Project Update Archive
Project 4: Application of comparative genomics, transcriptomics, and proteomics to optimize microbial reductive dehalogenation
This project seeks to develop and apply advanced tools based on molecular biology to analyze and optimize the In situ bioremediation of common Superfund groundwater pollutants, such as trichloroethene (TCE). In situ bioremediation, the use of microorganisms to convert hazardous pollutants to benign substances directly within the contaminated groundwater site, is one of the most promising and cost-effective strategies for cleaning up contaminated aquifers. Our research focuses on understanding the DNA, RNA, proteins and cellular metabolites of Dehalococcoides, the only known group of bacteria that can completely convert TCE to non-toxic ethene. The knowledge developed here will be used to design and optimize the engineering processes necessary to maximize the abundance and activity of these organisms for more efficient in situ remediation of TCE at Superfund sites.
Reductive dehalogenases, the enzymes responsible for destroying toxic chlorinated ethenes, use cobalamin (vitamin B12) as a crucial cofactor for achieving the catalytic function. Our previous research established the importance of cobalamin for the growth and TCE degradation activity of Dehalococcoides. This year we further investigated the cobalamin physiology of these organisms in order to identify the functional genes responsible for the uptake and utilization of cobalamin, given that Dehalococcoides are unable to synthesize cobalamin themselves. The total mRNA profiles of a model Dehalococcoides strain were examined during its growth with limited or excess concentrations of vitamin B12, as well as with cell-free spent medium from a chloroethene-degrading microbial community that is known to produce large amounts of vitamin B12. Both excess B12 and culture spent medium resulted in lower mRNA expression of the genes that are likely regulated by a cobalamin-sensing mechanism. In addition, only excess vitamin B12 triggered the lower mRNA copies of the genes encoding a cobalamin transport system. The environmental implication of these results is an improved strategy for encouraging the growth of Dehalococcoides both in situ and in laboratory cultures that can be applied for bioaugmentation of challenging remediation sites.
This year we initiated studies to identify and quantify genes and their expression profiles related to nitrogen and carbon metabolism in Dehalococcoides strain 195. Strain 195 is known to have the capability of using atmospheric nitrogen when there is no other ready-to-use or “fixed” nitrogen source available. Although this trait allows strain 195 to survive in broader environments, the growth rate and TCE degradation activity under nitrogen deficiency are significantly decreased. Our results show that the lack of fixed nitrogen causes significant increases in mRNA levels of the nitrogen-fixing genes. On-going experiments are being conducted to determine the total protein profile for strain 195 grown under nitrogen-fixing conditions, which will provide a holistic understanding on its physiological stress induced by nitrogen limitation. Finally, in order to understand the carbon metabolism of strain 195, we employed stable isotopomer tracer experiments to map out the amino acid biosynthesis pathways of strain 195. Several uncommon and novel pathways in isoleucine and citrate biosynthesis were identified from 13C labeling profiles of relevant amino acids in these pathways. On-going experiments focus on identification and characterization of genes encoding these pathways.
This project seeks to develop new tools to optimize the application of microorganisms to biodegrade common Superfund pollutants such as trichloroethene (TCE) within contaminated groundwater aquifers, a process called in situ bioremediation. In situ bioremediation is a promising and cost effective method for cleaning these contaminants up without bringing them to the surface and risking human exposure. The work of Drs. Lisa Alvarez-Cohen and Gary Anderson focuses on Dehalococcoides, the only known bacteria that can completely convert TCE to non-toxic ethane gas. A better understanding of the DNA, RNA and proteins of Dehalococcoides will greatly improve the understanding of how to effectively grow these finicky organisms, so that their abundance and activity at bioremediation sites can be maximized.
