Progress 09/01/04 to 08/31/08
Outputs
OUTPUTS: Primary outputs from this research were derived from laboratory research activities that have advanced fundamental knowledge biodegradation of organic pollutants. Over the course of this project, training and mentorship were provided for three Ph.D. students and one postdoctoral research associate. In addition, new collaborative research projects that were facilitated by this research included hosting of scientists from Pakistan, Korea, and China who have sent visiting students to my laboratory for periods of 6 months to 1 year. Significant research collaborations included Y.S. Kim (6 months) and a visiting faculty, Prof. Y.S. Chang (3 months) from Pohang University of Science and Technology, Korea, Dr. A. Khalid from the University of Agriculture, Faisalabad, Pakistan (1 year), and an upcoming student visit from China who will carry out new research on PAH degradation in my lab in 2009-2010. Products of this research include establishment of a memorandum of understanding between UCR and Zeijiang Academy of Agricultural Sciences in China which is facilitating continued cooperative research and scientific exchanges. Lastly, we have established a permanent poster display on petroleum degrading bacteria at the Page Museum of the Rancho La Brea Tarpits in Los Angeles. Specific events related to this research have included numerous invited seminars and lectures nationally and internationally. Major venues have included the following lectures: Keynote Address: Biodegradation of organic pollutants in the plant rhizosphere. International Conference: Rhizosphere 2004 - Perspectives and Challenges - A Tribute to Lorenz Hiltner, Munich, Germany. September 2004; Invited Presentation. Comprehensive analysis of organic pollutant degrading microbial populations in the plant rhizosphere. Federation of Korean Microbiological Societies. Seoul, Oct 2004; Oral Presentation. Identification of plants and plant derived substances that enhance biodegradation of polycyclic aromatic hydrocarbons in soils. 3rd Int. Conf. on Phytotechnologies. Atlanta, GA. April 2005; Oral Presentation. Comprehensive analysis of PAH degrading bacteria in the plant rhizosphere. USDA NRI Soil Biology Program PD Meeting. Newark, DE. Oct. 2005; Invited Presentation. Identification and biostimulation of PAH degrading bacteria in soils. 17th Ann Mtg of the Assoc. Environ. Health and Sciences. San Diego, CA March 19-22. 2007; Invited Talk. Bioremediation of PCB and PAH Contaminated Soils. Zeijiang Academy of Agricultural Sciences. Hangzhou, China Nov. 26, 2007; Invited Talk. Applications of Microbial Community Analysis for Evaluation of Soil Quality and Bioremediation. Nanjing Agricultural University, Nanjing China. Dec 1, 2007; Invited Talk. Applications of Novel Genes and Microorganisms from the Rancho La Brea Tarpits for Petroleum Biotechnology, Synthetic Genomics. San Diego, CA Dec. 6, 2007; and an Invited Lecture on Bioremediation of Organic Pollutants in Soils. Universidad de la Fronterra, Temuco, Chile Nov. 2008 PARTICIPANTS: James Haakrho Yi, Graduate student researcher Joong Wook Park, Graduate student researcher Ian Balcom, Graduate student researcher Young-Mo Kim, visiting graduate student research Jong-Shik Kim, postdoctoral research associate George C Page Museum - La Brea Tar Pits, Los Angeles, Azeem Khalid, visiting scientist University of Agriculture, Faisalabad, Pakistan. Zeijiang Academy of Sciences - partner organization for future projects on bioremediation. TARGET AUDIENCES: Environmental consultants, companies involved in bioremediation and environmental cleanup. Department of Energy and EPA researchers concerned with bioremediation and technology development for bioprocessing of oil hydrocarbons. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Phytoremediation is a commercially developed technology that is widely used for cleanup of PAH contaminated soil, but advances in this technology have been limited by lack of understanding of why certain plants are more effective than others, and what processes are taking place in the rhizosphere. This research has provided new insight into the particular chemicals that are responsible for stimulating the degradation of PAH. The results of this research point to specific chemical compounds, linoleic acid and sodium linoleate that can be used to treat PAH contaminated soils. The observation also leads to the possibility that those plant species containing high concentrations of these compounds can be used to cleanup PAH contaminated soils by using them as a type of compost or green manure that is incorporated into the soil. This should result in a significant reduction in cleanup cost for treatment of soils that are contaminated with these pollutants. Related studies that were synergistically supported by this research have also lead to identification of novel strains of bacteria that can be used for bioremediation of soils that are contaminated with oil or polybrominated diphenyl ethers, both of which are important environmental pollutants that are of concern for human health and environmental toxicology. Studies that were conducted at the Rancho La Brea tar pits in Los Angeles identified many novel asphalt-degrading bacteria that naturally thrive in heavy oil and possibly in the rhizosphere of turf grasses that grow in oil permeated soils. An exhibit reporting this research was established at the Page Museum in Hancock Park in Los Angeles, and serves as an educational exhibit for the thousands of school children and visitors who come to the museum each year. The Rancho La Brea discovery was also publicized in the Los Angeles Times and was picked up by national and international media. During the last year of this research, we extended our methods to examine azo dyes, which are similar aromatic ring containing pollutants that are common in water and soil in third world countries that have dye product industries. The long-term outcome of this research has been the development of more effective biotechnology to clean up oil, PAH, and other recalcitrant organic pollutants that have become widely dispersed in soils both in the United States and many other areas of the world.
