| NSF Org: |
CBET Division of Chemical, Bioengineering, Environmental, and Transport Systems |
| Recipient: |
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| Initial Amendment Date: | August 25, 2012 |
| Latest Amendment Date: | July 23, 2014 |
| Award Number: | 1233087 |
| Award Instrument: | Continuing Grant |
| Program Manager: |
William Cooper
CBET Division of Chemical, Bioengineering, Environmental, and Transport Systems ENG Directorate for Engineering |
| Start Date: | September 1, 2012 |
| End Date: | February 28, 2017 (Estimated) |
| Total Intended Award Amount: | $214,266.00 |
| Total Awarded Amount to Date: | $214,266.00 |
| Funds Obligated to Date: |
FY 2013 = $72,802.00 FY 2014 = $72,819.00 |
| History of Investigator: |
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| Recipient Sponsored Research Office: |
105 JESSUP HALL IOWA CITY IA US 52242-1316 (319)335-2123 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
2 Gilmore Hall Iowa City IA US 52242-1322 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): | EnvE-Environmental Engineering |
| Primary Program Source: |
01001314DB NSF RESEARCH & RELATED ACTIVIT 01001415DB NSF RESEARCH & RELATED ACTIVIT |
| Program Reference Code(s): |
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| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.041 |
ABSTRACT
1233087/1233154
Mattes/Cupples
Vinyl chloride (VC) is a known human carcinogen and a common groundwater contaminant. This research project focuses on identifying and characterizing the key microorganisms involved in VC bioremediation at field sites. VC plumes are generated under anaerobic conditions via reductive dechlorination of chlorinated solvents, but often escape into downgradient aerobic groundwater zones. Aerobic, growth-coupled VC-oxidizing (i.e. VC assimilating) bacteria are readily isolated from VC-contaminated sites, supporting the idea that they play a role in the degradation. However, direct evidence that VC-assimilating bacteria are present and active in situ remains elusive. Further, it is unclear if VC-assimilating isolates are the dominant microbes in the subsurface or if their cultivation is a result of enrichment bias. Also, VC-assimilators cannot be distinguished from the larger related group of ethene-oxidizers, which are fortuitous VC oxidizers. To address these knowledge gaps, this research will use stable isotope probing (SIP) to differentiate VC-assimilating bacteria from bacteria that fortuitously oxidize VC (i.e. the cometabolizers). SIP involves microbial assimilation of 13C labeled substrates into nucleic acids during growth-coupled biodegradation. Following this, 13C labeled nucleic acids are analyzed to identify the organisms responsible for contaminant degradation. The central hypothesis is that SIP techniques, when used in conjunction with existing molecular tools, will differentiate between etheneotrophs and VC-assimilators in both laboratory and field-based applications. The research involves the following specific aims: i) to use soil and/or groundwater samples from VC-contaminated sites andapply DNA-SIP to reveal the presence, identity, and activity of VC-assimilating microorganisms, ii) to use both VC-assimilating and cometabolizing laboratory cultures to develop a SIP-based assay that differentiates between these closely related microbial groups and iii) to provide direct evidence that VC-assimilating bacteria are present and active in situ.
Groundwater contamination by toxic pollutants is a major societal problem that threatens human health and the environment. In situ bioremediation practice hold promise for restoring groundwater to pre-contamination levels. Studies aimed at better understanding the presence, abundance and activity of biodegrading microbial communities will serve to bridge fundamental science and the practice of bioremediation. The long-term goal of this research is to advance the bioremediation field by developing molecular tools to characterize pollutant-degrading microorganisms in the environment. The research will involve the training of undergraduate and graduate environmental engineering students to apply molecular biology tools to environmental problems. The work will increase the number of underrepresented minority students and will also involve the development of K-12 outreach activities. The research will benefit society by addressing anthropogenic deterioration of water quality by groundwater pollutants.
PUBLICATIONS PRODUCED AS A RESULT OF THIS RESEARCH
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PROJECT OUTCOMES REPORT
Disclaimer
This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.
Vinyl chloride (VC) is a known human carcinogen and a common groundwater contaminant. Although anaerobic reductive dechlorination of VC to ethene is a predominant bioremediation strategy for VC, aerobic VC-oxidizing bacteria likely participate in complete mineralization of VC. The two major groups of VC-oxidizing bacteria are VC-assimilators, which use VC as a carbon and energy source, and VC-cometabolizers, which fortuitously oxidize VC. This research project focused on applying the stable isotope probing (SIP) technique to identify VC-assimilating bacteria in environmental samples and to distinguish VC-assimilators from VC-cometabolizers in mixed laboratory cultures. Additionally, the transition of VC-cometabolizers into VC-assimilators was investigated in this project in ethene-fed groundwater microcosms.
