Bacterial community structure and bamA gene diversity in anaerobic degradation of toluene and benzoate under denitrifying conditions

Abstract

Aim: To characterize the microbial community structure and bamA gene diversity involved in anaerobic degradation of toluene and benzoate under denitrifying conditions. Methods and Results: Nitrate‐reducing enrichment cultures were established on either toluene, benzoate or without additional substrate. Bacterial community structures were characterized by 16S rRNA gene–based PCR‐DGGE analysis. bamA gene diversity was analysed using DGGE and cloning methods. The results showed that bamA gene related to bamA of Thauera chlorobenzoica was dominant in toluene and benzoate cultures. However, a greater diversity of sequences was obtained in benzoate cultures. Low diversity of bamA gene was observed, and some similarities of bamA were also found between active cultures and backgrounds, suggesting that potential natural attenuation of aromatic compounds might occur. Conclusions: The combined analysis of 16S rRNA and bamA genes suggests that the species related to genera Thauera dominated toluene‐ and benzoate‐degrading cultures. The combination of multiple methods (DGGE and cloning) provides a more complete picture of bamA gene diversity. Significance and Impact of the Study: To our knowledge, this is the first report of bamA gene in denitrifying enrichments using DGGE and cloning analysis.

Functional Gene Markers for Fumarate-Adding and Dearomatizing Key Enzymes in Anaerobic Aromatic Hydrocarbon Degradation in Terrestrial Environments

“…They have also been found in a number of anaerobic monoaromatic hydrocarbon-degrading pure cultures which do not have a published genome yet, for example G. toluolica [Staats et al, 2011], Desulfosarcina spp., Desulfobacterium anilini and strains of the Gram-positive Desulfotomaculum gibsoniae and Desulfotomaculum thermobenzoicum , in p -xylene-degrading enrichment cultures dominated by Desulfosarcina ovata [Higashioka et al, 2011], as well as in toluene-degrading denitrifying enrichments hosting bamA genes related to Thauera chlorobenzoica [Li et al, 2012]. In contrast, a diversity of different bamA genes has recently been reported for a number of sulphate-reducing, denitrifying and methanogenic toluene-degrading enrichments obtained from various soil and sludge samples [Sun et al, 2014a].…”

“…The ring-cleaving bamA genes were first shown to be detectable in a number of sulphate-reducing [Kuntze et al, 2008] or nitrate-reducing aromatic compound-degrading enrichment cultures [Li et al, 2012]. They have also been found in a number of anaerobic monoaromatic hydrocarbon-degrading pure cultures which do not have a published genome yet, for example G. toluolica [Staats et al, 2011], Desulfosarcina spp., Desulfobacterium anilini and strains of the Gram-positive Desulfotomaculum gibsoniae and Desulfotomaculum thermobenzoicum , in p -xylene-degrading enrichment cultures dominated by Desulfosarcina ovata [Higashioka et al, 2011], as well as in toluene-degrading denitrifying enrichments hosting bamA genes related to Thauera chlorobenzoica [Li et al, 2012].…”

“…Although this analysis is not a strong diagnostic tool, the identity of distinct terminal restriction fragments can cautiously be elucidated via the cross-referencing of fragment lengths to reference sequences digested in silico, possibly even based on dual digests to increase discriminative confidence [von Netzer et al, 2013]. Similarly, denaturing gradient gel electrophoresis fingerprinting of bamA gene pools has also been applied to analyse the community structure of anaerobic degraders [Andrade et al, 2012;Li et al, 2012].…”

“…For a wide range of bamA/bzdY/oah genes, the forward primer SP9F in combination with the reverse prim-er ASP1R [Kuntze et al, 2008], as well as the slightly modified combination oah_f/oan_r [Staats et al, 2011], has been successfully used in numerous studies, thus demonstrating its applicability for detecting a wide range of anaerobic monoaromatic compound degraders [Andrade et al, 2012;Higashioka et al, 2011;Li et al, 2012;Porter and Young, 2013;Sun et al, 2014a]. Two additional primer sets were designed using the same forward primer SP9F but different reverse primers ASP23R and ASP33R targeting ring-opening hydrolase subclusters, including Gram-positive anaerobic monoaromatic compound degraders, as described above .…”

“…were designed later to recover a wider diversity of degraders, including also Gram-positives . Thus, in the mean- time, the bamA gene was also established as a widely applied biomarker for anaerobic degradation of monocyclic aromatic compounds [Andrade et al, 2012;Li et al, 2012;Young, 2013, 2014;Sun et al, 2014a].…”