Characterization of toluene and ethylbenzene biodegradation under nitrate-, iron(III)- and manganese(IV)-reducing conditions by compound-specific isotope analysis

Highlights

• Toluene/ethylbenzene isotope effects were studied in 3 cultures with varying TEAs.

• BTEX biodegradation under solid Mn(IV) was investigated by CSIA for the first time.

• Solid Mn(IV) as TEA always caused significant lower C and H isotope enrichment.

• Always similar correlation of C–H-isotopes upon ethylbenzene activation was shown.

• Subtypes of benzylsuccinate synthase may cause varying correlation of C–H-isotopes.

Abstract

Ethylbenzene and toluene degradation under nitrate-, Mn(IV)-, or Fe(III)-reducing conditions was investigated by compound specific stable isotope analysis (CSIA) using three model cultures (Aromatoleum aromaticum EbN1, Georgfuchsia toluolica G5G6, and a Azoarcus-dominated mixed culture). Systematically lower isotope enrichment factors for carbon and hydrogen were observed for particulate Mn(IV). The increasing diffusion distances of toluene or ethylbenzene to the solid Mn(IV) most likely caused limited bioavailability and hence resulted in the observed masking effect. The data suggests further ethylbenzene hydroxylation by ethylbenzene dehydrogenase (EBDH) and toluene activation by benzylsuccinate synthase (BSS) as initial activation steps. Notably, significantly different values in dual isotope analysis were detected for toluene degradation by G. toluolica under the three studied redox conditions, suggesting variations in the enzymatic transition state depending on the available TEA. The results indicate that two-dimensional CSIA has significant potential to assess anaerobic biodegradation of ethylbenzene and toluene at contaminated sites.

Introduction

Benzene, toluene, ethylbenzene, and xylenes, commonly referred to as BTEX compounds, are components of crude oil and gasoline. They are released to the environment either from natural deposits or from spillings due to human handling and are of major concern because of their toxicity in combination with their chemical inertness, good water-solubility and mobility in the saturated and unsaturated zone (Wiedemeier, 1999). For this reason, understanding of their environmental fate is of high importance. Reduction in toxicity, mass, and mobility of these contaminants in the subsurface can be caused by physicochemical effects, such as diffusion and sorption, or by microbial degradation. The latter one is the primary process in natural attenuation (Röling and van Verseveld, 2002).

The redox potential of the environment exerts selective pressure on the prevalence of microbial species as well as dominant biochemical strategies for the degradation of BTEX compounds. According to the principle of cellular energy conservation, common terminal electron acceptors (TEAs) will be utilized hierarchically, subject to thermodynamic constraints, such that the most energetically favorable reactions supported by the environment will tend to dominate initially, followed by less favorable reactions. E.g., the depletion of oxygen is followed by the subsequent reductions of nitrate, Mn(IV), ferric iron, sulfate, and carbon dioxide via methanogenesis (SI-Table 1). At contaminated sites, the simultaneous use of different electron acceptors in narrow zones at the plume fringe may play a major role (Bauer et al., 2009).

Facultative anaerobic microorganisms are generally able to couple hydrocarbon oxidation to a variety of TEAs with similar redox potentials, e.g. oxygen, nitrate, Mn(IV), and ferric iron, and thereby adapt to varying environmental conditions (Nealson and Saffarini, 1994).

