Professor Alvarez-Cohen's research areas include environmental microbiology and ecology, biotransformation and fate of environmental contaminants, anammox processes for nutent removal from wastewaters and innovative molecular and isotopic techniques for studying microbial ecology of complex communities. Specifically, her research focuses on the application of omics-based molecular tools and isotopic techniques to understand and optimize the bioremediation of emerging and conventional environmental contaminants by naturally occurring microorganisms and to facilitate beneficial nutrient removal from wastewater. Bioremediation and nutrient removal are processes that rely upon complex mixed microbial communities that interact to catalyze important reaction pathways.
Alvarez-Cohen’s lab takes a systems-based approach to understand communities as holistic units of interacting species, capable of performing environmentally relevant reactions.
Ongoing research projects include:
- Metabolic Degradation of 1,4-dioxane by Pseudonocardia Dioxanivorans Strain CB1190: Genomic and Post-genomic Studies
- Oxygenase-Catalyzed Biodegradation of Emerging Water Contaminants: 1,4-Dioxane and N-Nitrosodimethylamine
- Quantifying Gene Expression to Predict and Optimize Reductive Dechlorination by Dehalococcoides spp.
- Application of Microarrays to Identify Biomarkers of Reductive Dehalogenating-Microbial Communities
- Using Molecular and Isotopic Tools to Characterize the Biodegradation of Chlorinated Ethenes and Ethanes
- Characterizing the fate and biotransformation of fluorochemicals in aqueous film forming forms (AFFF)
- Corrinoid coenzyme requirement for effective TCE bioremediation using Dehalococcoides
- Meta-omics of Microbial Communities Involved in Bioremediation
Past research projects include:
- Biodegradation of the Flame Retardants Polybrominated Diphenyl Ethers
- Characterizing and Evolving the Propane Monooxygenase for N-Nitrosodimethylamine Biodegradation and Green Chemistry
Metabolic degradation of 1,4-dioxane by Pseudonocardia dioxanivorans strain CB1190: genomic and post-genomic studies
1,4-dioxane (dioxane) is an emerging groundwater contaminant that is a probable human carcinogen and a confirmed animal carcinogen. Since it has historically been used as a chlorinated solvent stabilizer, dioxane is frequently found co-mingled with chlorinated solvents, such as 1,1,1-trichloroethane. Unlike chlorinated solvents, however, dioxane is not easily removed from the environment by volatilization or sorption processes, so alternative decontamination processes are required. Fortunately, several strains of aerobic bacteria with the capacity to degrade dioxane have been identified, so the bioremediation of dioxane-contaminated groundwater with such bacteria is a potential technology.
Our lab has been studying one of the few bacterial strains that can fully metabolize dioxane, using dioxane as both a carbon and energy source for growth. This bacterium, Pseudonocardia dioxanivorans strain CB1190, was originally isolated from industrial sludge from a dioxane-contaminated site in South Carolina. While the kinetics of dioxane-degradation by strain CB1190 have been extensively studied, our knowledge of the enzyme systems and gene-regulation mechanisms involved in growth on dioxane is currently very limited. For this reason, in conjunction with the Joint Genome Institute, we are having the genome of strain CB1190 determined by high throughput sequencing technologies. An insight into strain CB1190's genetic complement will permit genome-enabled studies to better understand the bacterium's dioxane-degrading capability. These studies include:
- 1. Identification of the enzymes (e.g. monooxygenases) involved in the activation of the dioxane ring and investigation of their expression and regulation in the presence of competing carbon sources such as formate or the related cyclic ether tetrahydrofuran.
- 2. In collaboration with Dr. Yinjie Tang at Washington University, using isotopomer technology to determine the biochemical pathway for dioxane incorporation into central metabolism, thus exploring strain CB1190's ability to grow (rather than co-metabolize) on dioxane.
- 3. Using full genome expression microarrays to investigate the genes involved in nitrogen metabolism, particularly strain CB1190's ability to fix dinitrogen (N2), which is potentially important for environments limited in fixed-nitrogen.
- 4. In collaboration with Dr. Rebecca Parales at UC Davis, developing a genetic system for strain CB1190 to permit gene knockouts for confirming the involvement of candidate genes in dioxane metabolism.
The results of this study will provide a mechanistic understanding of the degradation of 1,4-dioxane and the growth of Pseudonocardia dioxanivorans strain CB1190, further providing a foundation for developing tools to monitor and enhance the bioremediation of dioxane in natural and engineered systems.
