Progress 11/28/01 to 09/30/04
Hazardous chemicals, such as polyaromatic hydrocarbons are persistent environmental contaminants because of their highly stable chemical structure and their adherence to solid particles in the environment. The role of bacterial species in decontamination of tropical soils was studied. Bacterial strains were isolated from Hawaiian soils in association with pyrene degradation. Strains were purified and then characterized by phenotypic and genotypic methods. Phenotypic methods included gram stain, spore stain, cellular morphology, colony morphology, Microlog substrate utilization profiling, API biochemical tests ZYM and Coryne, oxidation/fermentation tests, and motility tests. The genotypic methods used to characterize soil isolates included 16S rDNA sequencing, melt-profile analysis of 16S rDNA on denaturing gradient acrylamide gels, and PCR amplification of catabolic genes. As determined by 16S rDNA sequencing, the genera present in pyrene-degrading consortia included
Achromobacter, Alcaligenes, Arthrobacter, Bacillus, Frateuria, Microbacterium, Micrococcus, Mycobacterium, Ochrobactrum, Paenibacillus, Pseudoxanthamonas, Ralstonia, and Rhodanobacter. Using an overlay method developed during this study, all isolates were characterized with regard to pyrene degradation. All strains in this study able to use pyrene as a sole carbon source belonged to the genus Mycobacteria. Those strains able to degrade pyrene were tested for PAH-degradative activity of benzo(a)pyrene, chrysene, fluoranthene, fluorene, and phenanthrene. Several strains could degrade the smaller compounds (phenanthrene and fluoranthene), but compounds with higher molecular weight generally are more persistent and more difficult for bacteria to degrade. Chrysene, and fluorene were not degraded by any of the isolates associated with pyrene degradation. Benzo(a)pyrene was degraded only by one of the isolates. PAH degradation as determined by the plate assays has been confirmed by
degradation studies in liquid culture systems. Degradation studies in bioreactor systems showed variable results due to incomplete extraction of pyrene from soil. Thus, modeling studies under defined parameters of pH and nutrient levels were carried out in liquid culture rather than soil. The impasse for obtaining quantitative results for pyrene degradation in soil has been the physical separation of bacterial microflora (which require an aqueous phase) from toxic hydrocarbons, which are miscible in oil. For the first time, pyrene degradation can be quantitatively measured, while simultaneously measuring changes in the bacterial population. A novel biphasic system was developed and genomic methods were to characterize mixed populations during the degradation process. The significance of this work was presented in several public seminars to increase general awareness of environmental pollution and bioremediation as a potential solution to chemical contamination in the environment.
One of the most promising solutions to organic contamination involves bacterial degradation of pollutants and understanding the role of specific bacteria in decontaminating tropical soils is essential to this process. For the first time, pyrene degradation can be quantitatively measured, while simultaneously measuring changes in the bacterial population. A novel biphasic system was developed and genomic methods were to characterize mixed populations involved in the degradation process.
- No publications reported this period
Progress 10/01/02 to 09/30/03
The goal of this project has been to evaluate the microbial factors involved in bioremediation of toxic wastes in pyrene-contaminated soils. Bacterial species isolated from Hawaiian soils were shown to degrade pyrene under defined conditions that enable simultaneous growth measurements of populations. The current study focused on the isolation and characterization of pyrene-degrading bacteria from Hawaiian soils contaminated with polycyclic aromatic hydrocarbons (PAHs). Four isolation protocols were compared for their capacity to recover PAH-degrading bacteria. Groups of bacterial species isolated by each protocol were characterized by bacteriological tests, density gradient gel electrophoresis, and 16S rDNA sequence analysis. Bacteria were identified by comparing bacterial genome sequences with the national BLAST database. Methods A and B resulted in recovery of several species including Methylobacterium extorquens, other Methylobacterium sp., and Sphingomonas sp.
Method C resulted in no useful strains. Method D resulted in isolation of Mycobacterium sp., Mycobacterium sp. Achromobacter xylosoxidans, and a member of the Gamma superfamily of the class Proteobacteria. Bacterial strains were incorporated into soil bioreactors to determine their ability to degrade pyrene in soil, but no degradation of pyrene occurred in reactors amended with bacteria in groups A or B compared to controls. A two-phase liquid system was then designed to permit quantitative degradation of pyrene with simultaneous measurement of bacterial growth. Using the two-liquid phase (TLP) flasks pyrene removal was positively associated with growth of Group D strains. A Mycobacterium sp. was most effective in removing pyrene from the TLP cultures. The TLP system will permit further evaluations of biological and environmental factors that accelerate or retard pyrene degradation.
The impasse for obtaining quantitative results for pyrene degradation has been the physical separation of bacterial microflora (which require an aqueous phase) from toxic hydrocarbons, which are miscible in oil. For the first time, pyrene degradation can be quantitatively measured, while simultaneously measuring changes in the bacterial population. A novel biphasic system was developed and genomic methods were to characterize mixed populations during the degradation process.
- Hennessee, C. 2003. Isolation, diversity assessment, and characterization of pyrene utilization capacities of bacteria isolated from Hawaiian soils. M.S. Thesis. Fachhochschule Mannheim and University of Hawaii, Manoa. 96 pages.
Progress 10/01/01 to 09/30/02
Issue: Many areas worldwide are highly contaminated with organic compounds and heavy metals. The U.S. has over 1,200 'superfund' sites that require remediation, and Hawaii has about a dozen. Little is known about the role of bacteria in degradation of PAH's in tropical soils. What has been done? Bacterial candidates (180 strains) were isolated from local samples of Wahiawa soils and the roots of a perennial grass, Paspalum sp., which is a major candidate for phytoremediation of PAH's in tropical soils. All candidates were screened for use in bioremediation. Four bacterial species capable of pyrene degradation in laboratory and greenhouse studies were identified. Pyrene degradation rates were quantified and degradation products were analyzed.
The discovery of local strains of bacteria for bioremediation is valuable because such strains will not require State Import Permits for bioremediation of Hawaiian soils. As so little is little known about the ecology of these organisms and the mechanisms of degradation, this project is providing a solid basis upon which to develop model systems for bioremediation and reclamation of contaminated tropical soils.
- No publications reported this period