Onc of the research group’s objectives is to study the growth of Dehalococcoides in complex microbial communities such as one that they enriched from a local Superfund site (ANAS) that is capable of rapid and complete conversion of TCE to ethene. The researchers compared the DNA and RNA of strains of Dehalococcoides in the ANAS microbial community with that of a well-studied isolate in the laboratory, and confirmed that the genes associated with central metabolism, including an apparently incomplete carbon fixation pathway, vitamin B12 salvaging system, nitrogen fixation pathway, and central respiratory enzymes are present and active in both. Also, although one gene known to be responsible for TCE conversion was detected, 13 of the 19 other potential genes that could carry out this reaction were not detected, suggesting the occurrence of horizontal gene transfer. Application of a high-density phylogenetic microarray to study the microbial population in the ANAS culture identified the dominant and active members, including fermentors, sulfate reducers and methane generators. Understanding the microbial community that composes this stable and efficient bioremediating culture furthers understanding of microbial community dynamics. Extensive examination of this model community is planned.
Some of the researchers’ work this year dealt with exploring mechanisms to grow Dehalococcoides more rapidly and to a higher density. Current studies are underway that evaluate the effects of degradation products vinyl chloride and ethene on cell growth. They also tested the effects of nitrogen stress on the culture and showed that the isolate can actually obtain its nitrogen from atmospheric nitrogen gas. However, this reaction causes stress to the cells, resulting in slower growth and biodegradation. In contrast, when the isolate is grown in co-culture with a sulfate-reducing bacterium, it grows more rapidly and robustly to higher densities. Analysis of the RNA and proteins generated by the co-culture confirm that there are important interrelationships between the organisms, allowing them to grow more effectively together than alone.
The significance of this work is that the research group has developed molecular tools that allow them to study both the fundamental and the applied aspects of Dehalococcoides, a bacterium capable of bioremediating chlorinated solvents. These tools have allowed the researchers to make new discoveries about the growth of this organism, so that engineering processes may be designed for more successful in situ bioremediation.
This project seeks to optimize the microbial detoxification of common Superfund pollutants, perchloroethene (PCE) and trichloroethene (TCE) by focusing on the only genus of bacteria, Dehalococcoides, known to completely reduce PCE and TCE to ethene. A better understanding of the genome, transcriptome and proteome of Dehalococcoides will greatly improve the scientific communities understanding of the physiology of these difficult to grow organisms, so that their abundance and activity at bioremediation sites can be maximized. Chlorinated solvents are the most common groundwater contaminants at Superfund sites. In situ bioremediation is a promising and cost effective method for remediation of these contaminants. The aim of The Application of Comparative Genomics, Transcriptomics, and Proteomics to Optimize Microbial Reductive Dehalogenation project is to improve prediction and monitoring which will ultimately optimize system performance at remediation sites.
One of the main goals involved an enriched anaerobic microbial community (ANAS) that reductively dechlorinates TCE to ethene. Dr. Lisa Alvarez-Cohen and Dr. Gary Andersen compared the strains of Dehalococcoides in the ANAS community with the genome of D. ethenogenes 195. The analysis confirmed that the genes associated with central metabolism including an apparently incomplete carbon fixation pathway, cobalamin salvaging system, nitrogen fixation pathway, and five hydrogenase complexes are present in both 195 and ANAS. Although the gene encoding the TCE reductase tceA was detected, 13 of the 19 reductive dehalogenase genes present in 195 were not detected in ANAS. Additionally, 88% of the genes in predicted integrated genetic elements in strain 195 were not detected in ANAS, suggesting the occurrence of horizontal gene transfer. Interestingly, sections of the tryptophan operon and an operon encoding an ABC transporter in 195 were also not detected in ANAS.
Alvarez-Cohen and Anderson measured the transcriptomic effects of cobalamin (vitamin B12)-limited growth conditions and identified a cobalamin regulon in 195. In addition to cobalamin stress, the investigators tested the effects of nitrogen stress on strain 195. The research group established that strain 195 is capable of fixing atmospheric dinitrogen and the nitrogenase operon is stringently regulated according to the availability of the fixed nitrogen source ammonium. Physiologically, the lack of a fixed nitrogen source caused strain 195 to make an early transition into stationary phase characterized by decoupled growth and dechlorination activity and resulting in reduced overall dechlorination activity and cell density.
