Grantee Research Project Results

1999 Progress Report: Phylogenetic Analysis of Microbial Communities in Contaminated Nearshore Marine Sediments

EPA Grant Number: R826108
Title: Phylogenetic Analysis of Microbial Communities in Contaminated Nearshore Marine Sediments
Investigators: Herwig, Russell P.
Institution: University of Washington
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 1997 through August 31, 2000 (Extended to September 30, 2001)
Project Period Covered by this Report: September 1, 1998 through August 31, 1999
Project Amount: $370,446
RFA: Exploratory Research - Environmental Biology (1997) RFA Text |  Recipients Lists
Research Category: Aquatic Ecosystems , Biology/Life Sciences

Objective:

Little is known about the composition of microbial communities that live in contaminated nearshore marine sediments and how the composition in contaminated sediments compares to that found in clean reference sites. Environmental pollutants not only impact the composition and health of animals and plants that live in or on marine sediments, but also may influence the composition of the microbial communities. In many areas of the United States, urbanized or industrialized estuaries and marine sites are contaminated with polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and heavy metals. Because microorganisms that live in sediments are largely responsible for the transformation and bioremediation of environmental pollutants and play a significant role in geochemical cycling, it is important to understand the composition of the microorganisms that live in contaminated sites. Our laboratory has developed molecular protocols for the analysis of the microbial communities in marine sediments. With support from the U.S. Environmental Protection Agency (EPA), we continue to develop our protocols and examine the microbial composition of: (1) intertidal and subtidal marine sediments contaminated with creosote, a contaminant composed largely of PAHs; and (2) clean intertidal and subtidal reference marine sites. Samples for our investigation were retrieved from Eagle Harbor and Blakely Harbor, WA. Both of these harbors are located on the western shores of Puget Sound. Eagle Harbor is the site of a former wood treatment facility that was the source of creosote found in the nearby marine sediments. The primary objective of our research is to develop and test protocols for performing "large-scale" analyses on the composition of the microbial communities in nearshore marine sediments from Puget Sound and other marine sites around the United States using 16S rRNA phylogenetic techniques.

Progress Summary:

Determining the composition of the microorganisms that live in contaminated areas traditionally has been performed using broth or solid bacteriological media that supports the growth of microorganisms. Unfortunately, only a very small fraction of the microorganisms that exist in an environmental sample can be cultivated using such methods. To avoid the requirement to culture or grow microorganisms, scientists have attempted to identify the microbial species that are present in a sample by extracting and characterizing distinctive fragments of DNA, such as the genes for the 16S ribosomal RNA (16S rDNA). The 16S rDNA can be used to identify different species of bacteria and to explore the evolutionary or phylogenetic relationship between diverse species of organisms. During this past year, we successfully developed the required steps of our protocols that will enable us to perform the analysis of microbial communities in contaminated marine sediments without the necessity to culture the microorganisms. The steps of the protocol that we developed and evaluated include: (1) high-yield extraction and purification of high-molecular weight microbial DNA; (2) 16S rDNA cloning and high-throughput plasmid preparations using 96-well microtiter plate configurations; (3) efficient large-scale screening of clone libraries using restriction fragment length polymorphism (RFLP) analysis; (4) selection of representative 16S rDNA clones by performing a cluster analysis using Gelcompar II software; (5) partial sequencing of 16S rDNA in representative clones using an ABI 377 Sequencer; and (6) sequence analysis and phylogenetic analysis of representative 16S rDNA clones. These protocols allow us to examine the composition of microbial communities in marine sediments based on the phylogeny of the microbial 16S rRNA that is present in a sample.

Different steps of a molecular protocol may introduce significant biases that need to be understood when interpreting results. We are developing a method to examine biases that may be introduced during the polymerase chain reaction (PCR) amplification step. This step follows the DNA extraction step. Biases may be introduced by the differential PCR amplification of DNA from heterogeneous target DNA. To explore these concerns, we are examining the terminal restriction fragment length polymorphisms (T-RFLP) of the PCR products of the amplified microbial 16S rDNA present in marine sediments. T-RFLP was previously described by Liu, et al., 1997. Briefly, in this technique one of the PCR primers is labeled with a fluorescent dye, resulting in one of the ends of the PCR product to be labeled. The PCR product is digested with restriction enzymes. The resulting digest is analyzed on an automated DNA sequencer that separates all of the DNA fragments, but only the terminal restriction fragments are detected and sized. This protocol permits the researcher to distinguish individual species, represented by specifically sized terminal DNA fragments, within a complex microbial community. Our goal is to minimize the amount of bias introduced during the PCR of the 16S rDNA present in an environmental sample.

Once completed, our research will provide a protocol to other microbial ecologists for the large-scale analysis of composition of microbial communities and yield valuable information about the microbial communities present in contaminated marine sediments. For years, environmental biologists have considered the impact of a variety of environmental pollutants on the composition of animal and plant communities. Using the molecular tools developed in our investigation, microbial ecologists will have a more thorough understanding about the composition of microbial communities in marine sediments, and therefore, will be able to examine microbial communities in a manner similar to that used by animal and plant ecologists.

Future Activities:

Our objectives for the coming year will be to complete experiments involving PCR amplification biases and determine the PCR parameters that will allow for a minimum amount of bias to be introduced into our protocol. Once this is complete, our goal will be to apply our developed protocols to characterize the microbial composition of creosote-contaminated intertidal and subtidal marine sediments from Eagle Harbor, WA.

Supplemental Keywords:

sediments, marine, estuary, polycyclic aromatic hydrocarbons, PAHs, PNAs, bacteria, bioremediation, biology, monitoring, Pacific Coast, Pacific Northwest, Washington, WA, EPA Region 10., Scientific Discipline, Geographic Area, Waste, Water, Toxics, Ecological Risk Assessment, State, Biology, HAPS, Contaminated Sediments, Environmental Microbiology, Ecology and Ecosystems, contaminant transport, microbial pollution, contaminated sediment, marine ecosytems, Washington (WA), contaminated marine sediment, ecology assessment models, creosote, aquatic ecosystems, indicator species

Relevant Websites:

http://faculty.washington.edu/herwig/

Progress and Final Reports:

Original Abstract

1998

2000 Progress Report

2001

Final