Salt selected for hydrocarbon-degrading bacteria and enhanced hydrocarbon biodegradation in slurry bioreactors

Highlights

• Salt (≤ 5%) enhanced hydrocarbon mineralization in the absence of nutrients.

• Total microbial population size decreased with salt addition.

• Abundance of a halo-tolerant hydrocarbon-degrading genus increased with salt.

• Higher expression of hydrocarbon-degradation related genes with salt addition.

Abstract

Hydrocarbon and salt contamination of surface and groundwater resources often co-occur from oil production activities. However, salt is often considered as a potential inhibitor of microbial activity. The feasibility of microbiome-based biotechnologies to treat the hydrocarbon contamination is contingent on the ability of the indigenous community to adapt to saline conditions. Here, we demonstrate enhanced hydrocarbon biodegradation in soil slurries under saline conditions of up to ~1 M (5%) compared to non-saline systems and the underlying causes. The mineralization extent of hexadecane was enhanced by salinity in the absence of nutrients. Salinity, similar to nutrients, enhanced the mineralization but through ecological selection. Microbial community analysis indicated a significant enrichment of Actinobacteria phylum and an increase in the absolute abundance of the hydrocarbon-degrading Dietzia genus, but a decrease in the total population size with salinity. Moreover, the in situ expression of alkane hydroxylases genes of Dietzia was generally increased with salinity. The data demonstrate that indigenous halotolerant hydrocarbon degraders were enriched, and their hydrocarbon degradation genes upregulated under saline conditions. These findings have positive implications for engineered biotreatment approaches for hydrocarbons in saline environments such as those affected with produced waters and oil sands tailing ponds.

Introduction

The release of produced waters in oil and gas production sites often lead to groundwater co-contaimantion with salt and hydrocarbons. Salinities in the range of 0.2-4 M (1-20%) have been reported in pore waters of shale formations (Waldron et al., 2007), oil sands (Hum et al., 2006), shale oil fields (An et al., 2017; Shrestha et al., 2017; Strong et al., 2014), and in flowback waters from shale gas fields (Daly et al., 2016). Despite efforts for water recycling, a large portion of process-affected waters end up in tailing ponds and evaporation pits. Reuse of process-affected waters without treatment leads to adverse environmental effects. For example, untreated, diluted saline-produced water used for irrigation resulted in decreased crop yield, and reduced soil health and microbial diversity (Miller et al., 2020). Efficient and sustainable management of significant volumes of process-affected waters and clay-rich fine tailings co-contaminated with high salt and hydrocarbon is challenging (Foght et al., 2017; Heyob et al., 2017) but is necessary to prevent further surface and groundwater contamination. Moreover, the natural migration of formation brines to shallow aquifers (Warner et al., 2012), saline oil and gas wastewater spills (Shrestha et al., 2017), and leaking oil wells also lead to surface and groundwater contamination by salts and hydrocarbons. Biotechnology that uses hydrocarbon-degrading microbial populations has the potential to provide sustainable treatment solutions if their metabolic activity under high salinity conditions can be maintained.

Salinity can alter biodegradation rates of hydrocarbons and organic matter and influence the biogeochemical cycles of carbon in natural or engineered systems. It is well known that salt can adversely affect microbial life, for example, by disrupting the cell membrane or denaturation of enzymes (Polonenko et al., 1981). Lozupone and Knight demonstrated that salinity is generally the most significant factor in shaping microbial communities as compared to other ecologically determinant environmental factors such as temperature, or pH (Lozupone and Knight, 2007). Microbial adaptation to salt stress is regulated by the amount of energy required and the mode of osmotic adaptation (Oren, 2011). Halophilic and halotolerant bacteria may maintain their osmotic equilibrium by accumulating high concentrations of “compatible” organic solutes such as glycine betaine to adapt to a wide range of salinities (Fathepure, 2014; Hu et al., 2020; Oren, 2011). Various pathways of encoding glycine betaine were identified in the microbial genome from fracking effluents. Glycine betaine itself was also detected in those samples (Daly et al., 2016). Low levels of salt maintained in the cytoplasm in the presence of compatible solutes enable halophilic and halotolerant bacteria to still perform some metabolic processes, at least under moderate salinities (0.1-2 M, 0.5-11%). However, there is limited literature to assess if hydrocarbon degraders in indigenous communities of contaminated saline environments can thrive and maintain their metabolic activity through adaptations.

Using observations of metabolic behaviour or predictions from genomics, previous studies have identified halotolerant, hydrocarbon-degrading bacterial strains (Daly et al., 2016; Fang et al., 2017). However, no study has evaluated the degradation activity and transcriptomic response of indigenous halotolerant, hydrocarbon-degrading species in natural low salinity environments once exposed to high salt levels. High concentrations of salt in oil-contaminated environments provide an ecological niche for halotolerant hydrocarbon-degrading bacteria. Identifying conditions to stimulate the metabolic activity of halotolerant hydrocarbon degraders, and thorough characterization of their growth and metabolic activity in their natural communities may provide new remediation strategies for saline hydrocarbon-contaminated sites.

In this study, we compared the effect of salt addition (2.5 and 5 wt% (0.4-0.9 M) added NaCl), which corresponds to the more frequently detected salinities in sites, and no salt addition on the mineralization extent of ¹⁴C-labelled hexadecane in hexadecane and diesel spiked into historically oil-contaminated soils, in slurry bioreactors. Hexadecane is representative of the poorly soluble, non-volatile, but biodegradable fraction of petroleum hydrocarbons (Chang et al., 2011). Moreover, the dynamics of the microbial community and biodegradation activity over time were characterized in microcosms containing pure liquid hexadecane or diesel. This allowed examination of how the multitude of more soluble and thus readily bioavailable substrates in diesel affects the diversity and activity of microbial communities. Moreover, the population size and the absolute abundance of the indigenous halotolerant hydrocarbon-degrading Dietzia maris were determined using Droplet Digital PCR. Also, the in situ activity of the strain in the microbial community in terms of the expression of alkane hydroxylase genes was assessed with quantitative reverse transcription PCR (qRT-PCR).