Microbial selenate reduction in membrane biofilm reactors using ethane and propane as electron donors

Highlights

• Microbial Se(VI) reduction was achieved using C₂H₆ or C₃H₈ as electron donors.

• Zero valent Se was the final product formed from Se(VI) bio-reduction.

• PHAs generated in the biofilms contributed to Se(VI) bio-reduction.

• Mycobacterium/Rhodococcus and potential Se(VI) reducers were enriched.

Abstract

Selenate (Se(VI)) contamination in groundwater is one of major concerns for human health, in particular in shale gas extraction sites. Microbial selenate reduction coupled to methane (CH₄) oxidation has been demonstrated very recently. Little is known whether ethane (C₂H₆) and butane (C₃H₈) are able to drive selenate reduction, although they are also important components in shale gas. In this study, we demonstrated Se(VI) bio-reduction could be achieved using C₂H₆ and C₃H₈ as electron donors and carbon sources. Scanning electron microscopy coupled to energy dispersive X-ray spectroscopy (SEM-EDX) confirmed elemental Se (Se⁰) was the major final product formed from Se(VI) bio-reduction. Polyhydroxyalkanoates (PHAs) were generated in the biofilms as the internal electron-storage materials, which were consumed for sustaining Se(VI) bio-reduction in absence of C₂H₆ and C₃H₈. Microbial community analysis showed that two genera capable of oxidizing gaseous alkanes dominated in the biofilms, including Mycobacterium (in both C₂H₆ and C₃H₈-fed biofilms) and Rhodococcus (in C₃H₈-fed biofilm). In addition, several potential Se(VI) reducers (e.g., Variovorax) were detected in the biofilms. Investigation of Communities by Reconstruction of Unobserved States analysis supported that predictive genes associated with alkanes oxidation, denitrification and PHAs cycle were enriched in the biofilms. These findings offer insights into the process of selenate reduction driven by C₂H₆ and C₃H₈, which ultimately may help to develop a solution to use shale gas for groundwater remediation, especially near shale gas exploitation sites.

Introduction

Shale gas has attracted increasingly commercial interests and become economically recoverable over the last decades (Gregory et al., 2011). It is globally estimated that shale gas reserves are up to 7.3 × 10³ trillion ft³ (U.S. EIA et al., 2013). The price of shale gas is decreasing continuously due to new horizontal drilling and efficient extraction technologies. Shale gas is composed of various saturated gaseous alkanes, including methane (CH₄), ethane (C₂H₆), and propane (C₃H₈) (Ambrose et al., 2011; Jackson et al., 2013). While shale gas contains CH₄ as the primary component, the amounts of C₂H₆ and C₃H₈ could reach up to 20% and 10%, respectively, in shale gas (Umukoro and Ismail, 2017). However, accompanied with the exploitation, shale gas is readily to migrate to surrounding water systems including drinking water (Jackson et al., 2013; Osborn et al., 2011), groundwater (Llewellyn et al., 2015), and surface water (Zeng et al., 2013). For example, 23 times higher C₂H₆ was detected in drinking water wells when Marcellus shale gas was exploited (Jackson et al., 2013). The leakage of shale gas has become a public concern because the contained gaseous alkanes were potent greenhouse gases and they may cause photochemical pollution (Etiope and Ciccioli, 2009).

On the other hand, the shales commonly contain selenium (Se) (up to 50 mg/kg) (Tourtelot, 1962). The enrichment of Se in the shales could be a big risk of Se contamination to the surrounding water systems (especially when shale gas is exploited), which has been reported in Cretaceous black shales in Montana and Barnett Shales in Texas, USA (Mayland et al., 1989; Fontenot et al., 2013), and Bowland Shales in the north of England (Parnell et al., 2016). The concentration of dissolved Se in water systems near drilled shales (e.g., Colorado shale in central Montana) could reach up to 1 mg/L (Mayland et al., 1989). However, dissolved Se in aquatic ecosystems is a critical concern because of its acute toxicity. Se can replace sulfur (S) in proteins, reshape the structure of the proteins and damage their normal function (Lemly, 1998), thus disturbing normal metabolisms of organisms. There has been a serious Se pollution incident in Kesterson Reservoir, California, where high level of Se led to the deaths, deformities, and birth defects of a large number of waterfowls and fishes (Presser, 1994). In addition, Se could also cause cancer, cardiovascular disease, diabetes, and nervous disorder to human bodies (Sun et al., 2014). The U.S. Environmental Protection Agency (EPA) has set the maximum contaminant level (MCL) of Se at 50 μg Se/L (USEPA., 2015). In particular, selenate (SeO₄²⁻, Se(VI)) in the shales, with high toxicity and water solubility, is usually formed by weathering (Tuttle et al., 2014; Zawislanski et al., 2003). Butler et al. (1994) found Se(VI) was the dominant Se species (>97%) in groundwater samples from weathered Mancos Shale. Transformation of Se(VI) to elemental selenium (Se⁰) is beneficial for lowering Se toxicity and contamination, since Se⁰ formes as precipitates in aqueous phase thus can be easily removed.