Publications
- Khalid, A., M. Arshad, and D.E. Crowley. 2009. Biodegradation potential of pure and mixed bacterial cultures for removal of 4-nitroaniline from textile dye wastewater. Water Research. 43:1110-1116.
- Khalid, A. M. Arshad, and D.E. Crowley. 2008. Accelerated decolorization of structurally different azo dyes by newly isolated bacterial strains. Appl. Microbiol. Biotech. 78:361-369.
- Khalid, A., M. Arshad, and D.E. Crowley. 2008. Decolorization of azo dyes by Shewanella sp. under saline conditions. Appl. Microbiol. Biotech. 79:1053-1059.
- Balcom, I. and D.E. Crowley. 2009. Effects of pyrene on rhizoplane bacterial communities. Int. J. Phytoremediation. In Press.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: This research is examining the underlying mechanisms by which certain plant species promote the biodegradation of polycylic aromatic hydrocarbons (PAH) in contaminated soils. Possible ways that plants influence biodegradation have been hypothesized to include selective enrichment of PAH degrader species, growth-linked metabolism, production of co-substrates that induce cometabolism, and production of surfactants that increase PAH bioavailability to microorganisms. During this project year, we focused particularly on the identification of chemical substances in plant tissues that are responsible for stimulating the biodegradation of PAH by soil microorganisms. Other experiments were conducted to identify specific degrader organisms that respond to these plant substances, and to characterize the diversity of bacterial species that contribute to PAH biodegradation in the rhizosphere of different plants. Results of our research showed that the plant fatty acid, linoleic acid is
the most important chemical compound in the rhizosphere of plants that promote the biodegradation of the model PAH compounds, pyrene and benzo [a] pyrene. Addition of crushed plant tissues from species containing high concentrations of linoleic acid or culture of plants containing this chemical were both as effective as adding pure linoleic acid to the soil. Among plants that contain high amounts of linoleic acid are several tuberous plants such as carrot, radish, and celeriac. Addition of crushed root tissues of celeriac or cultivation of celery in PAH contaminated soil resulted in near complete removal of pyrene and benzyl [a] pyrene in 9 and 12 weeks respectively. Antibiotic inhibitor studies showed that gram positive bacteria, namely mycobacteria, were the predominant bacteria that responded to linoleic acid additions to soil. To further study the role of mycobacteria, we designed PCR primers for the nidA mycobacterial gene that encodes naphthalene inducible dioxygenases for PAH
degradation. PCR products derived from this primer set were then subjected to RFLP analysis to identify gene variants and to cluster similar groups of nidA like genes. Several hundred gene variants were identified, which could be clustered into 5 broad groups based on sequence analysis. Probes designed to discriminate each of these groups were designed for quantitative detection of gene copy numbers following soil amendment with linoleic acid. Additional studies examined culturable bacteria that are associated with the rhizosphere of 4 plant species that are used for phytoremediation. A wide range of pyrene degrading bacterial species representing diverse genera and divisions were shown to be present in the rhizosphere. The types of bacteria were dependent on the plant species with which they were associated, and also differed for soils having a history of exposure to PAH. The data suggest that the ability to degrade PAH is common among many taxa of bacteria, but that mycobacteria
play a pivotal role in PAH degradation.
