Physiological Attributes of Microbial BTEX Degradation in Oxygen-Limited Environments

Abstract

Our work has focused on the determination of physiological traits that may facilitate in situ degradation of xenobiotic compounds by indigenous microorganisms. For this our interests center on the following questions: What are the ambient conditions in a benzene, toluene, ethylbenzene, and xylene (BTEX)-contaminated aquifer? What is the behavior of indigenous bacteria under these conditions? What are the attributes of bacterial strains that are functional under hypoxic conditions? How do these strains compare with other BTEX-degrading strains? -Environ Health Perspect 103 (Suppl 59:4-51 (1995) Key words: biodegradation, hypoxia, Pseudomonas picketti, benzene, toluene, ethylbenzene, xylenes, groundwater, nitrate, oxygen Aquifer CharacteristicsFor several years we sampled three sites in which groundwater was contaminated by petroleum products, including benzene, toluene, ethylbenzene, and xylene (BTEX) components (1,2). All three of these contaminated aquifers were characterized by their low dissolved oxygen levels in the contaminated regions (-1-2 mg/l ), in contrast to uncontaminated adjacent regions in the aquifer (-3-4 mg/1). The occurrence of dissolved nitrate also showed a corresponding relationship when the contaminated and uncontaminated regions were compared. The nitrate concentrations were low or undetectable in the contaminated regions, but were higher in the uncontaminated regions. These relationships were observed for all three aquifers, leading to the conclusion that oxygen growth on BTEX were observed when cores of aquifer material that had been sampled vertically were tested for microbial activity as a function of depth and location within the plume regions. Bacterial strains growing on BTEX aerobically were observed for all samples independent of depth or BTEX concentration. However, when such samples were incubated under hypoxic conditions (i.e., < 2 mg/l dissolved oxygen), bacterial growth and denitrification matched BTEX degradation observed for microcosm cultures inoculated with bacteria eluted from the soil core samples. Therefore, differences were observed between bacteria that would degrade BTEX under hypoxic-denitrifying conditions and those that grow on BTEX under aerobic conditions. Diversity of Bacteria from Hypoxic EnvironmentsSamples from the BTEX-contaminated aquifers were incubated in mineral salts buffer overnight to elute bacteria from particles. These eluates were then plated on solid mineral salts medium followed by incubation aerobically or under hypoxic conditions in the presence of BTEX vapors. Aerobic cultures were incubated 48 hr and hypoxic cultures were incubated 1 week. Colonies were replated onto the same medium and incubated as before. These cultures then became the source for subsequent tests for the mineralization of individual BTEX components under both aerobic and hypoxic conditions. For this work, p-xylene was used as a representative xylene. The colonies selected for such tests were selected on the basis of their distinctive morphologies and colony size to fa...

Dynamics of an Oligotrophic Bacterial Aquifer Community during Contact with a Groundwater Plume Contaminated with Benzene, Toluene, Ethylbenzene, and Xylenes: an In Situ Mesocosm Study

“…Oxygen inhibits the activity of anaerobes but is typically only available at the fringe of the contaminant plume (28). Rapid consumption of the incoming oxygen possibly occurs, sustaining aerobic BTEX degradation (32,33). Furthermore, the concentration of nitrate was low at the contaminated area but high at the uncontaminated well, indicating that the rapid consumption of incoming nitrate occurred in the contaminated area, which might have sustained the oxygen-requiring but nitrateenhanced degradation of BTEX under hypoxic conditions, i.e., conditions with low concentrations of dissolved oxygen, as recently described for Ralstonia pickettii PKO1 and other aerobic BTEX degraders (25).…”

Characterization and Role of tbuX in Utilization of Toluene by Ralstonia pickettii PKO1

“…In addition, the expression of tbuX appears to be regulated in a manner such that low levels of TbuX are always present within the cell, whereas upon toluene exposure these levels dramatically increase, even more than those of toluene-3-monooxygenase. This expression pattern may relate to the possible role of TbuX as a facilitator of toluene entry into the cell.Ralstonia pickettii PKO1 has been investigated by our laboratory for several years as a model microorganism representative of those bacteria capable of metabolizing alkylaromatic hydrocarbons in oxygen-limited (hypoxic) aquifer environments (23,34,42,43). The tbu pathway of R. pickettii PKO1, which encodes enzymes for utilization of benzene, toluene, and related alkylaromatic hydrocarbons as well as enabling this strain to transform trichloroethylene (TCE), has been cloned as a 26.5-kbp DNA fragment designated pRO1957 (41).…”

“…Ralstonia pickettii PKO1 has been investigated by our laboratory for several years as a model microorganism representative of those bacteria capable of metabolizing alkylaromatic hydrocarbons in oxygen-limited (hypoxic) aquifer environments (23,34,42,43). The tbu pathway of R. pickettii PKO1, which encodes enzymes for utilization of benzene, toluene, and related alkylaromatic hydrocarbons as well as enabling this strain to transform trichloroethylene (TCE), has been cloned as a 26.5-kbp DNA fragment designated pRO1957 (41).…”

Microbial Toluene Removal in Hypoxic Model Constructed Wetlands Occurs Predominantly via the Ring Monooxygenation Pathway

“…The monooxygenation pathway has been also proposed as the predominant mechanism for the degradation of benzene-contaminated groundwater in a bench-scale constructed wetland (63). The dominant role of this pathway in the PFRs may be due to the high oxygen affinity of enzymes such as toluene monooxygenases compared to methyl-monooxygenases or toluene dioxygenases, which would allow the microorganisms harboring this type of enzymes to be adapted to grow on some aromatic compounds in this and other low oxygen environments (66,67). Likewise, the catechol 2,3-dioxygenases encoded in the catabolic operons, together with this type of monooxygenase, have also been reported to perform efficiently under low-oxygen conditions (68).…”