One of the most perplexing problems facing environmental engineering practitioners is stalls in biodegradation at TCE-contaminated groundwater sites. In spite of injection of a suitable electron donor and a microbial consortium (e.g., KB-1) able to mineralize TCE to CO2, sometimes the chlorinated solvents persist. The PI's recent research has shown that indigenous groundwater protists (i.e., 2-3 ìm nanoflagellates) can prey upon members of the microbial consortia, potentially preventing them from establishing a sufficient abundance in situ to accomplish complete TCE mineralization. The proposed research will determine if, and under what conditions, the protists can selectively prey upon key bacterial species, preventing the consortia from accomplishing the series of biodegradation reactions necessary to mineralize TCE and its progeny. Specifically, the research will determine if: (1) protistan predation and organic carbon availability have an influence on the microbial community structure; (2) protists selectively graze on key species in the TCE-degrading consortia, and if so, at what rates (nL of water cleared of bacteria/protist?h); (3) TCE and/or its progeny, at environmentally-relevant concentrations (10-1000 ìg/L), inhibit indigenous protists; (4) similar changes in the microbial community structure are observed in a TCE-contaminated aquifer; and (5) similar distributions of protists are observed in other TCE-contaminated sites. The ultimate goal of the research is to develop strategies to avoid any adverse effects of predation on TCE bioremediation. The proposed research will be conducted using: (1) microcosms and molecular techniques; (2) batch feeding experiments; (3) classic protistan toxicity tests and continuous flow ex situ diffusion chambers. The project PI has helped to develop the primary techniques used to study aquifer predator-prey relationships that are used today. Bringing these resources to bear on the question of whether protistan predation of key bacterial species can disrupt in situ TCE bioremediation/bioaugmentation has the potential to greatly increase our understanding of groundwater ecosystem dynamics and suggest possible predator avoidance strategies to prevent stalling (e.g., surface association, change in cell surface characteristics of consortia to limit prey preference).
Broader Impacts The project will result in the training of one master?s student and will include two undergraduate assistants and up to ten undergraduates completing individual projects related to this research to fulfill their capstone senior project requirement. This project will also include up to four middle-school students who will learn how environmental engineering depends upon multidisciplinary teams of scientists (microbiologists, hydrologists, and engineers) complementing each others skills and approaches to experimental design, data analysis and problem solving. (The middle-schoolers will include girls and Hispanic and/or African-American students). The research will result in the development of a molecular database for TCE-contaminated groundwater aquifers that will include eukaryotes and prokaryotes. This will serve as the foundation for future community libraries for TCE contaminated aquifers, and will represent one of the first attempts to characterize the protistan community in TCE-contaminated aquifers using molecular techniques. The source of the indigenous microbes and contaminated sediments is Pease International Tradeport (Formerly Pease Air Force Base) in Portsmouth, NH, a fractured bedrock aquifer containing a TCE plume. TCE contamination of precious groundwater resources continues to be a problem in spite of development of chemical and biological amendments to stimulate in situ bioremediation. This proposal, building on recent findings regarding the impact of ubiquitous naturally-occurring protists in groundwater, aims to address a likely cause of stalling in some TCE contaminated aquifers.
Trichloroethene (TCE) was one of the most commonly used industrial solvents of the 20th century. As its use increased, so did the incidence of releases to the environment. As a result, TCE is one of the most common groundwater contaminants. In situ bioremediation has become a popular TCE cleanup option as interest in low-energy, sustainable methods increases. This method uses bacteria to degrade TCE into carbon dioxide (Figure 1). While other cleanup methods are able to reduce TCE levels, they do have limitations and can come with a large environmental cost in energy consumption or widespread changes underground. Other methods are more costly, labor intensive, and produce more waste. Bioremediation requires some energy inputs, but has a lesser impact on the subsurface ecosystem because it uses naturally-occurring microbes. In situ bioremediation cleans the soil and groundwater in place, without removing and disposing it. However, if TCE is not degraded to carbon dioxide compounds such as vinyl chloride (VC) that are more toxic than the TCE remain (Figure 1). Although this can occur for many reasons, it is often attributed to inadequate bacteria and biodegradable organic carbon. We hypothesized that some unexplained failures of TCE biodegradation may be due to undesired changes in the amount and type of bacteria as a result of predation by microorganisms called protists (Figure 2). Hence, protistan predation must be better understood, so strategies can be developed to boost the TCE degradation rate and success. This research was performed at the University of New Hampshire and a TCE-contaminated fractured bedrock site at the former Pease Air Force Base in Portsmouth, NH. The objectives were to evaluate the impact of predation on the amount and type of bacteria, the rate at which TCE is degraded, the toxicity of TCE and related compounds to protists, and the protists’ feeding rates and prey preferences. The research showed that when protists were removed, complete degradation was achieved in less than 45 days if an adequate amount of biodegradable organic carbon was added to the system. When protists were present, complete degradation required more than 160 days. This only occurred when the protists died, possibly due to the increased concentration of VC or a combination of TCE by-products. Protistan predation clearly has an impact on the bacterial community composition and the success and rate of biodegradation of TCE. This may explain the "hit or miss" nature of in situ TCE bioremediation. The key to success may be implementing methods that counteract protistan predation. This may be accomplished through control of the protists or continual introduction of the necessary bacteria to replace those that have been removed. Predation rates were significantly lower when the bacteria were able to stick to each other and form clumps that were too large for the protists to ingest. When underground, the bacteria may be able to adhere to surfaces or each other and avoid some predation, so the predation rate underground may be significantly lower than observed in the laboratory. Changes in the population of microbes present were monitored using metagenomics. Complex shifts were observed in the type of bacteria and protists present when predation occurred. This study funded partial salary of one full-time staff member to oversee the project, partial salary for two full-time professors, one full time master’s student, one part-time master’s student for two summers, and 10 part-time undergraduate students. Undergraduate student activities included performing toxicity studies and designing/constructing flow-through chambers. Three middle school and high school students met monthly with the graduate student and were trained in tasks that allowed them to understand the importance of the research, and gain experience and spark interest in environmental science. As a result of this grant, the University of New Hampshire purchased a Gas Chromatograph, which has been used to analyze over 3,000 samples. Staff and students have been trained on this instrument, and it is an important teaching tool for many Environmental Engineering undergraduates. This instrument has also been used to demonstrate key concepts in a Sampling and Analysis course. It provided many students’ first exposure to gas chromatography. The contamination at the Pease International Tradeport is of concern to the City of Portsmouth, NH. The city uses groundwater underlying the Tradeport as a potable water supply. We have provided outreach to the city and will develop fact sheets on bioremediation of TCE. Publication of work associated with this grant is ongoing. One manuscript has been drafted, with at least one more anticipated. As genomics data is generated, it is also being included in public databases used by the scientific community.
