Microflora Involved in Phytoremediation of Polyaromatic Hydrocarbons

Description:

RESEARCH GOAL:

WORK COMPLETED

1. Information from GenBank on the internet was used to design several new primers to be tested in PCR studies. Sets of primers were designed for both specific replication of selected genes and others for universal replication of conserved regions of DNA associated with dioxygenases reported in the literature for several different polyaromatic compounds, including both PAHs and PCBs.
2. Fast DNAŽ SPIN Kits purchased from Q-BIOgene company for extraction and purification of DNA from soil samples were tested in comparison to various published methods that had been used previously in this laboratory.
3. Considerable time was devoted in repeated efforts to develop a manual means to conduct quantitative replication of nah genes using equipment currently available in the laboratory. This effort involved repeated sampling from PCR reacation tubes during the course of 30-cycle PCR runs followed by DNA separation and staining with conventional methods.
4. Bacterial counts made with the most probable number method on microtitor plates were compared to those made with conventional plating techniques in parallel experiments conducted on the same soil extracts.
5. A survey was made of equipment available to perform quantitative PCR measurements known as "real-time PCR." Sales representatives from Fisher Scientific meet with investigators from the Univ. of Oklahoma to describe the operation and capabilities of the "Smart Cycler", a real-time PCR instrument that is new on the market since May 2000. This instrument is approximately 1/3 the cost of earlier real-time PCR equipment, but has all the features necessary to use PCR methods to quantify genetic information in environmental/soil samples.
6. An equipment proposal requesting partial funding from the Univ. of Oklahoma to purchase the Smart Cycler instrument was prepared and submitted. These funds in conjunction with funds already set aside in the IPEC grant for PCR equipment will be used to purchase this state of the art piece of equipment.

RESULTS

1. The DNA prepared with Fast DNAŽ SPIN Kits was demonstrated in PCR analyses to be of superior quality to DNA prepared with other methods.
2. Using a very primitive manual method to conduct real-time PCR analyses it was shown that 17 cycles were necessary to detect a product from mulberry root zone DNA extracts whereas 30 cycles were necessary for Bermuda grass samples. Although it is impossible with this approach and available equipment to estimate the amount/number of zero-time nah genes in the samples, it is possible to conclude that the mulberry soil samples possessed a much higher level than the Bermuda grass, a result that is consistent with the counts of PAH-degrading bacteria recovered from these same samples.
3. The most probable number method was shown to be a fast and accurate method of determining total microbial counts, but quantification of low numbers of degrading bacteria was problematic because of unexpected growth in control wells where no substrate was provided.

SIGNIFICANCE

Major strides have been made in developing methods to use real-time PCR to quantify the enzymatic potential of the rhizosphere of different plant species to degrade polyaromatic PAH and PCB compounds. Successful purchase and use of the Smart Cycler will permit this IPEC sponsored research to be at the forefront of rhizosphere research with implementation of state of the art equipment and ideas.

PUBLIC COMMUNICATION OF RESULTS

Abstracts submitted for meetings

1. The plant root, a natural injector system to stimulate microbial degradation of recalcitrant soil contaminants. ISEB 2001 Phytoremediation meeting, Leipzig, Germany, May 15-16, 2001.
2. Rhizosphere remediation of organic soil contaminants. NATO Workshop, Prague, Czech Republic, May 24-30, 2001.
3. Vegetation and fungi at Czech PCB-contaminated sites as bioremediation candidates. Battelle Conference, San Diego, June 4-7, 2001.
4. Remediation of recalcitrant soil contaminants through the action of nature's injection system, the root. Organic Soil Contaminants Conference, Copenhagen, Denmark, Sept. 2-5, 2001.

Three of the above presentations were invited.