Understanding the limitations of microbial metabolism of

ethers used as fuel octane enhancers

Joseph P Salanitro

Shell Development Company, Houston, USA

Recent reports on the microbial degradation of alkyl ether octane enhancers

indicate that the metabolism of these compounds in soils and biosludges is

uncommon and relatively slow under aerobic and anaerobic conditions. A

mixed bacterial culture has now been isolated that can completely mineralize

the branched alkyl ether methyl tertiary butyl ether, and the ether-cleaving

activity of this culture appears to be subject to feedback regulation by

metabolites. In addition, this type of alkyl ether degradation appears to

be different from both alkyl-aryl ether cleaving and aromatic hydrocarbon

oxygenase activities reported in other microbial systems.

Current Opinion in Biotechnology 1995, 6:337-340

Introduction

Reformulated motor vehicle &els are currently mar- keted in the USA in response to the Federal Environ- mental Protection Agency (EPA) Clean Air Act amend- ments of 1990 aimed at reducing urban air pollution. These fuels have decreased levels of sulfur and olefins, reduced benzene and total aromatics, and are supple- mented with 5-15X (v/v) oxygenated chemicals (e.g. methanol, ethanol, tertiary butyl alcohol (TBA) and/or methyl tertiary butyl ether [MTBE]) as octane enhancers [1,2]. Oxygenates are usually blended in gasoline to pro- vide 2% (w/w) oxygen (which improves engine combus- tion efficiency), to reduce evaporative and engine emis- sions of hydrocarbons, and to decrease carbon mono- xide and ozone in the atmosphere. Many studies have investigated vehicle emissions from reformulated fuels (e.g. [3]; LG Anderson, P Wolfe, R Barre& JA Lan- ning, abstract, Annual Meeting of the American Chemi- cal Society, Chemistry, Denver, Colorado, USA, March 28th-April 2nd, 1993), but relatively little attention has been devoted to the environmental fate of water-soluble oxygenates (alcohols and ethers) from gasoline spills in media such as soil, groundwater and wastewater treat- ment systems. In contrast, numerous studies in the sci- ence literature on ground water discuss the transport and biodegradability of the more water-soluble hydrocarbons (e.g. benzene, toluene and xylenes) from fuel spills to the subsurface [4]. Oxygenates can migrate to the subsurface similarly from tank releases or they can be discharged to

sewage plants from process-unit or tank-cleaning oper- ations. In general, the 6te of organic chemicals in soils, groundwater and activated sludge systems is primarily determined by their susceptibility to microbial transfor- mation and biodegradation. It is important, therefore, to understand the biodegradability of fuel oxygenates in estimating impacts of persistence and toxicity in the environment. The more common oxygenates (e.g. aliphatic alcohols, methanol and ethanol) are known to be metabolized readily by bacteria in sewage sludges and soils [5-71. Even so, alcohols with quaternary carbon structures, such as the tertiary aliphatic alco- hols, TBA and t-amyl alcohol, are usually resistant or slowly degraded by microbes in sewage sludges and soils [6-8,9*,10,11]_ MTBE is currently the most widely used ether that is blended (-1 l-l 2% v/v) as an octane en- hancer in gasoline grades. As a chemical class, alkyl-alkyl ethers are generally resistant to mammalian and micro- bial metabolism, and little information is available on the degradation of these compounds in environmental me- dia.

It has long been known that MTBE is the most water- soluble and persistent compound in groundwater fol- lowing accidental release of a fuel to the subsuhce [12]. Diisopropyl ether (DIPE), ethyl tertiary butyl ether (ETBE) and methyl tertiary amyl ether (MTAE), which behave similarly to MTBE, have also been considered by the oil industry as octane enhancers for motor fuels. In this review, my comments are restricted to our limited knowledge of the microbial metabolism of ethers in gen-

Abbreviations DIPEAiisopropyl ether; DME-dimethyl ether; EPA-Environmental Protection Agency; ETBE-ethyl tertiary butyl ether;

MBE-methyl butyl ether; MTAE-methyl tertiary amyl ether; MTBE-methyl tertiary butyl ether; TBA-tertiary butyl alcohol; TBF-tertiary butyl formate.

0 Current Biology Ltd ISSN 0958-1669 337

338 Environmental biotechnology

eral, the characteristics of an isolated MTBE-degrading enrichment, and finally, some perspectives on the poten- tial of, and limits to, alkyl ether breakdown in soils and biosludges.

