Sulfide Generation by Dominant Halanaerobium Microorganisms in Hydraulically Fractured Shales

Abstract

Hydraulic fracturing of black shale formations has greatly increased United States oil and natural gas recovery. However, the accumulation of biomass in subsurface reservoirs and pipelines is detrimental because of possible well souring, microbially induced corrosion, and pore clogging. Temporal sampling of produced fluids from a well in the Utica Shale revealed the dominance of Halanaerobium strains within the in situ microbial community and the potential for these microorganisms to catalyze thiosulfate-dependent sulfidogenesis. From these field data, we investigated biogenic sulfide production catalyzed by a Halanaerobium strain isolated from the produced fluids using proteogenomics and laboratory growth experiments. Analysis of Halanaerobium isolate genomes and reconstructed genomes from metagenomic data sets revealed the conserved presence of rhodanese-like proteins and anaerobic sulfite reductase complexes capable of converting thiosulfate to sulfide. Shotgun proteomics measurements using a Halanaerobium isolate verified that these proteins were more abundant when thiosulfate was present in the growth medium, and culture-based assays identified thiosulfate-dependent sulfide production by the same isolate. Increased production of sulfide and organic acids during the stationary growth phase suggests that fermentative Halanaerobium uses thiosulfate to remove excess reductant. These findings emphasize the potential detrimental effects that could arise from thiosulfate-reducing microorganisms in hydraulically fractured shales, which are undetected by current industry-wide corrosion diagnostics. IMPORTANCE Although thousands of wells in deep shale formations across the United States have been hydraulically fractured for oil and gas recovery, the impact of microbial metabolism within these environments is poorly understood. Our research demonstrates that dominant microbial populations in these subsurface ecosystems contain the conserved capacity for the reduction of thiosulfate to sulfide and that this process is likely occurring in the environment. Sulfide generation (also known as "souring") is considered deleterious in the oil and gas industry because of both toxicity issues and impacts on corrosion of the subsurface infrastructure. Critically, the capacity for sulfide generation via reduction of sulfate was not detected in our data sets. Given that current industry wellhead tests for sulfidogenesis target canonical sulfate-reducing microorganisms, these data suggest that new approaches to the detection of sulfide-producing microorganisms may be necessary.

Keywords: Halanaerobium; shale; thiosulfate.

FIG 1

(A) Geochemical measurements of sulfur species from produced waters (~120-day time series) in Utica Shale. Sulfur isotope measurements were used to measure the reduction of oxidized sulfur species (sulfate and sulfonate from thiosulfate). The dashed line indicates down-hole injection of input fluids. Following the hydraulic fracturing process, the well was sealed for 86 days, during which no sampling could occur. (B) Changes in the relative abundance of Halanaerobium microorganisms in the period after hydraulic fracturing of the well. The dashed line indicates relative Halanaerobium abundance in input fluids prior to down-hole injection. (C) Number of reconstructed genes linked to thiosulfate transformations from metagenomic data sets collected during the monitoring period. Red indicates genes linked to Halanaerobium.

FIG 2

Phylogenetic placement of the environmental Halanaerobium isolate used in this study (WG8; highlighted in blue) relative to the reconstructed genomes of dominant Halanaerobium bacteria in Utica Shale-produced water samples (highlighted in orange). The eight Utica Shale Halanaerobium isolates are WG1, WG2, and WG5 to WG10. The numbers of rhodanese-encoding genes and anaerobic sulfite reductase subunits in each genome are illustrated to the right.

FIG 3

(A) Hydrogen sulfide production via thiosulfate reduction in live-cell incubations. (B) Growth curves of Halanaerobium WG8 in the presence or absence of thiosulfate. The calculated growth rates were 0.20 and 0.21 h⁻¹, respectively. (C) Changes in the concentrations of glucose and major Halanaerobium fermentation products when bacteria are cultured in the presence (+) or absence (−) of thiosulfate at both mid-log (Mid) and stationary (Stat) growth phases. In all cases, error bars represent the standard deviation of the mean of triplicate biological replicates.

FIG 4

(A) Relative abundances of key proteins identified via shotgun proteomics that are implicated in sulfur transformations by Halanaerobium bacteria grown in the presence (+thio) or absence (−thio) of thiosulfate. (B) Reactions catalyzed by the rhodanese and anaerobic sulfite reductase enzymes.