Source: UNIV OF HAWAII submitted to
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Nov 28, 2001
Project End Date
Sep 30, 2004
Grant Year
Project Director
Alvarez, A.
Recipient Organization
Performing Department
Non Technical Summary
Polycyclic aromatic hydrocarbons are priority pollutants and are classified as extremely hazardous substances by the United States Environmental Protection Agency due to their toxicity, mutagenicity and carcinogenicity. The goal is to understand the fundamental processes of bioremediation during the interactions between plants and microbial communities in tropical environments. The focus will be on pyrene degradation in Hawaiian soils.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Goals / Objectives
1) Determine which bacterial species are capable of biodegrading polycyclic aromatic hydrocarbons (PAHs) in Hawaiian soils. 2) Determine the PAH degradation rates and associate the DNA profiles of specific bacterial populations with respective degradation curves. 3) Determine parameters (cell density, temperature, moisture, soil pH and nutrient levels) for which PAH-degrading bacteria are most effective in bioreactor systems. 4) Model the interactions between microbial populations and environmental conditions to predict biodegradation rates of PAHs and reduce PAH contaminants to environmentally safe threshold levels. 5) Increase public understanding of environmental pollution and implications of bioremediation research.
Project Methods
1) Bacterial species capable of biodegrading PAHs will be isolated from soil and plant roots from ten PAH-contaminated sites in Hawaii. Bacteria will be tested for PAH catabolism using microplates. Strains capable of utilizing PAHs as sole carbon sources will be identifed using standard bacteriological tests, metabolic fingerprints, fatty acid analysis, 16S rDNA analysis and rep-PCR. 2) PAH-degradation rates will be determined and the DNA profiles of specific bacterial populations will be associated with respective degradation curves for Hawaiian oxisols and typic eutandepts to which pyrene has been added at appropriate levels. Population profiles will be related to periods of rapid decreases in pyrene concentration on the degradation curves and functionally dominant bacterial populations will be identified. Since conventional cultivation methods tend to skew our view of bacterial diversity towards culturable species, we will use 16S rRNA (rDNA) sequence amplification followed by denaturing gradient gel electrophoresis (DGGE) to profile both the culturable and non-culturable components of bacterial populations in the environment. Fluorometry will be used to follow the degradation of pyrene over time. 3) Parameters (cell density, temperature, moisture, soil pH and nutrient levels) for which pyrene-degrading bacteria are most effective will be examined in bioreactor systems. Selected strains capable of pyrene degradation will be inoculated into bioreactors on a pilot-scale, and the time and efficiency of pyrene degradation will be evaluated. Bacterial communities will be combined to favor complete metabolism and prevent metabolite accumulation and substrate inhibition effects. The bioreactors will be small-scale, aerobic, open systems. Each parameter will be examined as as a limiting factor of pyrene degradation. Correlations between factors will be evaluated to optimize the degrading activity of the selected bacteria and to develop cultivation strategies to supply sufficient biomass for pyrene-contaminated soil remediation. 4) Interactions between microbial populations and environmental conditions will be analyzed to determine biodegradation rates needed to reduce PAH contaminants to environmentally-safe levels. Regression equations subject to the assumptions and cautions inherent with such analyses will be used with direct and indirect methods for calculating microbial numbers. Microbial growth rates and pyrene-degradation rates will be related to the environmental conditions of temperature, pH, moisture, and nutrient concentrations, and secondary level models will be developed to predict the effects of different management procedures on microbial activity during pyrene degradation. 5) We will make an effort to increase public understanding of principles and benefits of bioremediation research through newspaper and magazine articles when appropriate. Extension of information to the public will be achieved through linkages with extension specialists in TPSS and PEPS departments.

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