PARTICIPANTS: James Haakrho Yi, Graduate student researcher Joong Wook Park, Graduate student researcher Ian Balcom, Graduate student researcher Young-Mo Kim, visiting graduate student research Jong-Shik Kim, postdoctoral research associate George C Page Museum - La Brea Tar Pits, Los Angeles
TARGET AUDIENCES: Environmental consultants, companies involved in bioremediation and environmental cleanup. Department of Energy and EPA researchers concerned with bioremediation and technology development for bioprocessing of oil hydrocarbons.
Impacts
Phytoremediation is a commercially developed technology that is widely used for cleanup of PAH contaminated soils. PAH are both mutagenic and carcinogenic are among the most common of all organic chemical pollutants in soils, including most superfund sites. Nonetheless, there has been little understanding of why certain plants are more effective than others, and what processes are taking place in the rhizosphere. This research provides new insight into the particular chemicals that are responsible for stimulating the degradation of PAH. The results of this research point to specific chemical compounds, linoleic acid and sodium linoleate that can now be used to treat PAH contaminated soils. The observation that these chemicals also occur in high concentrations in plants tissues also leads to the possibility that such plants can be used to cleanup PAH contaminated soils by using them as a type of compost or green manure that is incorporated into the soil. This should
result in a significant reduction in cleanup cost for treatment of soils that are contaminated with these pollutants. Related studies that were synergistically supported by this research have also lead to identification of novel strains of bacteria that can be used for bioremediation of soils that are contaminated with oil or polybrominated diphenyl ethers, both of which are important environmental pollutants that are of concern for human health and environmental toxicology. In particular studies that were conducted at the Rancho La Brea tar pits in Los Angeles identified many novel asphalt-degrading bacteria that naturally thrive in heavy oil and possibly in the rhizosphere of turf grasses that grow in oil permeated soils. An exhibit reporting this research was established at the Page Museum in Hancock Park in Los Angeles, and serves as an educational exhibit for the thousands of school children and visitors who come to the museum each year. The Rancho La Brea discovery was also
publicized in the Los Angeles Times and was picked up by national and international media. The long-term outcome of this research is the development of more effective biotechnology to clean up oil, PAH, and other recalcitrant organic pollutants that have become widely dispersed in soils both in the United States and many other areas of the world.
Publications
- Yi, H. and D.E. Crowley, 2007. Biostimulation of PAH degradation with plants containing high concentrations of linoleic acid. Environmental Science and Technology 41:4382-4388.
- Kim, J.S. and D.E. Crowley. 2007. Microbial diversity in natural asphalts of the Rancho La Brea tarpits. Applied and Environmental Microbiology 73:4579-4591.
- Kim, Y.-M. I.-H. Nam, K Murugesan, S. Schmidt, D.E. Crowley, and Y.S. Chang. 2007. Biodegradation of diphenyl ether and transformation of selected brominated congeners by Sphingomonas sp. PH-07. Applied Microbiology and Biotechnology 77:187-194.
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Progress 01/01/06 to 12/31/06
Outputs
Prior research reported last year had shown that biodegradation of high molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) by mycobacteria can be stimulated by amendment of soil with plant tissues containing high amounts of linoleic acid. Research conducted here identified the specific bacterial taxa that respond to the linoleic acid, and characterized gene expression patterns for nidA-related dioxygenase genes after augmentation of PAH contaminated soils with plant tissues or pure linoleic acid. Results of antibiotic inhibitor studies showed that mycobacteria were the predominant HMW-PAH degraders were corroborated by DNA expression profiles for mycobacterial dioxygenase genes. To investigate nid-A gene diversity, a degenerate primer set was designed and used to construct clone libraries from 6 soils. A total of 51 RFLP patterns were observed from 476 clones, indicating a high diversity of nid-A related genes. The 51 gene groups were then further sorted
into five major groups for which specific probes were designed. Using Northern hybridization and real-time PCR methods, RNA expression for three of the five groups was shown to be upregulated in a similar fashion by addition of either sodium linoleate or celery root crushates. The results suggest that linoleic acid and plant tissues containing high amounts of linoleic acid both stimulate mycobacterial populations, but that there are differences at the subpopulation level in which groups carrying different variants of the dioxygenase gene respond to linoleic acid and plant tissues containing high amounts of this biostimulant. The development of degenerate primers for nidA genes and identification of mycobacteria as a prominent group responding to linoleic acid provides insight into the mechanisms by which plants may enhance PAH degradation, and tools for monitoring HMW-PAH degrader activity in soils.
