Kirk T. O'Reilly

Characterization of the Initial Reactions during the Cometabolic Oxidation of Methyl tert-Butyl Ether by Propane-Grown Mycobacterium vaccae JOB5

ABSTRACT The initial reactions in the cometabolic oxidation of the gasoline oxygenate, methyl tert-butyl ether (MTBE), by Mycobacterium vaccae JOB5 have been characterized. Two products, tert-butyl formate (TBF) and tert-butyl alcohol (TBA), rapidly accumulated extracellularly when propane-grown cells were incubated with MTBE. Lower rates of TBF and TBA production from MTBE were also observed with cells grown on 1- or 2-propanol, while neither product was generated from MTBE by cells grown on casein-yeast extract-dextrose broth. Kinetic studies with propane-grown cells demonstrated that TBF is the dominant (≥80%) initial product of MTBE oxidation and that TBA accumulates from further biotic and abiotic hydrolysis of TBF. Our results suggest that the biotic hydrolysis of TBF is catalyzed by a heat-stable esterase with activity toward several other tert-butyl esters. Propane-grown cells also oxidized TBA, but no further oxidation products were detected. Like the oxidation of MTBE, TBA oxidation was fully inhibited by acetylene, an inactivator of short-chain alkane monooxygenase in M. vaccae JOB5. Oxidation of both MTBE and TBA was also inhibited by propane (Ki = 3.3 to 4.4 μM). Values for Ks of 1.36 and 1.18 mM and for Vmax of 24.4 and 10.4 nmol min−1 mg of protein−1 were derived for MTBE and TBA, respectively. We conclude that the initial steps in the pathway of MTBE oxidation by M. vaccae JOB5 involve two reactions catalyzed by the same monooxygenase (MTBE and TBA oxidation) that are temporally separated by an esterase-catalyzed hydrolysis of TBF to TBA. These results that suggest the initial reactions in MTBE oxidation by M. vaccae JOB5 are the same as those that we have previously characterized in gaseous alkane-utilizing fungi.

Cometabolism of Methyl tertiary Butyl Ether and Gaseous n-Alkanes by Pseudomonas mendocina KR-1 Grown on C5 to C8 n-Alkanes

ABSTRACT Pseudomonas mendocina KR-1 grew well on toluene, n-alkanes (C5 to C8), and 1° alcohols (C2 to C8) but not on other aromatics, gaseous n-alkanes (C1 to C4), isoalkanes (C4 to C6), 2° alcohols (C3 to C8), methyl tertiary butyl ether (MTBE), or tertiary butyl alcohol (TBA). Cells grown under carbon-limited conditions on n-alkanes in the presence of MTBE (42μ mol) oxidized up to 94% of the added MTBE to TBA. Less than 3% of the added MTBE was oxidized to TBA when cells were grown on either 1° alcohols, toluene, or dextrose in the presence of MTBE. Concentrated n-pentane-grown cells oxidized MTBE to TBA without a lag phase and without generating tertiary butyl formate (TBF) as an intermediate. Neither TBF nor TBA was consumed by n-pentane-grown cells, while formaldehyde, the expected C1 product of MTBE dealkylation, was rapidly consumed. Similar Ks values for MTBE were observed for cells grown on C5 to C8n-alkanes (12.95 ± 2.04 mM), suggesting that the same enzyme oxidizes MTBE in cells grown on each n-alkane. All growth-supporting n-alkanes (C5 to C8) inhibited MTBE oxidation by resting n-pentane-grown cells. Propane (Ki = 53 μM) and n-butane (Ki = 16 μM) also inhibited MTBE oxidation, and both gases were also consumed by cells during growth on n-pentane. Cultures grown on C5 to C8n-alkanes also exhibited up to twofold-higher levels of growth in the presence of propane or n-butane, whereas no growth stimulation was observed with methane, ethane, MTBE, TBA, or formaldehyde. The results are discussed in terms of their impacts on our understanding of MTBE biodegradation and cometabolism.

Induction of methyl tertiary butyl ether (MTBE)-oxidizing activity in Mycobacterium vaccae JOB5 by MTBE.

