Redox fluctuations shape the soil microbiome in the hypoxic bioremediation of octachlorinated dibenzodioxin- and dibenzofuran-contaminated soil

Highlights

• A rapid fluctuating redox cycling stimulates soil microbiome at hypoxic conditions.

• The evolved microbiome shows a marked degradation of PCDD/Fs at hypoxic conditions.

• Higher degradation of PCDD/Fs associates with redox fluctuated narrowly than widely.

Abstract

The biodegradation of polychlorinated-p-dioxins and dibenzofurans (PCDD/Fs) has been recently demonstrated in a single reactor under hypoxic conditions. Maintaining hypoxic conditions through periodic aerations results in a marked fluctuation of reduction–oxidation (redox) potential. To further assess the effects of redox fluctuations, we operated two fed-batch continuously stirred tank reactors (CSTRs) with sophisticated redox controls at different anoxic/oxic fluctuations to reduce PCDD/Fs in contaminated soil. The results of long-term reactor operation showed that the CSTR with redox fluctuations at a narrow range (−63 ± 68 mV) (CSTR_A) revealed a higher substrate hydrolysis level and PCDD/F degradation rate than did the CSTR with a redox potential that fluctuated at a broad range (−13 ± 118 mV) (CSTR_B). In accordance with analyses of bacterial 16S rRNA genes, the designated hypoxic conditions with added compost supported survival of bacterial populations at a density of approximately 10⁹ copies/g slurry. The evolved core microbiome was dominated by anoxic/oxic fluctuation-adapted Bacteroidetes, Alphaproteobacteria, and Actinobacteria, with higher species diversity and functionality, including hydrolysis and degradation of dioxin-like compounds in CSTR_A than in CSTR_B. Taken together, the overall results of this study expand the understanding of redox fluctuations in association with the degradation of recalcitrant substrates in soil and the corresponding microbiome.

Introduction

Polychlorinated-p-dioxins and dibenzofurans (PCDD/Fs) are toxic, bioaccumulative, and have been identified as persistent organic pollutants. Among their 210 congeners, those that substituted with chlorines at the 2,3,7,8 positions are the most harmful and regulated due to significant adverse human health and environmental effects (Cole et al., 2003; Fiedler, 2007). PCDD/F impurities are commonly produced in the manufacturing of various chlorinated pesticides, such as pentachlorophenol (Masunaga et al., 2001). With the historic boom of the chlorine industry in the 20th century and the release of large amounts of chlorinated chemicals, inventory sites of PCDD/F contamination are numerous worldwide (Weber et al., 2008). In these sites, the soil is usually contaminated with high concentrations of PCDD/Fs, in particular the fully chlorinated congeners octachlorodibenzo-p-dioxin (OCDD) and octachlorodibenzofuran (OCDF) (Lee et al., 2006; Persson et al., 2007; Li et al., 2012; Rodenburg et al., 2017). Because the degradability and decomposition rates of OCDD/F are extremely low, corresponding site remediation remains an ongoing challenging.

PCDD/Fs can be degraded aerobically and anaerobically through microbial activities with the involvement of a series of enzymes, including dehalogenases, oxygenases, dehydrogenases, and hydrolases. (Kurihara and Esaki, 2008; Sakaki and Munetsuna, 2010). Complete degradation of PCDD/Fs to CO₂, namely mineralization, is usually difficult within a pure culture; however, the transformation or detoxification of PCDD/Fs occurs in a metabolic or cometabolic microbial process (Field and Sierra-Alvarez, 2008). The produced dechlorinating metabolites (usually lower chlorinated congeners) can be potentially degraded in the pathway downstream, through which bacterial populations can conserve energy for growth (Yoshida et al., 2007). The “degraders” of PCDD/F congeners were found to be distributed in a broad phylogenetic spectrum. Numerous bacterial species under the phyla Actinobacteria, Proteobacteria, and Firmicutes with various ring-hydroxylating dioxygenases have been reported to degrade low-chlorinated congeners (chlorine number < 6) aerobically (Hiraishi, 2003). Under absolutely anaerobic conditions, members of the genus Dehalococcoides and their relatives under the phylum Chloroflexi are the only known organisms able to dechlorinate PCDD/Fs (Hiraishi, 2008). Several bacterial groups, such as Pseudomonas, Burkholderia, Clostridium, and Alcaligenes, require a primary substrate for growth and cometabolism of dioxin-like compounds (Hiraishi, 2008; Parsons and Storms, 1989; Furukawa et al., 2004; Haddock et al., 1995).

