Versatile transformations of hydrocarbons in anaerobic bacteria: substrate ranges and regio- and stereo-chemistry of activation reactions†

Abstract

Anaerobic metabolism of hydrocarbons proceeds either via addition to fumarate or by hydroxylation in various microorganisms, e.g., sulfate-reducing or denitrifying bacteria, which are specialized in utilizing n-alkanes or alkylbenzenes as growth substrates. General pathways for carbon assimilation and energy gain have been elucidated for a limited number of possible substrates. In this work the metabolic activity of 11 bacterial strains during anaerobic growth with crude oil was investigated and compared with the metabolite patterns appearing during anaerobic growth with more than 40 different hydrocarbons supplied as binary mixtures. We show that the range of co-metabolically formed alkyl- and arylalkyl-succinates is much broader in n-alkane than in alkylbenzene utilizers. The structures and stereochemistry of these products are resolved. Furthermore, we demonstrate that anaerobic hydroxylation of alkylbenzenes does not only occur in denitrifiers but also in sulfate reducers. We propose that these processes play a role in detoxification under conditions of solvent stress. The thermophilic sulfate-reducing strain TD3 is shown to produce n-alkylsuccinates, which are suggested not to derive from terminal activation of n-alkanes, but rather to represent intermediates of a metabolic pathway short-cutting fumarate regeneration by reverse action of succinate synthase. The outcomes of this study provide a basis for geochemically tracing such processes in natural habitats and contribute to an improved understanding of microbial activity in hydrocarbon-rich anoxic environments.

Metabolism of Hydrocarbons in <b><i>n</i></b>-Alkane-Utilizing Anaerobic Bacteria

“…These results appear to indicate that the substrate range of thermophilic sulfate-reducing strain TD3 is even broader than those of the two denitrifying strains. A detailed evaluation of the observed product patterns provided further insight into the apparent preferences with respect to certain structural features of transformable hydrocarbons [Jarling et al, 2015]. On the other hand, no evidence was obtained for the addition of aliphatic hydrocarbons to fumarate by any of the eight tested alkylbenzene utilizers.…”

“…The product mixture obtained from racemic hexan-2-ol containing all four diastereoisomers was separated into diastereomeric pairs of enantiomers by fractional recrystallization. The biomimetic addition of the 4-isopropylbenzyl and 1-phenylethyl radical, generated from the corresponding iodides, to fumarate and dimethyl fumarate yielded (4-isopropylbenzyl)succinate [Strijkstra et al, 2014] and dimethyl (1-phenylethyl)succinate [Jarling et al, 2015], respectively ( fig. 3 b).…”

“…A different radical-generating procedure had previously been used for biomimetic synthesis of dimethyl cyclopentylsuccinate ] and dimethyl n -propyl-and isopropylsuccinate [Kniemeyer et al, 2007]. Various phenylalkylsuccinates, as their corresponding 1-methyl esters, were synthesized in a two-step procedure starting with a Stobbe condensation of dimethyl succinate and a suitable ketone followed by a reduction of the resulting 1-methyl itaconate using blue samarium diiodide [Jarling et al, 2015] ( fig. 3 c).…”

“…The above-mentioned derivatization method to achieve gas chromatographic separation of stereoisomers of substituted succinates has been optimized and applied systematically to succinates deriving from a wide range of hydrocarbons [Jarling et al, 2015]. The results indicate that all tested n -alkane-utilizing anaerobic bacteria generally produce mixtures consisting of two stereoisomers with an opposite configuration at C2 of the arylalkyl-/ alkylsuccinate regardless of the type of hydrocarbon activated, be it an alkane or an alkylbenzene, while all tested alkylbenzene-utilizing anaerobic bacteria always produce only one stereoisomer, i.e.…”

“…These findings prompted detailed studies with numerous hydrocarbon-oxidizing bacteria and crude oil or defined substrate mixtures. For comparison, both n -alkane-and alkylbenzene-utilizing bacteria were included in these investigations [Jarling et al, 2015;Rabus et al, 2011]. In a targeted study it was shown that three tested nalkane utilizers were able to convert toluene to benzylsuccinate when supplemented with a growth-supporting nalkane .…”