ABSTRACT Alkane-grown cells of Mycobacterium vaccae JOB5 cometabolically degrade the gasoline oxygenate methyl tertiary butyl ether (MTBE) through the activities of an alkane-inducible monooxygenase and other enzymes in the alkane oxidation pathway. In this study we examined the effects of MTBE on the MTBE-oxidizing activity of M. vaccae JOB5 grown on diverse nonalkane substrates. Carbon-limited cultures were grown on glycerol, lactate, several sugars, and tricarboxylic acid cycle intermediates, both in the presence and absence of MTBE. In all MTBE-containing cultures, MTBE consumption occurred and tertiary butyl alcohol (TBA) and tertiary butyl formate accumulated in the culture medium. Acetylene, a specific inactivator of alkane- and MTBE-oxidizing activities, fully inhibited MTBE consumption and product accumulation but had no other apparent effects on culture growth. The MTBE-dependent stimulation of MTBE-oxidizing activity in fructose- and glycerol-grown cells was saturable with respect to MTBE concentration (50% saturation level = 2.4 to 2.75 mM), and the onset of MTBE oxidation in glycerol-grown cells was inhibited by both rifampin and chloramphenicol. Other oxygenates (TBA and tertiary amyl methyl ether) also induced the enzyme activity required for their own degradation in glycerol-grown cells. Presence of MTBE also promoted MTBE oxidation in cells grown on organic acids, compounds that are often found in anaerobic, gasoline-contaminated environments. Experiments with acid-grown cells suggested induction of MTBE-oxidizing activity by MTBE is subject to catabolite repression. The results of this study are discussed in terms of their potential implications towards our understanding of the role of cometabolism in MTBE and TBA biodegradation in gasoline-contaminated environments.

Indirect aluminum toxicity to the green alga Scenedesmus through increased cupric ion activity

Additions of aluminum and copper to chemically defined media resulted in inhibition of growth of Scenedesmus and of alkaline phosphatase activity. The alkaline phosphatase activity was assayed both on commercially available purified enzyme from bacteria and on the enzyme present in whole Scenedesmus cells. The effect of metal additions was compared to the total aluminum added and to the computed free ion activities for both copper and aluminum. In all three systems (algal growth, purified enzyme, and algal enzyme) the observed toxicity with increased total aluminum was mostly due to an increase in cupric ion activity. The algal growth response was affected for the range of cupric ion activities from 10/sup -6/ to 10/sup -12/. The toxic dose response of aluminum was largely due to indirect competitive effects of Al in the medium that displaced copper from the chelator. 33 references, 4 figures.

Parsing pyrogenic polycyclic aromatic hydrocarbons: Forensic chemistry, receptor models, and source control policy

A realistic understanding of contaminant sources is required to set appropriate control policy. Forensic chemical methods can be powerful tools in source characterization and identification, but they require a multiple-lines-of-evidence approach. Atmospheric receptor models, such as the US Environmental Protection Agency (USEPA)s chemical mass balance (CMB), are increasingly being used to evaluate sources of pyrogenic polycyclic aromatic hydrocarbons (PAHs) in sediments. This paper describes the assumptions underlying receptor models and discusses challenges in complying with these assumptions in practice. Given the variability within, and the similarity among, pyrogenic PAH source types, model outputs are sensitive to specific inputs, and parsing among some source types may not be possible. Although still useful for identifying potential sources, the technical specialist applying these methods must describe both the results and their inherent uncertainties in a way that is understandable to nontechnical policy makers. The authors present an example case study concerning an investigation of a class of parking-lot sealers as a significant source of PAHs in urban sediment. Principal component analysis is used to evaluate published CMB model inputs and outputs. Targeted analyses of 2 areas where bans have been implemented are included. The results do not support the claim that parking-lot sealers are a significant source of PAHs in urban sediments.

Identification of ester metabolites from petroleum hydrocarbon biodegradation in groundwater using GC×GC‐TOFMS

In an effort to understand the nature and toxicity of petroleum hydrocarbon degradation metabolites, 2-dimensional gas chromatography linked to a time-of-flight mass spectrometer (GC×GC-TOFMS) was used to conduct nontargeted analysis of the extracts of 61 groundwater samples collected from 10 fuel release sites. An unexpected result was the tentative identification of 197 unique esters. Although esters are known to be part of specific hydrocarbon degradative pathways, they are not commonly considered or evaluated in field studies of petroleum biodegradation. In addition to describing the compounds identified, the present study discusses the role for nontargeted analysis in environmental studies. Overall, the low toxicological profile of the identified esters, along with the limited potential for exposure, renders them unlikely to pose any significant health risk.

