The UC SBRP is excited to announce a new book by one of our researchers:

Environmental Microbiology
Principles and Applications
Patrick K. Jjemba
Biological Sciences Department, University of Cincinnati, Cincinnati, Ohio, USA
ISBN 1-57808-348-6; July 2004; c. 376 pages, paperback
This book was written for an audience that has a basic understanding of microbiology. Often microbiologists tend to overzealously focus on bacteria, inadvertently ignoring other microbes (i.e., algae, fungi, protozoa, and viruses). This discrepancy is redressed herein. Read the complete review, and order here!

Research Goals: To study the structure and activity of bacteria that degrade PAHs in soil.

Overview: Microorganisms are important in the degradation of organic pollutants in the environment. The degrading microbial communities have traditionally been described by isolation and culturing. However, only a small proportion of bacterial species in the natural environment are culturable. Thus, the activity of most potential degraders that have not been cultivated still remains unknown. Under this project, the identity of bacteria that degrade PAHs in soil is being determined using culture-independent techniques.

Progress to date: Soil was obtained from a site west of Cincinnati that has a long history of polycyclic aromatic hydrocarbon (PAH) contamination. Total bacteria were extracted from the contaminated soil and the bacteria that could utilize added pyrene as a growth substrate were quantified. The soil mixture also contained nalidixic acid, piromidic acid or ciprofloxacin. These three antibiotics inhibit DNA gyrase, the enzyme that is responsible for the coiling and relaxation of DNA. Once DNA replication is inhibited, the active bacterial cells cannot divide, ending up in distinctively elongated forms. Thus, by providing pyrene as a carbon source, in the presence of these antibiotics, the response to the added pyrene is determined by counting morphologically elongated cells (Table 1). This approach is widely referred to as Substrate-Responsive Direct Viable Counting (SR-DVC).

Table 1: The response of the indigenous soil microbial population to 0.5 mg pyrene/L after 24 and 53 hours in a PAH-contaminated soil.

Cell elongation, which is used as an indication of growth response to the substrate, depended on the concentration of the inhibitors and the duration for which the cells are exposed to the inhibitor. With ciprofloxacin as the inhibitor, the elongation of most cells is delayed up to 53 h.

We have combined SR-DVC and fluorescent in situ hybridization (FISH) with a probe (Eub338) that specifically targets most bacteria, to probe the cells and confirm that they are bacteria and not some other microorganism. Probes such as Eub338 are commercially synthesized single strands of nucleic acids that are attached to a fluorescent dye and have sequences that are specific to complementary highly conserved ribosomal RNA (rRNA) sequences within intact microbial cells. This approach links phylogenetic information with physiological function in situ without the conventional cultivation of bacteria. We are interested in knowing who these bacteria are and are currently using more specific probes to identify them further. Nucleic acid probes that complement rRNA have been developed for a wide range of microorganisms, including those that have never been cultivated.

Application of Knowledge: Once known, management strategies that enable these organisms to thrive abundantly in contaminated sites can be developed as to enhance bioremediation efforts. The approach that we are using can also be coupled with more innovative methods to enhance the growth of previously non-cultivated PAH degraders and further stimulate their metabolic potential. This approach will also help us understand the genetic structure and activity of microbial populations found in PAH-contaminated soils and sediments and how they interact with each other to transform these contaminants in the environment. The techniques used in this study are applicable to a variety of environments such as biofilms and groundwater.