The researchers characterized the proteome of Dehalococcoides ethenogenes strain 195 during the transition period between the exponential-growth and stationary phases. The proteome was characterized by analyzing trypsin-digested peptides with two-dimensional liquid chromatography coupled to tandem mass spectrometry. In total, both peptides and transcripts were detected for 790 unique genes, which is 52% of the total non-redundant protein-coding genome. Of these, high expression was measured for three reductive dehalogenases, two hydrogenases, and a formate dehydrogenase. Also notable was the high expression measured for corrinoid ABC-type transport and salvage systems.
As part of the expanded scope with emerging contaminants, Alvarez-Cohen and Anderson studied anaerobic debromination of the flame retardants polybrominated diphenyl ethers (PBDEs). Studies with the ANAS culture and two other anaerobic species Dehalobacter restrictus and Desulfitobacterium hafniense demonstrated similar PBDE degradation capabilities across different species. Two-dimensional chromatography was then used to identify the specific debromination products for seven environmentally-relevant PBDEs.
This project is improving the ability to cleanup groundwater contamination using naturally occurring microorganisms with a process called in situ bioremediation. In situ bioremediation is an effective and desirable approach since contaminants are destroyed directly in the ground, rather than transferred to the surface where human exposure may occur. The specific objectives of this study are to develop new tools to reliably monitor the in situ bioremediation of chlorinated solvents and emerging groundwater contaminants. The focus is on advanced molecular tools and isotopic analyses that can be applied quantitatively. Reliable monitoring methods will decrease the uncertainty and cost of bioremediation while enabling the application of more effective risk assessment and risk management techniques.
Over the past year the research teams of Drs. Alvarez-Cohen and Conrad have made significant progress on both molecular biology and stable isotopic approaches. During this year they have developed quantitative polymerase chain reaction (qPCR) techniques to track the specific bacterial strains and their specific proteins involved in in situ bioremediation. The practical application of this work is that a simple, rapid test can be used to determine whether key bacteria and the appropriate enzymes are present at a contaminated groundwater site for in situ bioremediation to be successfully applied. Because this technique is more accurate and faster than previous alternatives, it should greatly facilitate site assessment, application design, and bioremediation monitoring. This year the researchers have also substantially expanded their molecular capabilities to include the use of whole-genome microarrays to apply genomics and transcriptomics to track not only the presence, but also the activities of the bacteria involved in in situ bioremediation.
Beyond their work with chlorinated solvent degrading bacteria, they have also studied the biodegradation of emerging water contaminants N-nitrosodimethylamine, 1,4 dioxane and most recently, polybrominated diphenyl ethers. They have successfully identified naturally-occurring bacteria that are capable of degrading each of these compounds, and are currently working out the biochemical pathways for their degradation. These studies should result in the development of methods for the in situ bioremediation of these compounds in the environment.
This project is improving the project investigators’ ability to reliably monitor the in situ bioremediation of groundwater contaminants by developing advanced molecular tools and isotopic analyses that can be applied quantitatively. Reliable monitoring methods will decrease the uncertainty and cost of bioremediation while enabling the application of more effective risk assessment and risk management techniques.
Over the past year project investigators have continued improving tools based upon both molecular biology and stable isotopic signatures. Molecular techniques have been applied to monitor the degradation of trichloroethene (TCE) and perchloroethene (PCE) at two large-scale field sites. Previously, the researchers had shown that one of the sites lacked a key species necessary for complete bioremediation and that subsequent introduction of that species had been successful. Over the last year they have tracked the spread of the key species and shown that its presence correlates well with areas where PCE is completely degraded. The practical application of this study is that a simple molecular test for the presence of a key organism has been successfully used both to identify the limiting factors for bioremediation and to monitor the progress of the adopted solutions. Because this technique is more accurate and faster than previous alternatives, it should greatly facilitate site assessment, application design, and bioremediation monitoring.
