Chromium Tolerant Microbial Communities from the Chesapeake Bay Watershed

Chromium tolerant bacteria were enumerated from portions of the Chesapeake Bay watershed and examined for their potential to reduce Cr(VI). Water and sediment samples were collected from various locations in Baltimore Harbor and Bear Creek, as well as Sandy Point State Park in Maryland and the Anacostia River in Washington, DC. Samples were spread onto agar plates with Cr042-(5 ppm) as the sole terminal electron acceptor. Plates were incubated anaerobically and colony forming units (CFU) enumerated. CFU arising on minimal-Cr042medium ranged from 10 -10 mC or f 1 and community estimates from sites in proximity to Baltimore City were approximately 6-30X higher than distal sites. Bacterial identification by BIO LOGTM or l 68 rRNA sequencing indicated the presence of bacteria of the genera Klebsiella, Pseudomonas, Burkholderia, Kluyvera and others. Typical Cr(VI) reduction rates by these isolates were significantly lower than Shewanella oneidensis, a known metal-reducing bacterium. Results suggested that microbial communities in the Chesapeake Bay watershed, particularly in Baltimore Harbor and Bear Creek, had a high tolerance for Cr(VI) and/or could grow slowly with Cr(VI) as a terminal electron acceptor. However, the isolates did not rapidly degrade Cr(VI) in the laboratory. INTRODUCTION The Chesapeake Bay is the largest estuary in the U.S. and is fed by a broad watershed that includes six states (New York, Pennsylvania, Delaware, Maryland, Virginia and West Virginia) and the District of Columbia, encompassing an area of approximately 12,000 km (Pritchard and Schubel, 2001). Forests, cultivated and abandoned agricultural land, wetlands and residential areas surround the Bay and its adjacent watershed. It is home to a wide range of aquatic wildlife and has regional economic importance in the fishery and shipping industries (Lippson and Lippson, 1997). In addition, the Chesapeake Bay is ·a popular site for recreational boaters and tourists. * To whom correspondence should be addressed. E-mail: jonesmee@cc(nrl.navy.mil; Telephone: (202)404-6361; Fax: (202)404-8515 142 VIRGINIA JOURNAL OF SCIENCE As a result of past and recent human activities, pollutants and other contaminants (i.e., pesticides, herbicides, organophosphates, polychlorinated biphenyls [PCBs], petroleum products and heavy metals) have accumulated in the Bay (Lynch, 2001). Pollutants reach the Bay through river drainage, runoff and direct discharge ( Curtin, 2001 ). One of the more problematic contaminants is chromium, which was mined north of the Bay in the 19th and 20th centuries. Chromium is an important industrial metal used in the manufacture of many diverse products, including ferrous and nonferrous alloys, paints, pigments, wood preservatives and corrosion inhibitors (Fendorf et al., 2000). Such manufacturing industries have operated in and around the Bay region during the past two centuries. Chromium is a redox active transition metal with a wide range of possible oxidation states, although, only two (+6 and +3) are stable in the environment. It is a widespread contaminant in the environment and is recognized as a toxic substance and carcinogen (Kimbrough et al., 1999). Cr(VI) is highly water soluble and is easily transported through aquatic environments. In contrast, Cr(III) is much less soluble and precipitates as a hydroxide above pH 5.5. Due to its lower solubility, Cr(III) is considered less toxic and is, in fact, a necessary micronutrient for humans and other animals (Hamilton and Wetterhahn, 1987). Chromium tolerance may occur by several potential mechanisms including plasmid-encoded resistance, transport mechanisms and reduction (Wang, 2000; Cervantes et al., 2001 ). Reduction of soluble (more toxic) Cr(VI) to less soluble (less toxic) Cr(III) is influenced by several factors ( e.g., pH, temperature 2 redox potential) and can be mediated by various chemical species (i.e., Fe(II), S ), some plants and several microorganisms (Fendorf et al., 2000; Lytle et al., 1998; Wang, 2000). A metal-reducing microorganism, Shewane/la oneidensis, has been shown in laboratory experiments to reduce Cr(VI) at high rates (Daulton et. al., 2001 ). Thus, one potential strategy for environmental Cr(VI) removal would be the addition of S. oneidensis into contaminated sites. However, it is not known at this time whether S. oneidensis can compete with native microflora at Cr(VI) contaminated sites. Therefore, a possible remediation plan would be to stimulate naturally-occurring Cr-tolerant and Cr(VI)-reducing bacteria (CRB) in contaminated environments by fertilization or other environmental manipulation. Alternatively, wastewater treatment schemes could be developed using naturally-occurring CRB in bioreactor systems. In situ, naturally-occurring CRB may have Cr(VI) reduction capabilities superior to those of S. oneidensis. To assess the feasibility of such bioremediation strategies, we evaluated the prevalence of Cr-tolerant and other bacterial communities in the Chesapeake Bay watershed, which includes regions previously shown to contain high levels of contaminants including chromium (Baker et. al., 199,7). Environmental isolates were identified and tested for their ability to reduce Cr(VI) in the laboratory.