Biodegradation of single or multiple BTEX compounds from petroleum-contaminated aquifers under iron-reducing conditions has been demonstrated at several contaminated sites (Anderson and Lovley, 1999, Botton and Parson, 2006, Rooney-Varga et al., 1999). Ferric iron is generally considered the most abundant terminal electron acceptor for organic matter oxidation in many sediments (Lovley, 1991, Luu and Ramsay, 2003). Cultivation of environmental samples in laboratory microcosms under iron-reducing conditions has led to a variety of BTEX-degrading enrichment cultures and isolates. Microbial toluene mineralization coupled to ferric iron respiration has been identified for (i) different members of the genus Geobacter (Coates et al., 2001, Kunapuli et al., 2010, Lovley et al., 1993, Lovley and Lonergan, 1990), (ii) for Georgfuchsia toluolica from the newly discovered genus Georgfuchsia (Weelink et al., 2009), and (iii) Desulfitobacterium aromaticivorans from the class Clostridia (Kunapuli et al., 2010). To the knowledge of the authors, only three laboratory cultures have been reported to grow on ethylbenzene while respiring ferric iron (Jahn et al., 2005, Weelink et al., 2009). Cultivation of BTEX-degrading enrichment cultures with Mn(IV) as TEA has been described only rarely (Langenhoff et al., 1997a, Langenhoff et al., 1997b, Villatoro-Monzón et al., 2003). Contrasting to Fe(III) and Mn(IV), BTEX degradation under nitrate-reducing conditions has been studied more frequently. Most toluene or ethylbenzene degrading denitrifying bacteria belong to the Azoarcus/Thauera/Aromatoleum cluster within the Betaproteobacteria (Weelink et al., 2010). Also the strict anaerobic freshwater strain Georgfuchsia toluolica G5G6 belongs to this class and is able to reduce nitrate while using toluene or ethylbenzene as electron donors and carbon sources (Weelink et al., 2009).

The anaerobic activation of inert aromatic hydrocarbons has revealed several biochemical mechanisms comprising (i) fumarate addition catalyzed by the glycyl-radical enzyme benzyl succinate synthase (BSS) or related enzymes, (ii) oxygen-independent hydroxylation, which has been described e.g., for the activation of ethylbenzene by the molybdenum-cofactor containing enzyme ethylbenzene dehydrogenase (EBDH) (reviewed in Fuchs et al., 2011, Heider, 2007), and (iii) carboxylation which has been reported for naphthalene (Mouttaki et al., 2012) and assumed for benzene (Abu Laban et al., 2010, Luo et al., 2014).

Changes in isotope composition of substrates during biochemical reactions can give insight in enzymatic reaction mechanisms and thus can help to understand prevailing processes at contaminated sites. Compound-specific isotope analysis has been demonstrated to be a powerful tool for the assessment of in situ biodegradation of BTEX compounds. The great advantage of this technique is to characterize and to quantify degradation processes, on condition that isotope fractionation factors are known (Meckenstock et al., 2004). Molecules just differing in the isotopic composition at the reactive site have slightly different reaction rates in the rate-limiting transformation step. As a result, molecules with the less preferred isotope in the reactive position become enriched. This process in unidirectional reactions is referred to as isotope fractionation or kinetic isotope effect (KIE) (Bigeleisen and Wolfsberg, 1958). KIEs are caused by differences in activation energies required for a bond change in a chemical reaction depending on the presence of a light or heavy isotope of an element in the bond. Thus, the KIE measures the ratio of the reaction rates of two isotopologues, and the degree of enrichment is itself an indicator of the reaction mechanism. As the KIE reflects the isotope-sensitive step in the transition state, it characterizes the mode of bond change during the chemical transformation.

However, the apparent kinetic isotope effect (AKIE) can be considerably smaller than the intrinsic KIE. In biological systems, the bioavailability, the uptake of the substrate into the cell or the binding of the substrate to the enzyme can control the overall kinetics and mask the isotope fractionation. When the rate-limiting step is prior to the catalytic isotope-sensitive step, substrate molecules can be converted almost irrespective of their isotope composition at the reactive site (Northrop, 1981). Consequently, the AKIE or a single isotope fractionation factor contains information about the bond cleavage and previous rate limitations.

