Evaluation of two carbon sources and Pseudomonas putida F1 for use in the bioremediation of chromate: A whole soil microbial community approach

Stephanie Thieman, Purdue University

Abstract

Chromate [Cr(VI)] is a highly soluble, toxic heavy metal, posing a wide range of environmental clean-up obstacles. Reduction of Cr(VI) to the less soluble, less toxic Cr(III) state via microorganisms has shown potential as a cost-effective, non-invasive remediation procedure. This study examines the effects of chronic chromium exposure on native microbial populations and shifts in community dynamics when amended with combinations of substrate, Cr(VI), and a known chromate reducer (Pseudomonas putida F1). Chromate contaminated soil from the Department of Energy’s Hanford site in Washington State was used to construct benchtop microcosms to study the effect of the addition of various carbon sources, additional chromate, and P. putida F1, a bacterium that we have determined to be an excellent chromate reducer, on the autochthonous microbial community. Triplicate microcosms amended with glucose, lactate, Cr(VI), or P. putida F1, as well as combinations of these, were incubated for either 36 or 63 days during which time the evolved CO2 and remaining Cr(VI) levels were periodically measured in order to assess the physiological response of the community in terms of aerobic respiration and chromate reduction. In the case of microcosms incubated 36 days, one microcosm from each treatment was sacrificed, and a subset of these was chosen for molecular phylogenetic analysis. The respiration data indicated that in the absence of exogenous P. putida F1, respiration levels were highest for lactate-ammended microcosms, but respiration rates were nearly identical on lactate and glucose after 2 weeks incubation. Addition of P. putida F1 to lactate- or glucose-treated microcosms yielded higher respiration levels with lactate (3.5 x 10-2 mmole versus 1.2 x 10-2 mmole total CO2; all values based on 50 g soil/microcosm and standardized to milligrams carbon added); however, respiration rates on lactate slowed while they increased on glucose, narrowing the gap between the two curves (CO 2 levels of 6.6 x 10-2 mmole on glucose, as compared to 8.3 x 10-2 mmole on lactate). Phylogenetic analysis of microcosm microbial communities revealed treatment-specific differences in clustering patterns. Chromate reduction in P. putida F1 pure culture revealed a lactate phenomenon where rapid reduction occurs in the presence of lactate, even when cells are absent, followed by extensive fluctuations in Cr(VI) levels. Combined, the pure culture work and microcosm soil respiration and reduction analysis present a hierarchy of carbon amendments, where glucose is far superior to lactate in terms of ability to support growth, stabilize reduction, and immobilize Cr(III) in a chromate contaminated system.

Degree

M.S.

Advisors

Applegate, Purdue University.

Subject Area

Microbiology|Environmental engineering

COinS