This year project investigators have also substantially expanded their molecular toolkit for tracking microorganisms and their activity by developing quantitative polymerase chain reaction (qPCR) for application to both gene copy numbers and for quantification of expression. The investigators have developed an internal standard technique that uses luciferase genes from fireflies to allow them to quantify gene expression in an absolute sense. In addition, the investigators have begun to study the transcriptomics of bacteria that reductively dechlorinate PCE, TCE, and VC. To this end they have designed a full genome microarray for quantitative analysis in comparative genomics and transcriptomics.
During this past year the investigators have also continued their studies of the mechanisms and kinetics of NDMA and 1,4 dioxane degradation by oxygenase-expressing microorganisms. Both of these contaminants are water-soluble carcinogens that are becoming prevalent drinking water contaminants. NDMA finds its way into drinking waters as contamination associated with aerospace facilities and as a byproduct of the chlorination of wastewaters; 1,4 dioxane is a solvent stabilizer that is found co-mingled in solvent plumes. Over the past year the investigators have shown that a common strain of laboratory bacteria that normally cannot degrade NDMA gains degradation activity when a specific monooxygenase enzyme is engineered into it. In addition, they have used radioactive labeling to show that an organism they have designated Pseudonocardia dioxanivorans uses dioxane as a growth substrate, and likely uses monooxygenases to convert more than 50% of the dioxane to CO2. The practical application of this study is to identify and characterize species that will be useful for the future bioremediation of these and similar toxins.
This project is making major contributions towards improving our ability to reliably monitor the in situ bioremediation of groundwater contaminants by developing molecular and isotopic analyses that can be applied quantitatively. Reliable monitoring methods will decrease the uncertainty and cost of bioremediation while enabling the application of more effective risk assessment and risk management techniques.
Over the past year, project investigators have made significant progress toward improving and applying tools based upon both molecular biology and stable isotopic signatures. Molecular techniques were applied to compare the effect of lactate injection at two large-scale field sites contaminated with TCE and PCE. These techniques diagnosed a difference in the indigenous microbial community that resulted in successful bioremediation occurring at only one of the two sites. Subsequently, molecular analyses showed that a pilot bioaugmentation study undertaken to overcome the microbial limitation was successful. The practical application of these findings is that a simple molecular test for the presence of key organisms can predict whether biostimulation or bioaugmentation processes are more suitable, improving application design and potentially decreasing remediation time by months.
Over the past year, these researchers have also made significant progress in applying stable isotope analysis for monitoring, assessing, and validating bioremediation processes. They have succeeded in measuring the carbon isotopic fractionations associated with the aerobic biodegradation of vinyl chloride (VC), trichloroethylene (TCE), and cis-1,2-dichloroethylene (cDCE) by both metabolic and cometabolic processes. For all three of the tested chlorinated ethenes, isotopic fractionation associated with aerobic degradation was significantly smaller than that reported for anaerobic reductive dechlorination, suggesting that analysis of compound-specific isotopic fractionation could be useful for determining whether aerobic or anaerobic degradation of VC and DCE predominates in field applications of in situ bioremediation.
Finally, Dr. Alvarez-Cohen’s team has achieved notable progress in the expanded goal of elucidating the mechanisms and kinetics of NDMA and 1,4 dioxane degradation by oxygenase-expressing microorganisms. Both of these contaminants are water-soluble carcinogens that are becoming prevalent drinking water contaminants. NDMA finds its way into drinking waters as contamination associated with aerospace facilities and as a byproduct of the chlorination of wastewaters; 1,4 dioxane is a solvent stabilizer that is found co-mingled in solvent plumes. A strong case has been made that monooxygenase enzymes are responsible for the degradation of these two compounds. The investigators found five dissimilar bacterial strains that, under conditions that encourage monooxygenase enzyme production, are capable of biodegrading NDMA. In addition, several monooxygenase-expressing cultures were found to degrade 1,4 dioxane. Also, it was shown that acetylene gas, known to inhibit monooxygenase enzymes, completely inhibits the degradation of NDMA or 1,4 dioxane in most strains. Kinetic measurements, biochemical studies, and experiments to identify degradation intermediates are underway for both molecules.