Se(VI) could be microbially reduced to Se⁰ by phylogenetically diverse microorganisms. Specific selenate-reducing bacteria, e.g., Thauera selenatis (Schroeder et al., 1997), Enterobacter cloacae SLD1a-1 (Watts et al., 2003), and Sulfurospirillum barnesii (Watts et al., 2005), are capable of reducing Se(VI) to Se(IV) using their periplasmic selenate reductases or membrane-bound selenate reductase. Some nitrate reducers, e.g., Paracoccus pantotrophus and Ralstonia eutropha, could also carry out Se(VI) reduction using their periplasmic (Nap) or membrane-bound (Nar) nitrate reductases (Sabaty et al., 2001). Se(IV) could be further reduced to Se⁰ by periplasmic nitrite reductase (Nir) (DeMoll-Decker & Macy, 1993) or hydrogenase I (Yanke et al., 1995).

Electron donors are required to drive microbial selenate reduction. To date, lactate (Lenz et al., 2009), acetate (Navarro et al., 2015), and hydrogen (Lai et al., 2014) have been used as electron donor to reduce Se(VI). Very recently, Lai et al. (2016) found Se(VI) could be reduced to Se⁰ using methane (CH₄) as the sole electron donor and carbon source. They proposed methanotrophs could oxidized CH₄, in which intermediates such as organic metabolites would be released and used by methanotrophs or other selenate-reducing bacteria for converting Se(VI) to Se⁰. In another study, Luo et al. (2018) reported Se(VI) reduction could be mediated by nitrite/nitrate-dependent anaerobic methane oxidation microorganisms (including Candidatus Methanoperedens and Candidatus Methylomirabilis) in a CH₄ based membrane biofilm reactor (MBfR).

Although microbial selenate reduction coupled to methane oxidation has been reported (Lai et al., 2016; Luo et al., 2018), it is still unknown whether C₂H₆ and C₃H₈ are able to drive selenate reduction as electron donors. It has been well known that C₂H₆ and C₃H₈ can be aerobically oxidized in a similar metabolic pathway of CH₄ oxidation, in which C₂H₆ and C₃H₈ can be oxidized into ethanol or propanol, followed by conversion to aldehydes and acids (Shennan, 2006). Considering the highly positive redox potential of Se(VI)/Se(IV) (+440 mV) and Se(IV)/Se⁰ (+210 mV) (Nancharaiah and Lens, 2015; Doran, 1982), and the negative redox potential of C₂H₆ (−270 mV) and C₃H₈ (−277 mV) (Rittmann and McCarty, 2001), C₂H₆ or C₃H₈ coupled to Se(VI) reduction is energetically feasible. Thus, it is proposed that proper application of these C₂H₆ and C₃H₈ might be beneficial to develop solutions to utilize shale gas to eliminate selenate contamination in aquatic systems near shale gas exploitation sites. In addition, the application of shale gas as electron donor could be a preferred alternative for bio-reduction of Se(VI), since shale gas is much cheaper (∼$0.08–1.00/m³) (Markets Insider., 2020) than pure CH₄ (∼$1.34/m³) (Global Petrol Prices, 2020).

Thus, the objective of this study was to test the feasibility of Se(VI) bio-reduction using C₂H₆ and C₃H₈ in shale gas as the electron donors. For proving the concept, we set up two independent MBfRs, in which non-porous polypropylene fibers were used to deliver C₂H₆ or C₃H₈ directly to the biofilms attaching on the fiber walls. By utilizing MBfR systems, a high gaseous electron donor utilizing efficiency can be achieved (e.g., 96% of methane utilization efficiency in Cai et al., 2018), thus minimizing the off-gassing (Liu et al., 2019; Rittmann et al., 2014; Zhou et al., 2019). Both long-term operation and batch tests were conducted to study Se(VI) removal pattern with these two gaseous alkanes. The dominant microorganisms involved in C₂H₆ or C₃H₈ oxidation and selenate reduction were analyzed by using high-throughput 16S rRNA gene sequencing. In addition, key functional genes involved in C₂H₆/C₃H₈ oxidation and selenate bio-reduction were predicted by applying Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) pipeline.