Funded by the Strategic Environmental Research and Development Program and in collaboration with Shaily Mahendra at UCLA.
Oxygenase-Catalyzed Biodegradation of Emerging Water Contaminants: 1,4-Dioxane and N-Nitrosodimethylamine
This research examines the biodegradation of the emerging water contaminants 1,4-dioxane and N-nitrosodimethylamine (NDMA). Both 1,4-dioxane and NDMA are probable human carcinogens and confirmed animal carcinogens. Neither is significantly attenuated in the environment by volatilization or sorption processes. Due to its widespread use as a solvent stabilizer, 1,4-dioxane is frequently found comingled with chlorinated solvents at DoD and DoE sites; while NDMA is found as a degradation byproduct in proximity to aerospace facilities that used hydrazine-based rocket fuel.
Although the carcinogenic threats of 1,4-dioxane and NDMA have been understood for many years, they have not historically been considered important water quality issues, mostly due to lack of awareness about their potential occurrence in drinking water supplies. However, with recent advances in analytical methods and growing public awareness of their occurrence in drinking water supplies, 1,4-dioxane and NDMA are emerging as important water contaminants. Consequently, a better understanding of the effects of bacterial degradation on the fate and persistence of 1,4-dioxane and NDMA in the environment is needed.
This study will identify organisms, and more importantly a class of enzymes, capable of aerobically biodegrading 1,4-dioxane and NDMA. Furthermore, this study will elucidate the biochemical pathways responsible for 1,4-dioxane and NDMA degradation, quantify reaction kinetics, and develop models to predict those kinetics. It will also explore the effects of co-contaminants (e.g. 1,1,1-TCA, DCE, toluene, and chloroform) and inducing substrates (e.g., methane, propane, butane) on the contaminant degradation rates.
The results of this study will provide a mechanistic understanding of degradation of 1,4-dioxane and NDMA by aerobic microorganisms and as such it will provide a foundation for the bioremediation of these contaminants in natural and engineered systems.
Funded by the Strategic Environmental Research and Development Program.
Mass spectrometry for dioxane biodegradation pathway
Quantifying Gene Expression to Predict and Optimize Reductive Dechlorination by Dehalococcoides spp.
This study will apply gene expression analysis techniques to evaluate, predict, and optimize Dehalococcoides spp. reductive dechlorination activity in complex laboratory and field-site microbial communities. Based on the observation that Dehalococcoides spp. grow and dechlorinate less robustly in pure culture than in mixed communities, molecular techniques will be used to compare increasingly complex communities, ranging from pure and enrichment cultures to soil microcosms and field samples, in order to identify expression-based markers that correlate with robustness of dechlorination activity. First, the per-cell dechlorination rate will be compared across a suite of communities to determine the predictive value of several fundamental metrics such as the cell density of Dehalococcoides spp., the identity and quantity of reductase genes present, and the expression level of each reductase gene. Second, whole-genome microarrays of D. ethenogenes 195 will be used to compare the suite of communities and identify novel genes whose expression is closely correlated with per-cell dechlorination rates. Genes identified by microarrays to be potentially good expression markers will be further analyzed by real time quantitative PCR (qPCR). Third, identified predictive markers will be tested on field-site samples from the Idaho National Environmental Engineering Laboratory, where lactate injection to promote in situ bioremediation of trichloroethene (TCE) is ongoing, to seek correlations of degradation activity gradients with gene expression across space and time. Finally, predictive kinetic models for reductive dechlorination will be developed that incorporate the presence, copy number, and expression level of specific genes and the expression of validated and field-tested correlative genes.
Funded by the National Science Foundation, BES-01-0504244.
Hierarchical clustering of genes differentially expressed as Dehalococcoidesethenogenes transitions from the early-exponential (EE) to the late-stationary (LS) growth phases. The color gradient from blue to yellow represents increasing gene expression.
Application of Microarrays to Identify Biomarkers of Reductive Dehalogenating-Microbial Communities
(In collaboration with Gary Andersen and Eoin Brodie at Lawrence Berkeley National Laboratory and Stephen Zinder at Cornell University)
Background. Tetrachloroethene (PCE) and trichloroethene (TCE) have been widely used as industrial solvents and, as a result of poor storage and disposal practices, are now common contaminants of groundwater resources. Fortunately, PCE and TCE can be effectively biodegraded in anaerobic environments by reductive dechlorination processes. Recent progress has been made towards exploiting these processes for bioremediation applications. There remains a need, however, for appropriate and cost-effective biomarkers for assessing, monitoring, and optimizing the performance of these processes. Biomarker development has primarily focused on identifying nucleic acid sequences, peptides, proteins, or lipids of organisms that catalyze biodegradation reactions of interest. Although promising, such approaches are limited in that they do not address the roles of organisms that support and/or enhance the activity of dechlorinating organisms. Novel biomarkers that quantify the presence, abundance, and activity of supporting organisms are therefore needed to more effectively assess and optimize dechlorination processes.
