Current Research Goals:

Introduction

The assessment of intrinsic rates of hydrocarbon degradation in subterranean matrices is still problematic.  Renewed interest in the development of quantitative indicators of bioremediation activity by anaerobic microbial communities stems from a better understanding of the metabolic fate of contaminating compounds such as hydrocarbons.  Despite the inert chemical nature of these molecules, biodegradation under aerobic and anaerobic conditions has been well-documented.  For example, aerobic degradation of alkanes involves incorporation of molecular oxygen into the hydrocarbon, by mono- or dioxygenases, to create metabolites that can be oxidized by central catabolic pathways [1].  However, pathways that occur under anaerobic conditions are not fully elucidated, and it was not until the late 1980’s that anaerobic hydrocarbon decay was shown [2].  We currently understand two mechanisms for the initiation of alkane degradation in anoxic environments, specifically fumarate addition and carboxylation.  During fumarate addition, the subterminal carbon of the alkane is added across the double bond of fumarate to form alkylsuccinate derivatives [3, 8].  For carboxylation, it has been proposed that CO2 is added to the C3 position, with the subsequent release of the terminal and subterminal carbons [4].  Following the activation of the alkane by fumarate addition or carboxylation, the presence of the polar functional group allows the molecule to enter metabolic pathways related to β-oxidation and fatty-acid catabolism.  Pure cultures have now been characterized that are capable of alkane degradation using the fumarate addition mechanism under denitrifying [3] and sulfate-reducing conditions [5], as well as a sulfate-reducer that is capable of alkane carboxylation [6].  According to thermodynamic calculations and experimental evidence including the detection of signature metabolites from enrichment cultures [7], we now understand that the occurrence of one alkane activation mechanism does not exclude the alternative.  However, which of these two processes represents the predominant reaction mechanism in situ is unknown.  Furthermore, the factors that may influence the in situ rates of anaerobic hydrocarbon decay have not been elucidated and questions remain as to the complete biochemical and enzymatic mechanisms of the reactions involved.

Fractionation of Alkanes by Anaerobic Bacteria

Little is known about the isotopic fractionation of alkanes by anaerobic organisms that are capable of hydrocarbon oxidation.  For example, are fractionation factors for fumarate addition different from those for carboxylation?  It is known however that 13C/12C  ratios for saturated hydrocarbons and PAHs are stable under aerobic conditions, leading to the conclusion that fractionation does not occur when oxygen is present in the environment [9].  Several studies have investigated δ 13C values for enrichment cultures.  Wilkes et al. [10] found that alkylbenzene carbon isotopic fractionation values ranged from between -26 to -33% in a sulfate-reducing enrichment culture from a North Sea oil tank.  However, δ 13C values for n-alkanes ranging in length from C-23 to C-35 were constant for at least 23 years in a peat bog environment [11].  Therefore, to better appreciate the fate of aliphatic hydrocarbons in the environment, it is important to interrogate the rates of carbon and hydrogen fractionation by pure cultures of sulfate-reducing bacteria that utilize both types of alkane-activating strategies. 

Protein Identification in Desulfoglaeba alkanexedens

Seminal work on the characterization of alkane degradation began when the activating enzyme for toluene degradation, benzylsuccinate synthase (Bss), was described in the denitrifying Betaproteobacterium Thauera aromatica [12].  From this work, it was discovered that aromatic hydrocarbon activation takes place using a glycyl radical enzyme with a molecular mass of 220 ± 20 kDa [13].  From our current understanding of glycyl radical enzymes, activating proteins must be present to extract a hydrogen molecule from the reactive glycine residue.  These activating enzymes have been identified as S-adenosylmethionine-dependent radical generators, or SAM-radical enzymes [14].  The Bss system from T. aromatica produces optically pure (R)-(+)-benzylsuccinate [12] but notably the activating enzyme for alkanes, which has also been shown to be a glycyl radical containing enzyme [15], produces diasteromers of the resulting alkylsuccinates [16].  The production of diasteromers is rare in biologically catalyzed reactions.  Researchers have recently had success in identifying proteins that are important in a nitrate-dependent alkane degradation pathway by using 2-dimensional (2D) gel electrophoresis [14].  By comparing the protein profiles under contrasting growth conditions, for instance when D. alkanexedens is grown on butyrate and decane, we should be able to identify proteins that are differentially expressed and therefore associated with alkane metabolism.

Literature Cited

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3.            Wilkes, H., et al., Anaerobic degradation of n-hexane in a denitrifying bacterium: Further degradation of the initial intermediate (1-methylpentyl)succinate via C-skeleton rearrangement. Arch Microbiol, 2002. 177: p. 235-243.
4.            So, C.M. and L.Y. Young, Initial Reactions in Anaerobic Alkane Degradation by a Sulfate Reducer, Strain AK-01. Appl Environ Micro, 1999. 65(12): p. 5532-5540.
5.            Davidova, I.A., et al., Desulfoglaeba alkanexedens gen. nov., sp. nov., an n-alkane-degrading, sulfate-reducing bacterium. International J System Evol Microbiol, 2006. 56(12): p. 2737-2742.
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8.           Morasch, B., et al., Stable Isotope Fractionation Caused by Glycyl Radical Enzymes during Bacterial Degradation of Aromatic Compounds. Appl Environ Micro, 2004. 70(5): p. 2935-2940.
9.           Mazeas, L., H. Budzinski, and N. Raymond, Absence of stable carbon isotope fractionation of saturated and polycyclic aromatic hydrocarbons during aerobic bacterial biodegradation. Org Geochem, 2002. 33(11): p. 1259-1272.
10.          Wilkes, H., et al., Anaerobic degradation and carbon isotopic fractionation of alkylbenzenes in crude oil by sulphate-reducing bacteria. Org Geochem, 2000. 31(1): p. 101-115.
11.          Huang, Y., et al., Absence of carbon isotope fractionation of individual n-alkanes in a 23-year field decomposition experiment with Calluna vulgaris. Org Geochem, 1997. 26(7-8): p. 497-501.
12.          Leutwein, C. and J. Heider, Anaerobic toluene-catabolic pathway in denitrifying Thauera aromatica : activation and B-oxidation of the first intermediate, (R)-(M)-benzylsuccinate. Microbiology, 1999. 145: p. 3265-3271.
13.          Leuthner, B., et al., Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism. Mol Microbiol, 1998. 28(3):p. 615-628.
14.          Sofia, H.J., et al., Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucl Acid Res., 2001. 29(5): p. 1097-1106.
15.          Grundmann, O., et al., Genes encoding the candidate enzyme for anaerobic activation of n-alkanes in the denitrifying bacterium, strain HxN1. Environ Microbiol, 2008. 10(2): p. 376-385.
16.          Kropp, K.G., I.A. Davidova, and J.M. Suflita, Anaerobic Oxidation of n-Dodecane by an Addition Reaction in a Sulfate-Reducing Bacterial Enrichment Culture. Appl Environ Micro, 2000. 66(12): p. 5393-5398.