Letter commenting on “Primary sources and toxicity of PAHs in Milwaukee‐area streambed sediment”—To the editor

Baldwin et al. [1] sought to identify sources of polycyclic aromatic hydrocarbons (PAHs) in sediment samples collected from streams in Milwaukee, Wisconsin, USA. The authors used a number of approaches that rely on comparing the PAH chemistry of sediment samples to those of proposed sources. These approaches are only valid if appropriate sources are correctly identified and sufficiently characterized. Unfortunately, Baldwin et al. rely almost entirely on source profiles initially published by Van Metre and Mahler [2]. The work in which Van Metre has been involved has focused on implicating refined tar pavement sealants (RTS) as a significant source of PAHs in urban sediments [2–4]. The RTS source profile used by Baldwin et al. is not the PAH profile of RTS but was derived by Van Metre from parking lot dust sample data specifically to be statistically consistent with urban sediment rather than with actual RTS [2]. This results in a circular argument where the similarity of urban sediment samples with urban dust is used by Baldwin et al. to suggest that RTS is a source. Baldwin et al. [1] claim that PAH concentrations in some of the sediments samplers were too high to be urban background. However, a comparison of background PAH concentration in urban soil [5] with sediments in the Baldwin et al. study indicates soils can have sufficient PAH concentrations to account for those measured in sediments. Soil is a source of solids that become creek sediments, but soils are less likely to have been impacted by runoff from paved surfaces. The goal of the Kay et al. study [5] was to characterize PAH concentrations resulting from atmospheric deposition. To compare the concentration of the sediment in the Baldwin et al. and the soil PAH data from Kay et al., [5] we used ProUCL 4.1 [6] to calculate a 95% upper tolerance limit background for the soil samples of 120mg/kg PAH16. The upper tolerance limit is 5 times greater than the assumed background of 20mg/kg by Baldwin et al. Six of the 40 samples exceeded 120mg/kg, in the Baldwin et al. study, but only 2 exceeded 130mg/kg. Polycyclic aromatic hydrocarbon diagnostic ratios do not support the study conclusions of Baldwin et al. [1]. Although the ratios of benzo[b]fluoranthene to benzo[k]fluoranthene and fluoranthene to pyrene of urban sediment may be similar to urban parking lot dusts, they are not similar to RTS. Figure 1 shows these 2 ratios for the sediment samples fromBaldwin et al., the parking lot dusts from Van Metre et al. [7], fresh RTS data from Mahler et al. [3], and fresh RTS data from Scoggins et al. [8]. Only 1 of the 18 RTS samples plotted near the sediment samples. Baldwin et al. noted an average benzo[b]fluoranthene to benzo[k]fluoranthene ratio of 2.54 for Milwaukee sediment, but the average ratio is only 1.38 for the RTS samples. Given the variability in the PAH profiles of different categories of sources, minor differences in the statistical similarity (X) between sediment samples and literature derived profiles does not support claims of causation. Technical issues concerning thenon-RTSsource profiles usedbyBaldwinet al. [1] are described indetail elsewhere [9,10].Manyof the profileswere derived from the work of Li et al. [11], who had combined data from at least 22 different papers dating as far back as 1976. Some of the papers Li et al. [11] used had compiled data from other sources, so it is impossible to adequately evaluate their validity or variability. A comparison between the traffic tunnel profile used byBaldwin et al. and data from1of the source documents fromLi et al. [11] (Venkataraman et al. [12]) shows a poor relationship with R of 0.17 to 0.38, indicating source profiles may not represent the stated sources (Figure 2). We agree with the caveat fromLi et al. [11] concerning the use of published source profiles:

Establishing the Chemical Footprint of Potential Injury from Petroleum Product Releases at Fuel Terminals

ABSTRACT Owners and operators of waterfront fuel terminals can be liable for natural resource damages (NRD) caused by releases from their facilities. While NRD assessments typically focus on measuring exposure and predicting injury, there is often insufficient consideration of two key issues: 1) adequate and specific consideration of the chemical background, and 2) establishing the release(s) footprint through linking sediment chemistry to the particular release(s). Recent changes in the legal framework used to assign and apportion remedial and NRD liability further increase the need to quantify a facilitys contribution to environmental harm. The goal of this paper is to discuss the changing legal framework and present a multiple lines of evidence approach both for characterizing the chemical conditions that would be expected in the absence of a release, and for identifying a facility-specific chemical footprint related to possible natural resource damages. Critical components include a thorough understa...

Oxygen Supply and Demand: Considerations for the Design of Groundwater Remediation Systems

Biodegradation of dissolved constituents in groundwater is often oxygen limited. A variety of remediation systems are available that act by supplying oxygen. The potential efficacy of these systems depends on their ability to overcome in situ oxygen demand. We present methods for evaluating and comparing the oxygen supply rate of remedial technologies. Analytical methods for measuring in situ oxygen demand are also presented. Specific terminology proposed to improve the understanding of how the results of these methods can be used in the design and operation of remedial systems. The use of these concepts in the implementation of in situ chemical oxidation technologies is also discussed.