As in heterogenic systems a number of processes can influence the observed change in isotope ratios, kinetic isotope effects for multiple elements can be correlated to control for these influences (Hunkeler et al., 2001, Kuder et al., 2004, Zwank et al., 2004). Isotopically non-sensitive kinetic effects prior to catalysis are thought to affect atoms of different elements at same extent. This way, the correlation of isotope fractionation again reflects the intrinsic bond change. For example, the first irreversible reaction of BTEX degradation includes a C–H bond cleavage. The correlation of ¹³C and ²H enrichment is called dual isotope analysis and can lead to characteristic Λ values for different degradation pathways in dependency of the substrate. Although Λ values support the interpretation of reaction mechanisms and subsurface processes, they are not robust for high hydrogen isotope fractionation. A recently developed corrective procedure helps to overcome this problem and is based on ζ *(rp) values (Dorer et al., 2014a).

The concept of dual isotope analysis has been applied for BTEX compounds in recent years. Previous studies have shown that Λ or ζ*(rp) values varied for different TEAs for the degradation of benzene (Fischer et al., 2008, Mancini et al., 2008), toluene (Vogt et al., 2008), and ethylbenzene (Dorer et al., 2014a, Dorer et al., 2014b). In the case of ethylbenzene, side-chain hydroxylation and fumarate addition most likely generate altered Λ and ζ*(rp) values (Dorer et al., 2014b). For toluene, only fumarate addition has been described as an initial activating step. Hence dual isotope values might reflect slightly different transition states of various subtypes of the same enzyme BSS (Kümmel et al., 2013, Vogt et al., 2008).

Notably, isotope enrichment factors (ε) as well as Λ or ζ*(rp) values for the degradation of a specific aromatic compound by a specific microbial culture using different electron acceptors have not yet been determined. Since microorganisms can switch between degradation pathways for the same compound in the case of shifting TEA's (Shinoda et al., 2004), even ε, Λ, and ζ*(rp) values drawn from the degradation of a single compound may vary due to variations in enzyme expression: Isoenzymes or completely different enzymes that catalyze a transformation step may vary in the reaction mechanism. Even if the same enzymes are expressed by a single organism, the availability of the electron acceptor can significantly influence ε factors, as previously shown for Geobacter metallireducens during toluene degradation in the presence of different forms of ferric iron (Tobler et al., 2008). Thus, although isotopic characterization may help to interpret data from contaminated sites and to make predictions on BTEX biodegradation, the isotopic fractionation must itself be investigated for each compound before any conclusion about the applicability of CSIA can be drawn.

When CSIA is applicable to track biodegradation of a certain pollutant, a variety of samples on transects from the plume to the fringes is collected and their isotopic signature measured. The comparison of ε, Λ, and ζ*(rp) values with laboratory data may allow for assessing the transformation process often supported by additional molecular or geochemical data. The application of CSIA in BTEX-contaminated sites was described in recent studies (Feisthauer et al., 2012, Fischer et al., 2009, Fischer et al., 2007, Griebler et al., 2004, Mancini et al., 2002).

The goal of this study was to determine whether manganese, iron, and nitrate as TEAs lead to mechanistic differences in toluene and ethylbenzene activation in a particular microorganism, indicated by specific correlations of hydrogen and carbon isotope fractionation. To investigate the variability of isotope fractionation effected by electron acceptors, three aromatics-degrading Betaproteobacteria were investigated: (i) Aromatoleum aromaticum EbN1 (Rabus et al., 2005, Rabus and Widdel, 1995), (ii) Georgfuchsia toluolica G5G6 (Weelink et al., 2009), and (iii) the enrichment culture DD-Anox1 (Dorer et al., 2014b). BSS catalyzes toluene activation in A. aromaticum and G. toluolica using nitrate, Fe(III) and Mn(IV), respectively (Kube et al., 2004, Oosterkamp, 2013, Weelink et al., 2009), and ethylbenzene dehydrogenase catalyzes ethylbenzene activation in the denitrifying A. aromaticum, G. toluolica and DD-Anox1 (Dorer et al., 2014b, Kniemeyer and Heider, 2001). For comparison of toluene and ethylbenzene fractionation factors in this study, the carbon and hydrogen fractionation pattern of the three cultures were characterized under nitrate-, Mn(IV)-, and iron-reducing conditions, and implications for environmental applications were drawn.