Impacts
PAHs are ubiquitous soil pollutants that are especially problematic in the ca 400,000 contaminanted brownfield sites across the US. This research is examining the underlying mechanisms by which certain plants are able to promote the degradation of PAHs by production of phytochemicals that enhance the biodegradation of PAHs by root associated bacteria.
Publications
- Park, J-W and D.E. Crowley. 2006. Dynamic changes in nahAc gene copy numbers during degradation of naphthalene in PAH-contaminated soils. Appl. Microbiol. Biotechnol. 17: 1322-1329.
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Progress 01/01/05 to 12/31/05
Outputs
The rhizosphere microbial community structure of plants grown in polycyclic aromatic hydrocarbon (PAH) contaminated soil from the March Air Reserve Base and EPA Superfund site was examined through molecular and enrichment culture methods to elucidate plant-promoted PAH biodegradation. Phylogenetic characterizations of the plant root associated microbial communities were achieved through direct extraction of nucleic acids from rhizosphere soil. Initial comparisons utilized low resolution profiling of community similarities using PCR-DGGE of 16S rRNA. The identities of bacteria associated with predominant DNA bands were then determined by DNA sequencing and comparison to the ribosomal DNA database at GenBank. Based on the database matches, the dominant DGGE bands had a high sequence similarity to species of Sphingomonas, Pseudomonas, Bacillus, Brevibacillus, Ochrobactrum, Ensifer, Rhodospirillaceae, and many strains of uncultured bacteria that have been isolated by
others from contaminated environments such as oil-contaminated soil. In complement to the molecular studies, a culture-based root fingerprinting technique was used to isolate high molecular weight (HMW) PAH-degrading microbial species using pyrene as a sole carbon substrate. Roots from plants grown in PAH contaminated soil were pressed on carbon-free minimal salts agar plates. A layer of pyrene crystals was deposited on the surface of the plates by sublimation. Colonies that were capable of using pyrene as a carbon source (indicated by clearing zones) were picked for phylogenetic analysis. Further characterization of individual members of the pyrene-degrading consortia that were obtained after transfer to alternative media were not able to grow on pyrene as pure cultures, indicating that degradation was facilitated only by mixtures of bacterial species in consortia. The results suggest an enriching-effect of the plant root for HMW PAH-degrading soil microbes in aged contaminated soil.
As bioaugmentation of contaminated soils for enhanced biodegradation is often limited by low activity and survival of the added culture, harnessing the priming effect and apparent selectivity of certain plant rhizospheres for PAH biodegrading consortia may a primary mechanism by which some plants enhance biodegradation rates of HMW PAHs in soil.
Impacts
This research is providing fundamental information on the mechanism by which certain plants promote the biodegradation of polycyclic aromatic hydrocarbons in contaminated soils. This information will be used to develop criteria for selection of plants that can be used in phytoremediation of one of the most important and widespread chemical contaminants that occur in soils from oil contamination, fire, and industrial activity.
Publications
- Crowley, D.E., H. Yi, J-W. Park, E.S. Gilbert, A. Singer, E. Luepromchai, K. Haathuc, I. Balcom. 2005. Biodegradation of organic pollutants in the plant rhizosphere. Proceedings of the Hiltner Centenial International Conference on the Plant Rhizosphere. Munich. Germany. Sept. 2004. pp 213-214.
- Park. J.W., and D.E. Crowley. 2005. Normalization of DNA extraction for accurate quantification of target genes by real-time PCR and DGGE. Biotechniques. 38:579-586.
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Progress 01/01/04 to 12/31/04
Outputs
Experiments have been initiated to identify and enumerate PAH degrading bacteria in the rhizosphere of different plant species in soils having a history of contamination with PAHs and in similar soils with no contamination. Other experiments are examining the role of specific substances contained in root exudates that may influence the bioavailability of PAHs to degrader organisms or that may selectively enrich for PAH degrading bacteria.
Impacts
This research is providing fundamental information on the mechanism by which certain plants promote the biodegradation of polycyclic aromatic hydrocarbons in contaminated soils. This information will be used to develop criteria for selection of plants that can be used in phytoremediation of one of the most important and widespread chemical contaminants that occur in soils from oil contamination, fire, and industrial activity.
Publications
- No publications reported this period
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