrate degradation (Michaelis-Menten equation) and cell growth (Monod equation) cannot adequately describe the behavior of these interacting effects. In this study, a modification of Michaelis-Menten/Monod kinetics is proposed that incorporates the effects of product toxicity, reducing energy limitation, and competitive inhibition together with cell growth and decay. This model is able to predict the kinetics of chlorinated organic degradation by oxygenase-expressing cultures over a range of substrate conditions (resting cells, cells with reducing energy substrate, cells with growth substrate). The effects of endogenous and external reducing energy sources on observed cometabolic degradation rates and transformation capacities (k c ob and T c ob ) are discussed and verified by experimental results.

Quantifying Genes and Transcripts to Assess the In Situ Physiology of Dehalococcoides spp. in a Trichloroethene-Contaminated Groundwater Site

ABSTRACT Quantitative PCR (qPCR) was coupled with reverse transcription (RT) to analyze both gene copy numbers and transcripts of the 16S rRNA gene and three reductive dehalogenase (RDase) genes (tceA, vcrA, and bvcA) as biomarkers of “Dehalococcoides” spp. in the groundwater of a trichloroethene-dense nonaqueous-phase liquid site at Fort Lewis, WA, that was sequentially subjected to biostimulation and bioaugmentation. Dehalococcoides cells carrying the tceA, vcrA, and bvcA genes were indigenous to the site. The sum of the three identified RDase gene copy numbers closely correlated to 16S rRNA gene copy numbers throughout the biostimulation and bioaugmentation activity, suggesting that these RDase genes represented the major Dehalococcoides metabolic functions at this site. Biomarker quantification revealed an overall increase of more than 3 orders of magnitude in the total Dehalococcoides population through the 1-year monitoring period (spanning biostimulation and bioaugmentation), and measurement of the respective RDase gene concentrations indicated different growth dynamics among Dehalococcoides cells. The Dehalococcoides cells containing the tceA gene consistently lagged behind other Dehalococcoides cells in population numbers and made up less than 5% of the total Dehalococcoides population, whereas the vcrA- and bvcA-containing cells represented the dominant fractions. Quantification of transcripts in groundwater samples verified that the 16S rRNA gene and the bvcA and vcrA genes were consistently highly expressed in all samples examined, while the tceA transcripts were detected inconsistently, suggesting a less active physiological state of the cells with this gene. The production of vinyl chloride and ethene toward the end of treatment supported the physiological activity of the bvcA- and vcrA-carrying cells. A clone library of the expressed RDase genes in field samples produced with degenerate primers revealed the expression of two putative RDase genes that were not previously monitored with RT-qPCR. The level of abundance of one of the putative RDase genes (FtL-RDase-1638) identified in the cDNA clone library tracked closely in field samples with abundance of the bvcA gene, suggesting that the FtL-RDase-1638 gene was likely colocated in genomes containing the bvcA gene. Overall, results from this study demonstrate that quantification of biomarker dynamics at field sites can provide useful information about the in situ physiology of Dehalococcoides strains and their associated activity.

Discrimination of multiple Dehalococcoides strains in a trichloroethene enrichment by quantification of their reductive dehalogenase genes.

ABSTRACT While many anaerobic microbial communities are capable of reductively dechlorinating tetrachloroethene (PCE) and trichloroethene (TCE) to dichloroethene (DCE), vinyl chloride (VC), and finally ethene, the accumulation of the highly toxic intermediates, cis-DCE (cDCE) and VC, presents a challenge for bioremediation processes. Members of the genus Dehalococcoides are apparently solely responsible for dechlorination beyond DCE, but isolates of Dehalococcoides each metabolize only a subset of PCE dechlorination intermediates and the interactions among distinct Dehalococcoides strains that result in complete dechlorination are not well understood. Here we apply quantitative PCR to 16S rRNA and reductase gene sequences to discriminate and track Dehalococcoides strains in a TCE enrichment derived from soil taken from the Alameda Naval Air Station (ANAS) using a four-gene plasmid standard. This standard increased experimental accuracy such that 16S rRNA and summed reductase gene copy numbers matched to within 10%. The ANAS culture was found to contain only a single Dehalococcoides 16S rRNA gene sequence, matching that of D. ethenogenes 195, but both the vcrA and tceA reductive dehalogenase genes. Quantities of these two genes in the enrichment summed to the quantity of the Dehalococcoides 16S rRNA gene. Further, between ANAS subcultures enriched on TCE, cDCE, or VC, the relative copy number of the two dehalogenases shifted 14-fold, indicating that the genes are present in two different Dehalococcoides strains. Comparison of cell yields in VC-, cDCE-, and TCE-enriched subcultures suggests that the tceA-containing strain is responsible for nearly all of the TCE and cDCE metabolism in ANAS, whereas the vcrA-containing strain is responsible for all of the VC metabolism.