Benzene Degradation by a Variovorax Species within a Coal Tar-Contaminated Groundwater Microbial Community

Abstract

Investigations of environmental microbial communities are crucial for the discovery of populations capable of degrading hazardous compounds and may lead to improved bioremediation strategies. The goal of this study was to identify microorganisms responsible for aerobic benzene degradation in coal tar-contaminated groundwater. Benzene degradation was monitored in laboratory incubations of well waters using gas chromatography mass spectrometry (GC-MS). Stable isotope probing (SIP) experiments using [13C]benzene enabled us to obtain 13C-labled community DNA. From this, 16S rRNA clone libraries identified Gammaproteobacteria and Betaproteobacteria as the active benzene-metabolizing microbial populations. Subsequent cultivation experiments yielded nine bacterial isolates that grew in the presence of benzene; five were confirmed in laboratory cultures to grow on benzene. The isolated benzene-degrading organisms were genotypically similar (>97% 16S rRNA gene nucleotide identities) to the organisms identified in SIP experiments. One isolate, Variovorax MAK3, was further investigated for the expression of a putative aromatic ring-hydroxylating dioxygenase (RHD) hypothesized to be involved in benzene degradation. Microcosm experiments using Variovorax MAK3 revealed a 10-fold increase in RHD (Vapar_5383) expression, establishing a link between this gene and benzene degradation. Furthermore, the addition of Variovorax MAK3 to microcosms prepared from site waters accelerated community benzene degradation and correspondingly increased RHD gene expression. In microcosms using uninoculated groundwater, quantitative (q)PCR assays (with 16S rRNA and RDH genes) showed that Variovorax was present and responsive to added benzene. These data demonstrate how the convergence of cultivation-dependent and -independent techniques can boost understandings of active populations and functional genes in complex benzene-degrading microbial communities. IMPORTANCE Benzene is a human carcinogen whose presence in contaminated groundwater drives environmental cleanup efforts. Although the aerobic biodegradation of benzene has long been established, knowledge of the identity of the microorganisms in complex naturally occurring microbial communities responsible for benzene biodegradation has evaded scientific inquiry for many decades. Here, we applied a molecular biology technique known as stable isotope probing (SIP) to the microbial communities residing in contaminated groundwater samples to identify the community members active in benzene biodegradation. We complemented this approach by isolating and growing in the laboratory a bacterium representative of the bacteria found using SIP. Further characterization of the isolated bacterium enabled us to track the expression of a key gene that attacks benzene both in pure cultures of the bacterium and in the naturally occurring groundwater microbial community. This work advances information regarding the documentation of microbial processes, especially the populations and genes that contribute to bioremediation.

Methylene blue decolorizing bacteria isolated from water sewage in Yogyakarta, Indonesia

“…Similar to Acidovorax sp., Variovorax sp. isolated from a coal tar-contaminated aquifer possess putative aromatic ring hydroxylation dioxygenase enzymes that enable bacteria to degrade benzene (Posman et al 2017). However, there is no information regarding the ability of Xylophilus to facilitate bioremediation.…”

Community dynamics and functional characteristics of naphthalene‐degrading populations in contaminated surface sediments and hypoxic/anoxic groundwater

“…A) and in the near identical pairwise average nucleotide identities between corresponding MAGs (> 99.4%; Supporting Information Table S1). Two strains of benzene‐degrading Rhodococcus previously isolated from the deep MW were identical across the full‐length 16S rRNA gene (‘MAK2’ and ‘MAK4’; Posman et al ., ). Similarly, near identical 16S rRNA gene clones for Stenotrophomonas (1261/1269 bases) and Ralstonia (1198/1201 bases) were present in an anaerobic SIP‐benzene experiment from surface sediments (accessions: EU499720.1 and EU499693.1; Liou et al ., ).…”

“…S1). The biogeochemistry and microbial ecology of both sites have been characterized for over two decades (Madsen et al, 1991;Madsen et al, 1996;Bakermans et al, 2002;Jeon et al, 2003;Liou et al 2008;Yagi et al, 2010a;Posman et al, 2017).…”

“…The presence of genes involved in biofilm formation, motility and electron shuttling were also assessed (Supporting Information). All functional genes and phylogenetic gene markers from previous research at Site 24 were recovered using accessions for surface sediment studies: Jeon and colleagues () and Liou and colleagues (), and groundwater studies: Yagi and Madsen (), Yagi and colleagues () and Posman and colleagues ().…”

Immune response characterization of mice immunized with Lactobacillus plantarum expressing spike antigen of transmissible gastroenteritis virus

“…Bacterial genomic DNA were extracted by bacterial genomic DNA kit according to the manufacturer's instructions (CW Biotech, China). For general bacterial identification, 16S rRNA universal primers were selected (Posman et al 2017). The most commonly used universal primer was 27F/1492R, 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, 1492R: 5′-GGTTACCTTGTTACGACTT-3′.…”