Objective. In this research, we will identify 16S-rRNA-based phylogenetic and mRNA-based functional biomarkers diagnostic of microbial communities that support the robust growth and activity of chlorinated ethene-degrading organisms, with particular emphasis on Dehalococcoides species. Members of the Dehalococcoides genus can degrade chlorinated ethenes completely to ethene and also degrade a wide range of other chlorinated aromatic and aliphatic pollutants.
Summary of Process/Technology. We will apply state-of-the-art microarray-based tools to identify relevant biomarkers. A 16S-rRNA microarray that targets 9000 unique microbial phylogenies will be applied to identify key Archaea and Bacteria that are present and active in a range of Dehalococcoides-containing microbial communities, including enrichment cultures, soil microcosms, and groundwater samples. We will then construct defined co- and tri-cultures that contain Dehalococcoides and one or more of the key supportive organisms. Constructed cultures that exhibit enhanced and sustained rates of dechlorination and/or novel metabolic capabilities, such as the ability to use organic acids as electron donors, will be identified and the 16S-rRNA genes of the supporting organisms will be selected as phylogenetic biomarkers. In addition, a genomic expression microarray that targets the Dehalococcoides genus will be applied to the constructed cultures, to robust enrichment cultures, and to environmental samples to identify functional biomarkers indicative of the activity and/or functional role of supporting organisms. Finally, quantitative PCR will be used to derive correlations between the quantitative detection of these biomarkers, chlorinated ethene degrading activity, and the metabolic requirements of the microbial communities. These models will provide novel tools for assessing the structure and total biodegradative potential of Dehalococcoides in uncharacterized microbial communities.
Benefits. The broadest significance of the proposed work is that it will lead to improved strategies for optimizing in situ bioremediation technologies. The biomarkers developed here could shorten the bioremediation process feedback cycle by replacing traditional diagnostics, such as microcosm responses that are monitored over weeks, with appropriate 16S-rRNA- and gene expression-based diagnostics that can be monitored within hours. Furthermore, the insights gained about important ecological interactions within reductive dechlorinating microbial communities will improve our ability to design, construct, and optimize bioaugmentation and biostimulation systems.
Funded by Strategic Environmental Research and Development Program (SERDP).
Shown in the figures above are preliminary data of bacterial and archaeal populations that exhibit the greatest changes over the course of treatment at Ft. Lewis, Seattle, a TCE-contaminated site that has undergone in-situ bioremediation.
Using Molecular and Isotopic Tools to Characterize the Biodegradation of Chlorinated Ethenes and Ethanes
This project focuses on the comprehensive understanding of the fate and transport of chlorinated solvents in the subsurface, specifically those transformation pathways resulting from biodegradation processes. Emphasis is placed on the use of various molecular and isotopic tools to measure the biological transformation of chlorinated ethenes and ethanes, as well as the expanded characterization of the microorganisms responsible for their degradation. Tools and techniques that are used to characterize the biodegradation include: carbon isotope fractionation analyses, microarrays, and (RT) – qPCR. These analyses aid in the understanding of the activity and distribution of dehalogenating microorganisms in both laboratory and field environments.
The collection and analysis of environmental samples from various contaminated field sites allows for the in situ examination of complex microbial communities. These studies seek to assess the impacts of an active remediation system on the biological degradation of chlorinated ethenes, as well as examine the in situ physiology and metabolism of Dehalococcoides spp. The various aforementioned molecular and isotopic tools are used on the samples to investigate certain physical and biological processes. This includes monitoring the presence and expression of applicable taxonomic and functional genes at various time points before, during, and after remediation activities. These field studies are also complemented with fundamental laboratory experiments that are designed to determine the variability and specificity of the information provided by these tools. For example, the variation in stable isotope fractionation patterns of chlorinated solvents in laboratory cultures has been examined to determine whether the physiological state of the microorganism impacts the observed fractionation pattern. Achievable growth conditions were systematically varied in order to determine whether there are potential variations in the subsequent isotopic fractionation. This is ultimately important for understanding field-derived fractionation data.
