Assessment of Anaerobic Toluene Biodegradation Activity by bssA Transcript/Gene Ratios

Abstract

Benzylsuccinate synthase (bssA) genes associated with toluene degradation were profiled across a groundwater contaminant plume under nitrate-reducing conditions and were detected in significant numbers throughout the plume. However, differences between groundwater and core sediment samples suggested that microbial transport, rather than local activity, was the underlying cause of the high copy numbers within the downgradient plume. Both gene transcript and reactant concentrations were consistent with this hypothesis. Expression of bssA genes from denitrifying toluene degraders was induced by toluene but only in the presence of nitrate, and transcript abundance dropped rapidly following the removal of either toluene or nitrate. The drop in bssA transcripts following the removal of toluene could be described by an exponential decay function with a half-life on the order of 1 h. Interestingly, bssA transcripts never disappeared completely but were always detected at some level if either inducer was present. Therefore, the detection of transcripts alone may not be sufficient evidence for contaminant degradation. To avoid mistakenly associating basal-level gene expression with actively degrading microbial populations, an integrated approach using the ratio of functional gene transcripts to gene copies is recommended. This approach minimizes the impact of microbial transport on activity assessment and allows reliable assessments of microbial activity to be obtained from water samples.T he use of molecular biological tools has led to significant advancements in the area of in situ bioremediation. Of particulate note is the use of quantitative PCR (qPCR) for the detection and quantification of functional genes associated with the degradation of contaminants. However, as has been repeatedly demonstrated in both column and field studies, functional gene abundance is not always predictive of contaminant degradation, nor does it consistently correlate with gene expression or contaminant concentration (1, 2). Furthermore, high gene copy numbers are often sustained when there is little or no active degradation (3, 4), making such measurements difficult to interpret.In some cases, microbial transport may provide an explanation for elevated gene copies in the absence of degradation. It has been suggested that genes recovered from groundwater samples may represent conditions upgradient of where they were sampled, because planktonic microorganisms are subjected to transport (5). As a result, organisms observed in groundwater samples may not reflect local chemical conditions; rather, their presence may be the result of transport from upstream locations where chemical conditions favored their activity and growth. The impact of microbial transport can be minimized by assessing activity with sediment sampling (6); however, the relative simplicity of groundwater sampling makes it more attractive from a practical standpoint.Functional gene transcripts have been suggested as a more reliable indicator of local contaminant biodegradation,...

Aerobic Toluene Degraders in the Rhizosphere of a Constructed Wetland Model Show Diurnal Polyhydroxyalkanoate Metabolism

“…As the catalytic activity of benzylsuccinate synthase is irreversibly destroyed by oxygen (51), its absence in the aerobic CW model makes sense; however, a surprise was that bssA transcripts were the highest of all quantified genes involved in toluene degradation. The presence of bssA transcripts without concomitant in situ anaerobic toluene degradation was previously observed (52). Basal expression of bssA was induced either by toluene or nitrate, which might explain the detection of bssA transcripts without protein translation in our system.…”

Identification of Biomarker Genes To Predict Biodegradation of 1,4-Dioxane

“…A previous study determined that the expression of tceA or vcrA remained elevated for more than 48 h after the disappearance of TCE (12). A more recent study on expression of the benzylsuccinate synthase gene, bssA, identified a rapid decline of mRNA transcripts by over 2 orders of magnitude upon depletion of nitrate (56). However, similar to our study showing maintained expression levels of DXMO and ALDH, bssA transcripts stabilized and maintained greater than 1.0 ϫ 10 4 copies/ml for over 3 weeks after nitrate was removed (56).…”

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

“…Already, Kazy et al [2010] have indicated a significant correlation between anaerobic toluene degradation rates and qPCR bssA counts in anaerobic microcosms prepared with material from a hydrocarbon-contaminated aquifer. More recently, toluene degradation activity stimulated by the addition of nitrate was quantitatively monitored via bssA transcript-to-gene ratios across a contaminant plume directly in the field [Brow et al, 2013]. Both represent relevant advances towards the future implementation of a marker gene-or transcript-based prediction of biodegradation rates in complex natural systems.…”