Microbial transformation of ether-type compounds

It is important to distinguish between the types of ether linkage that have been shown to be metabolized by microbes. The most readily utilized types of ‘ether- bond’ in compounds are those represented by ethy- lene glycol (CH3CH20CH$ZH20H), and low molec- ular weight polyethylene glycols (H[OCH2CH&,OH) which comprise the hydrophilic portion of common non-ionic surfactants [13]. Oxygenase-type enzyme sys- tems and/or aerobic and anaerobic cultures have been shown to metabolize the aryl ether linkages in aromatic compounds, such as methoxybenzoates [14], dimethoxy- benzyl alcohol [15], lignin ether subunits [lb], 2,4- dichlorophenoxyacetate [17], diphenyl ether [18] and the haloalkyl ethers his (2-chloroethyl) ether and 2- chloroethyl vinyl ether [19]. The ether bonds in many of these compounds are somewhat destabilized for attack by oxygenases or chemical hydrolysis because of electron- withdrawing groups and aromatic ring resonance. Other studies have shown that cyclic ethers (e.g. dioxane and tetrahydrofuran) can be utilized as a sole carbon source by both pure cultures of Rhodococcus and actinomycete species [20**,21]. Linear and branched-chain alkyl ether structures, however, are chemically stable at neutral pH, and relatively few biodegradation reports have demon- strated definitive ether cleavage and carbon utilization. Recently, Hyman et al. [22*] have provided evidence for the metabolism of dimethyl ether (DME), a gaseous ether. They found that DME (500 mgl-1 [v/v]) is both a substrate and inhibitor of the ammonia monoxygenase of Nitrosomonas europoea. In other studies, DME at higher levels (22% [v/v]) has been found to be a competitive inhibitor of methane oxidation by methylotrophic soil cultures [23]. It is possible, however, that similar to am- monia monooxygenase, the methyl monooxygenase of methane oxidizers may be capable of oxidizing DME. The capacity of ammonia monooxygenase or methyl monoxygenase to cleave other linear or branched alkyl ethers is unknown.

Our research group [24**] has reported the first definitive evidence for the complete mineralization of a branched alkyl ether by microbes. The isolated bacterial en- richment (termed BC-1) could cleave the ether link- age of MTBE with the transient formation of TBA. BC-1 can also metabolize other linear and branched ethers, including diethyl ether, methylbutyl ether (MBE), DIPE, ETBE and MTAE (JP Salanitro, unpub- lished data), suggesting that it may contain a common

ether-cleaving enzyme activity. The actinomycete iso- lated h-om biosludge by Parales et al. [20”], which grows on l,Cdioxane, can also utilize diethylether and MBE, but not DIPE, ETBE, MTBE or ethylene glycol ethers. The degradation of MBE (50mgl-1 of carbon) under methanogenic conditions has been reported by Suflita and Mormile [25**]. MBE decomposed to CHJ slowly in sediment-groundwater microcosms prepared from material sampled at an anaerobic aquifer. Neither MTBE nor any other alkyl ether tested was metabolized under the same conditions.

In a subsequent study by Mormile et al. [26**], most alkyl ethers tested under sulfate- and nitrate-reducing condi- tions were resistant to degradation either by anaerobic river and creek sediments or by pure cultures of aceto- gens (Acetobacferium and Eubaceterium species). Even so, these workers presented evidence for the cleavage of MTBE to TBA under anaerobic (methanogenic) con- ditions in one Ohio River sediment slurry incubated with 73 mgl-1 MTBE. TBA accumulated in the micro- cosm culture and was not metabolized. Yeh and Novak [9*] have presented preliminary data showing that ETBE and MTBE may be degraded by anaerobic subsoil mi- crocosms. They did not confirm ether degradation us- ing radiolabeled substrate, however, nor did they verify whether electron acceptor utilization was coupled to substrate metabolism under the anaerobic (denitritjling and methanogenic) conditions used.

Characteristics of a branched alkyl ether degrading culture

Continuous culture experiments carried out in our lab- oratory have revealed that high rates of ether degrada- tion in the mixed MTBE-degradinginitrifjring culture (BC-1) can be maintained when the unit is operated with a long cell residence time (~50 days) [24**]. Cell yields (0.2-0.3 g g-1) were low for an aerobic culture, but not unusual for cultures (e.g. industrial activated sludges) with long cell cycles. The continuous culture experi- ments suggested, but did not prove, that high rates of nitrification both were linked to ether cleavage and sta- bilized the MTBE-degrading population. BC-1 metab- olized I%-methoxy-labeled MTBE to CO2, with cell proliferation. Experiments on the effect of NH4+ oxi- dation on MTBE mineralization indicate that high con- centrations of NH4+ do not inhibit MTBE degradation. Preincubation of BC-1 with NH4+ or allylthiourea, a ni- trification inhibitor, also do not affect oxygen uptake in the presence of MTBE (JP Salanitro, unpublished data). The MTBE-degrading culture does not oxidize benzene or toluene (as measured by 02 uptake). These data sug- gest that the ether-cleavage system in BC-1 may be different 6om ammonia monooxygenase and aromatic hydrocarbon oxygenases.