Funded by the Chevron Energy Technology Company
Characterizing the fate and biotransformation of fluorochemicals in aqueous film forming forms (AFFF)
AFFF are complex mixtures of fluorocarbon surfactants, hydrocarbon surfactants, and solvents that were designed to spontaneously spread over hydrocarbon-fuel fires to extinguish flames and to prevent re-ignition. Repeated applications of AFFF at military fire-training sites have resulted in hundreds of fluorochemical-contaminated groundwater, sediment, and soil sites. The fate and transport of perfluorinated compounds (PFCs) in the environment is not well understood and the biodegradation potential for many of these pollutants has not been established. Additionally, because halogenated solvents, such as trichloroethene (TCE) were routinely ignited for fire-training activities, many of the sites may have significant co-contamination of chlorinated ethenes.
The goals of our research are to determine the biodegradability of fluorinated contaminants and other priority pollutants under redox conditions representative of groundwater at fire training sites. We are studying these processes using microcosms derived from both pristine and AFFF-contaminated soils as well as TCE-degrading enrichment cultures. Various molecular tools are being employed to characterize AFFF-utilizing microbial communities and identify active microorganisms, as well as to determine changes in microbial community structure upon exposure to AFFF. By developing a better understanding of the biotransformation of AFFF fluorochemical contaminants and their effects on the biodegradation of co-contaminants (e.g. TCE) in the subsurface, our study will contribute to remediation approaches that greatly decrease the time and cost required to monitor and treat contaminated soil and groundwater systems.
Corrinoid coenzyme requirement for effective TCE bioremediation using Dehalococcoides
Microorganisms of the Dehalococcoides (Dhc) genus play a crucial role in remediating groundwater contaminated by chlorinated solvents. Their unique metabolic ability to convert chlorinated solvents such as per- and tri-chloroethene to innocuous end product ethene is attributed to corrinoid-dependent reductive dehalogenases. Corrinoids, the required coenzymes for reductive dehalogenase activity, are a group of cobalt-containing molecules (including vitamin B12) that have wide structural variability in the lower axial ligand. The currently known Dhcstrains cannot synthesize corrinoids de novo and thus have to obtain them from the environment. It has been reported that corrinoid limitation, which can impact the robustness and efficiency of dechlorination, can be avoided either by adding exogenous vitamin B12 or by growing Dhcin microbial consortia containing microorganisms capable of corrinoid synthesis.
Currently, little is known about the actual corrinoid species that Dhcobtains from their environment and needs to support their physiological functions. The goal of this study is to investigate both the corrinoid forms present in effective TCE-dechlorinating microbial communities and the structural specificity of the corrinoid coenzymes required by Dhc. The corrinoid forms and concentrations are being determined in the effective TCE-dechlorinating microbial communities that have been enriched with no external B12 amendment. The Dhc physiological responses to these corrinoid forms are being characterized. This study contributes both to our understanding of the corrinoid syntrophic interactions in effective TCE-dechlorinating microbial communities and to the structure-function relationship of corrinoid coenzymes on the metabolic fitness of Dhc.
Meta-omics of Microbial Communities Involved in Bioremediation
This research seeks to advance our fundamental understanding of microbial communities that are capable of bioremediating environmental contaminants. We will apply systems biology approaches to study biodegradation abilities and interactions within microbial communities that remediate two water contaminants, trichloroethene (TCE) and 1,4-dioxane (dioxane), both of which are common problems at Superfund sites.
Despite broadly dissimilar biodegradation mechanisms, the bioremediation processes for TCE and dioxane both rely upon the activities of mixed microbial communities for achieving effective remediation goals. Consequently, there is a need for comprehensive and effective approaches to assess, predict, and optimize the performance of these complex microbial communities. Key functional degraders involved in these bioremediation processes, such as Dehalococcoides spp. for TCE dechlorination and Pseudonocardia spp. for dioxane degradation, have been studied in pure cultures, and mechanistic understanding of the degradation pathways have been elucidated. Although useful, such a reductionist understanding of key degrading microorganisms is not directly applicable to microbially diverse environmental samples. The lack of a comprehensive understanding of the effects of microbial community structure and its physiological characteristics on the capabilities of key degrading microorganisms is a barrier to progress in the field. In addition, there is a need to develop quantitative predictive tools for bioremediation assessment and process control.