The four major activities in this project:
1) Conduct SIP analysis on two different VC enrichment cultures
2) Investigate diversity of etnE (a gene associated with aerobic VC biodegradation) in environmental samples, VC-assimilating isolates and VC and ethene enrichment cultures.
3) Investigate adaptation to VC in mixed ethene enrichment cultures developed from contaminated groundwater
4) Conduct SIP with a mixture of two pure cultures – Mycobacterium strain JS622 (VC cometabolizer) and Nocardioides strain JS614 (VC-assimilator).
We report the following outcomes in terms of intellectual merit:
Major activity 1. SIP analysis on two different VC enrichment cultures revealed potentially novel bacteria involved in VC uptake. This includes the genera Nocardioides, Brevundimonas, Tissierella, Rhodoferax, Sediminibacterium, Aquabacterium, Variovorax, and Pseudomonas. These studies have expanded targets for investigating aerobic VC degradation potential at contaminated sites.
Major activity 2. The epoxyalkane:coenzyme M transferase (EaCoMT) gene (etnE) plays a critical role in bacterial assimilation of short-chain alkenes, such as ethene and VC, thus an improved understanding of EaCoMT gene diversity and distribution is significant to the field of bioremediation. In major activity 2, we described the diversity of etnE in environmental samples and uncovered new relationships between etnE abundance and VC concentration in groundwater samples. Expanision and curation of the EaCoMT gene database will facilitate improved design of environmental molecular diagnostic tools and high-throughput sequencing approaches for future bioremediation studies.
Major activity 3. The main finding of this activity was that a plasmid identical to plasmids founds in VC-assimilating Nocardioides strain isolates was present in contaminated groundwater. This plasmid was enriched along with the bacteria growing on ethene and VC in mixed cultures. Although plasmids have previously been implicated in VC-assimilating bacteria, there has been little evidence of the role of a particular plasmid with a microbial community degrading VC. Thus, the identification of a plasmid in the mixed culture metagenomics data is an important result and we believe this plasmid holds the key to the ability for these mixed cultures to degrade VC. There is also an important evolutionary aspect to consider. In these cultures, the presence of the plasmid appeared to be directly related to the ability for microbes to use VC as a carbon and energy source.
Major activity 4. The major finding of this activity was that a defined mixed culture containing a VC-cometabolizer and a VC-assimilator did not behave as expected. The SIP approach in theory should be able to distinguish between a VC cometabolizer and a VC-assimilator, but it appears that there is a novel interaction between these two specific strains (JS622 and JS614) that allows JS622 to obtain carbon and energy from VC that it otherwise cannot do in isolation. This has important implications for this project and for VC biodegradation in the environment because it suggests that VC-assimilators are more prevalent in the environment that currently appreciated.
Broader impacts
Two female graduate students at the University of Iowa, and one underrepresented female graduate student at Michigan State University were trained to conduct microbial culturing and molecular biology experiments in support of this project. These students are currently employed in an environmental consulting firm, an environmental microbiology lab, and as a postdoctoral scholar, respectively. Therefore, this project has contributed to workforce development with students normally underrepresented in Science Technology Engineering and Math (STEM) fields. Three high school students have also participated in the project in the past three summers. Their experience also contributes to broadening participation in STEM fields.
Project results were disseminated at national and international conferences and published in peer-reviewed journals. Current publications resulting from this project:
Paes, F., X. Liu, T.E. Mattes, and A. Cupples, 2016. Nocardioides, Sediminibacterium, Aquabacterium, Variovorax and Pseudomonas linked to carbon uptake during aerobic vinyl chloride biodegradation. Environmental Science and Pollution Research (23: 19062 – 19070).
X. Liu, T.E. Mattes, 2016. Diversity and distribution of Epoxyalkane:Coenzyme M transferase genes in groundwater from chloroethene-contaminated sites. Applied and Environmental Microbiology (82: 3269-3279).
Paes, F., X. Liu, T.E. Mattes, and A. Cupples, 2015. Elucidating carbon uptake from vinyl chloride using stable isotope probing and Illumina sequencing. Applied Microbiology and Biotechnology. (99: 7735-7743).
Last Modified: 05/31/2017
Modified by: Timothy E Mattes
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