Chlorinated solvents are the most common groundwater contaminants at Superfund sites. In situ bioremediation is a promising and cost effective method of remediation. One major limitation is the difficulty associated with predicting and monitoring progress over time. This project is making major contributions towards reliably monitoring this method by developing molecular and isotopic analyses that can be applied quantitatively. Reliable bioremediation monitoring methods will decrease the uncertainty and cost of this method while enabling the application of more effective risk assessment and risk management techniques. Isotope measurements were used to track and control the potential generation of harmful biological intermediates. This success will result in approximately $15 million cost savings for the cleanup of the field site, and will open the door for the emerging technique of stable isotope analysis to be applied more widely at a variety of groundwater contaminated sites. Two specific approaches are being used. The molecular biology approach aims to apply a suite of cutting-edge molecular tools in a quantitative manner to evaluate the relative contribution of different physiological groups of bacteria to the degradation of chlorinated solvents such as perchloroethylene (PCE) and trichloroethylene (TCE). The second approach is to quantify the fractionation of chlorine and carbon stable isotopes caused by microbial degradation processes to facilitate the identification of dominant pathways and redox conditions for specific contaminant biodegradation reactions.
Over the past year, the investigators made significant progress toward each project goal. The scope of the original goals has been expanded to reflect an increase in analytical capabilities within the UC-Berkeley laboratory. Work continues with an enriched anaerobic microbial community. The practical implication of Dr. Alvarez-Cohen’s findings is that enhanced bioremediation strategies that rely on electron donor addition have a high potential for success. Project investigators have also begun to apply these molecular techniques in the field, to evaluate microbial community shifts caused by enhanced bioremediation strategies conducted at large-scale field sites. Two field sites contaminated with TCE and PCE were chosen to test the efficacy of lactate injection to promote reductive dechlorination. A pilot study to test bioaugmentation at this site is planned for spring 2003.
Finally, a variety of techniques have been applied to elucidate the mechanisms and kinetics of NDMA and 1,4 dioxane degradation by oxygenase-expressing microorganisms. NDMA is a potent carcinogen and 1,4 dioxane is a carcinogen with a health advisory level of 3 parts per billion. NDMA, a component of rocket fuel, is also produced by the chlorination of wastewaters; 1,4 dioxane is a solvent stabilizer that is found co-mingled in solvent plumes. Three dissimilar bacterial strains were found that generate monooxygenase enzymes for growth on toluene and are each capable of biodegrading NDMA when induced with toluene. In addition, several monooxygenase-expressing cultures were found to degrade 1,4 dioxaney. Acetylene gas completely inhibits the degradation of NDMA or 1,4 dioxane in at least one strain each. Kinetic measurements and experiments to identify degradation intermediates are underway for both molecules.
Project investigators have made major progress in applying techniques that were developed in laboratory experiments to large-scale field studies. Changes in subsurface microbial communities at two field sites undergoing enhanced in-situ bioremediation of trichloroethylene (TCE) have been examined using molecular techniques. The two sites differ in that partially dechlorinated intermediates were present at one prior to enhanced bioremediation while no such intermediates were observed at the other. Major differences were observed in microbial community structure between the two sites, including the absence of a major known dechlorinating bacterium, Dehalococcoides ethenogenes, at the site where bioremediation was not successful. Current work involves the quantification of functional gene expression for TCE dechlorination. In addition, studies were initiated to evaluate the potential for aerobic biodegradation of N-nitrosodimethylamine (NDMA), a newly emerging carcinogenic water contaminant. From the literature on the activation mechanism of this compound in mammalian systems, we hypothesized that monooxygenase-expressing bacteria would degrade it. Preliminary studies have supported this hypothesis.