Therefore, this project will focus on a holistic understanding of the population dynamics, metabolisms and functional interactions that shape the bioremediation of Superfund contaminants. This research will pioneer meta-omics approaches to elucidate microbial community structure-function relationships within complex systems, leading to improved abilities to design and optimize bioremediation processes, which, in turn, will decrease the cost and time required for remediation and reduce the exposure associated with groundwater contamination.
In this research, we will examine communities using a variety of high-throughput molecular biology tools, such as metagenomic analysis of isotopically enriched DNA (combination of stable isotope probing and next generation sequencing technology) or application of microarray hybridization using custom-designed microarrays.
Funded by National Institute of Environmental Health Sciences (NIEHS - Superfund Research Program)
Biodegradation of the Flame Retardants Polybrominated Diphenyl Ethers
Polybrominated Diphenyl Ethers (PBDEs) are flame retardants that have been used for over thirty years in a wide range of consumer textile and plastic products, such as TVs, sofas and automobiles. Some PBDEs, in particular ones with five bromines, are endocrine disruptors and have thus been banned in certain states and countries. The fate of these compounds in the environment is unknown.
Our research studies the fate of PBDEs in anaerobic and aerobic environments though biotransformation by bacteria. We have found that PBDE can be reductively debrominated by anaerobic bacteria, whereby the bromines are progressively removed and replaced with hydrogen. The more highly brominated PBDEs are however less toxic and can become more toxic if this transformation occurs.
We are currently studying the pathways and kinetics of this process using two-dimensional chromatography in order to separate and identify all possible degradation products. Our goal is to understand whether toxic byproducts can potentially accumulate in the environment due to biotransformation by bacteria.
Funded by UC Center for Water Resources.
Two-dimensional GC chromatogram of a PBDE standard.
Characterizing and Evolving the Propane Monooxygenase for N-Nitrosodimethylamine Biodegradation and Green Chemistry
(in collaboration with William Mohn and Lindsey Eltis of University of British Columbia, Vancouver, and Thomas Wood at Texas A&M)
Recent advancements in analytical detection methods have led to a growing awareness of the presence of N-nitrosodimethylamine (NDMA) in many drinking water sources. NDMA is a member of extremely-potent carcinogens, nitrosamines, whose occurrence in the environment has been linked to decomposition of hydrazine-based rocket fuels and chlorination of water and wastewater.
Bacterial strains expressing specific types of monooxygenases have demonstrated the ability to degrade NDMA, with induction of the propane monooxygenase in Rhodococcus sp. RR1 leading to the fastest rates of degradation. In addition to NDMA, other environmental contaminants such as trichloroethene (TCE), methyl tert-butyl ether (MTBE), and chloroform can be degraded by bacterial strains induced on propane. Despite its apparent versatility as a bacterial enzyme system, very little is known about bacterial propane monooxygenases.
The goal of this project is to characterize and evolve the propane monooxygenase enzyme, to better understand the nature of this enzyme to degrade NDMA and other xenobiotics, and to enhance the transformation rates of those compounds.
Our approach is to first use the fully annotated genome of Rhodococcus sp. RHA1, a strain similar to RR1, to identify and heterologously express the gene cluster coding for propane monooxygenase in a recombinant clones.
The second goal is to purify the components of the propane monooxygenase from the recombinant clones to investigate enzyme activity reconstituted in vitro and for crystallization. Both the recombinant clones and the purified propane monooxygenase will identify the enzymes xenobiotic transformation abilities.
The final goal is to use random and site-specific mutagenesis for directed evolution of the propane monooxygenase in order to improve transformation of xenobiotics. This will be the first instance that protein engineering will be applied to increase the degradation abilities of a propane monooxygenase. Evolved enzymes will be evaluated for green chemistry applications, such as for producing dyes or pharmaceuticals.
Funded by National Institute of Health.
Transcriptomic evidence for the role of propane monooxygenase in NDMA degradation. Growth of Rhodococcus sp. RHA1 on propane greatly enhances the rate of NDMA degradation. A DNA microarray was used to determine which enzyme is responsible for NDMA degradation under propane growth conditions. The microarray plot (above) shows a 125-fold increase in expression levels of a propane monooxygenase gene (prmA) when RHA1 transcripts from growth on propane are compared to that on pyruvate. These results suggests that a propane monooxygenase is responsible for NDMA degradation. Additional molecular biology strategies, such as qRT-PCR, knockout phenotypes and recombinant clones, will be used to verify these microarray findings.