Characterization of anaerobic xylene biodegradation by two‐dimensional isotope fractionation analysis

Abstract

We determined stable carbon and hydrogen isotope fractionation factors for anaerobic degradation of xylene isomers by several pure and mixed cultures. All cultures initiated xylene degradation by the addition of fumarate to a methyl moiety, as is known from the literature or verified by the presence of methylbenzylsuccinates as metabolic intermediates. Additionally, the A subunit of benzylsuccinate synthase (bssA) was identified in the majority of the cultures by bssA-targeted primers. Xylene degradation was always coupled to a significant carbon and hydrogen isotope fractionation. The values of the apparent kinetic isotope effect (AKIE) for carbon and hydrogen indicate that the cleavage of a carbon-hydrogen bond is an isotope-sensitive step during fumarate addition to xylene isomers. The slopes of the linear regression for hydrogen (Δδ(2) H) versus carbon (Δδ(13) C) discrimination (Λ = Δδ(2) H/Δδ(13) C ≈ εHbulk /εCbulk ) ranged from 12 ± 4 to 29 ± 5 and were comparable to Λ values previously determined for anaerobic toluene degradation. The results suggest that combined carbon and hydrogen isotope fractionation analyses can be used to monitor anaerobic xylene degradation at contaminated sites.

Diversity of Benzylsuccinate Synthase-Like ( bssA ) Genes in Hydrocarbon-Polluted Marine Sediments Suggests Substrate-Dependent Clustering

“…Sequences closely related to this hypothetical D. gibsoniae bssA gene were also found in environmental samples where methanogenesis was prevalent and where a Clostridium counterpart was suggested to be involved in toluene degradation (32,53). Moreover, several sequences in cluster II were retrieved from environments dominated by sulfatereducing conditions, where hydrocarbon oxidizers were associated with communities where Proteobacteria were dominant (35,52).…”

“…Cluster II included the previously described clusters T1 and T2 of unidentified BssA homologues, which constituted two distinct groups within this cluster (35). Sequences included in cluster II were associated with the in situ oxidation of BTEX compounds, including p-xylene, in sediment enrichments under sulfate-reducing conditions (35,52). Only 26 of our sediment sequences clustered within this group and were retrieved primarily from the Atlantic Islands contaminated sediments.…”

“…Eight bssA gene libraries were constructed from DNA isolated from each sediment sample, and 70 clones from each library were sequenced. As previously described, non-bssA-related DNA fragments of the same size were also obtained by using these primers, which represented up to one-third of the analyzed sequences (see Table S4 in the supplemental material) (35,52).…”

Stable Isotope Probing Approaches to Study Anaerobic Hydrocarbon Degradation and Degraders

“…In the cultures tested so far, xylene is also activated via fumarate addition by BSS. The key players and enzymatic disposition of an m -xylene-degrading sulfate-reducing culture enriched from BTEX-contaminated groundwater have been elucidated by DNA-SIP and protein-SIP using partially 13 C-labeled m -xylene [Bozinovski et al, 2012;Herrmann et al, 2009]. These studies showed that a phylotype affiliated to the Desulfobacteraceae was the main m -xylene-assimilating organism due to the dominance of its 16S rRNA gene in heavy DNA, and the high percentage of identified 13 C-labeled proteins affiliated to this family.…”

Combined Application of PCR-Based Functional Assays for the Detection of Aromatic-Compound-Degrading Anaerobes

“…Moreover, a recent study indicated that the bssA-targeting assay appears not to be applicable for all bssA-like enzyme-containing organisms (11).…”