Although the PCDD/Fs can persist in the environment for a long time, laboratory and field testing studies have suggested that PCDD/Fs can be degraded by specialized microbial communities in contaminated soil and sediment, and that degradation rates can even be enhanced with biostimulated and bioaugmented microbial consortia (Hiraishi, 2003). The bioreactor method, in combination with the assembly of microbial populations cultivated with appropriate conditions or the particular degraders in the bioreactor, can be used to speed up PCDD/F degradation, thus achieving remediation goals. PCDD/F-degrading microbial activities can be induced using bioreactors under anaerobic and aerobic conditions to achieve detoxification (reductive dechlorination) and ring decomposition, respectively (Narihiro et al., 2010; Tu et al., 2014). In 2014 and 2016, our team demonstrated that potential degraders grown at various reduction–oxidation (redox) levels coexisted at oxygen-deficient or hypoxic conditions (<2 mg/L) and that their collaborative interactions facilitated the simultaneous dechlorination and oxidative decomposition of OCDD/F and metabolites in the microcosm and reactor systems (Chen et al., 2014; Chen et al., 2016).

In the reactor environment, molecular oxygen as the preferred electron acceptor is first used by microbes for the aerobic degradation of organic substances. Once molecular oxygen is no longer available, alternative electron acceptors are then used with the redox potential decreasing from the oxidizing status (positive redox values) to the reducing state (negative redox values). Microbial metabolism shifts from aerobic to facultative aerobic, and eventually to anaerobic respiration. Redox potential is an integrated parameter that reveals microbial activities under varied redox conditions and is mainly controlled by redox reactions (abundance of electron donors and acceptors) and surrounding environmental factors (pH, temperature, etc.) (Fiedler et al., 2007). Numerous studies have shown that the fluctuating redox potential can affect the transformation processes of element (C, N, and metals) cycling, such as methane production (Satpathy et al., 1997), glucose degradation (Picek et al., 2000), nitrification and denitrification (Eriksson et al., 2003), nutrient removal (Zhao et al., 1999; Białowiec et al., 2012), and arsenic speciation (Masscheleyn, 1991). These fluctuating patterns can occur on shorter (hourly to daily) or longer time scales (weekly to seasonally) and change the microbial activities and community composition in association with these periods. Hypoxic conditions span over a broad redox range outside the ranges of aerobic and anaerobic conditions and are usually subject to highly dynamic fluctuations. This feature plays a key role in determining the diversity and stability of a microbial community and its efficiency in degrading organic substances, such as dioxin-like compounds (Hiraishi, 2008). However, our understanding of redox effects on the hypoxic degradation of PCDD/Fs and the relevant microbial community remains incomplete.

In this study, two continuously stirred tank reactors (CSTR) were operated in fed-batch mode to study the effects of dynamic redox on the hypoxic degradation of PCDD/Fs and the acclimation of microbial communities in PCDD/F-contaminated soil. A fed-batch CSTR reactor provides excellent mixing of microbial populations and soil substrates and enables the precise control of growth conditions (e.g., temperature, pH, and redox) for fastidious microorganisms, as well as for the reproducible nondestructive sampling of microbial consortia for the analyses of PCDD/Fs and microbiomes. The two CSTR reactors were maintained at hypoxic conditions of specific redox ranges through different methods of controlling aeration. The performance of hypoxic bioreactors was evaluated with organic hydrolysis and OCDD/F degradation. The relevant reactor microbiomes were studied with 16S rRNA gene amplicon sequencing analysis on the Illumina sequencing platform.