Microbial metabolism of ethers used as fuel octane enhancers Salanitro 339

Fig. 1. A possible aerobic microbial degradative pathway for MTBE. The proposed scheme involves the initial oxidation of MTBE to t-butyl formate and subsequent cleavage to TBA and formaldehyde or formate. TBA is then metabolized via isopropyl alcohol, acetone and pyruvate to acetate and CO*. Confirmation of this pathway for MTBE degradation will require experiments on the utilization of the proposed metabolic products by the MTBE-degrading culture.

The mechanism and/or product of the initial enzy- matic attack on alkyl-alkyl ethers is not known. Studies on the atmospheric decomposition of the MTBE and ETBE by hydroxyl radical initiated reactions have shown that tertiary butyl formate (TBF; [HCOOCC(CH3)3]) and formaldehyde are primary products [27,28]. Un- published experiments in our laboratory have shown that TBF is a better substrate than MTBE in oxy- gen uptake assays with BC-1. TBF may be the ini- tial product of the microbial oxidation of MTBE. We also know that TBA is a transient intermediate in the metabolism of MTBE by BC-1 [24**]. TBA has been shown to be a major metabolite both in MTBE in- halation experiments using rats and in studies of liver microsome P450-linked monooxygenase activity in the presence of MTBE [29,30]. We speculate, therefore, that formaldehyde or formate and TBA might be the ether cleavage products of MTBE produced by the BC-1 culture. The metabolic pathway for TBA has not been confirmed in microbial systems. But in the atmospheric oxidation of TBA (mediated by hydro- xyl radicals), acetone and formaldehyde are formed in equimolar amounts [27]. Acetone may also be an inter- mediate in the mammalian metabolism of MTBE. Rats exposed to MTBE by feeding and inhalation excrete 2- methyl-2,2-propanediol and a-hydroxyisobutyric acid

[29]. These compounds might be readily metabolized to isopropyl alcohol and/or acetone. Preliminary (02 uptake) experiments with BC-1 also indicate that this ether-degrading culture can utilize TBA and poten- tial downstream metabolites of TBA, namely isopropyl alcohol, acetone, pyruvate and acetate (JP Salanitro, un- published data). A proposed biodegradation pathway for MTBE by BC-1 is shown in Figure 1.

Conclusions and future considerations

At this time, it is difficult to assess the potential of, and limits to, the bioremediation of wastes, soils and water containing alkyl ether octane enhancers. Too few cul- tures have been isolated and little information is known on the physiology and biochemistry of alkyl ether bio- transformation and the ability to sustain ether-degrading activity in soils and activated sludges. Although some ev- idence suggests aerobic and anaerobic cleavage of linear alkyl ethers can take place, this decomposition is slow. In the case of the branched alkyl ether MTBE, only one bacterial enrichment has been obtained for study. Cur- rent experience with this slow-growing MTBE-degrad- ing system (BC-1) suggests that the requisite oxygenase (ether-cleaving enzyme) may be subject to considerable feedback regulation by metabolites (e.g. TBA, isopropyl alcohol and acetone); moreover, it is possible that ethers interfere with ATP synthesis required for cell growth. For example, TBA or acetone may also be competitive substrates for the same ‘MTBE oxygenase’. Precedence for this type of metabolic regulation with MTBE was provided by Savolainen et al. [30]. In their study, MTBE and TBA conferred apparently different substrate speci- ficities for the same monooxygenase complex in rat liver metabolism. TBA was oxidized more slowly and its metabolism was the rate-limiting step in MTBE degra- dation. In BC-1, either the rate of TBA degradation can also be regulated by the same ether-cleaving enzyme or other species in the mixed culture can metabolize TBA, which in turn, is regulated by MTBE. The stability of mixed cultures containing multiple substrate interactions needs to be assessed when inoculated into heterogeneous microbial systems, such as soils and biosludges for biore- mediation. More studies on ether-degrading cultures, es- pecially branched alkyl ethers, are needed to understand enzyme induction of ether cleavage, stability and main- tenance of mixed populations, substrate specificity, and metabolite control mechanisms.

References and recommended reading

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JP Salanitro, Shell Development Company, 3333 Highway 6

South, Houston, Texas 77082, USA.