Nitrate-assisted biodegradation of polycyclic aromatic hydrocarbons (PAHs) in the water-level-fluctuation zone of the three Gorges Reservoir, China: Insights from in situ microbial interaction analyses and a microcosmic experiment

Highlights

• Higher PAH concentrations occurred in the urban section.

• The anaerobic pathway dominated in the biodegradation of PAHs.

• Nitrate was the primary electron acceptor for the anaerobic biodegradation of PAHs.

• Anaerolineaceae, Dechloromonas, Bacteroidetes & Geobacter were the keystone species.

Abstract

An increase in polycyclic aromatic hydrocarbon (PAH) pollution poses significant challenges to human and ecosystem health in the Three Gorges Reservoir (TGR) of the Yangtze River. Based on the combination of PAH analysis with qPCR and high-throughput sequencing of bacteria, 32 topsoil samples collected from 16 sites along the TGR were used to investigate the distribution and biodegradation pathways of PAHs in the water-level-fluctuation zone (WLFZ). The results indicated that the concentrations of PAHs were 43.8–228.2 and 30.8–206.3 ng/g soil (dry weight) under the high- and low-water-level (HWL and LWL) conditions, respectively. The PAH concentration in urban areas was higher than that in rural areas. Under both the HWL and LWL conditions, the abundance of the bamA gene, a biomarker of anaerobic PAH biodegradation, was significantly higher than that of the ring-hydroxylating-dioxygenase (RHD) gene, a biomarker of aerobic PAH biodegradation. The abundance of the bamA gene was significantly positively correlated with PAHs (R² = 0.8), and the biodegradation percentage of PAHs incubated anaerobically was greater than that in the aerobically incubated microcosm experiments. These data implicated a key role of the anaerobic pathway in PAH biodegradation. Co-occurrence network analysis suggested that anaerobic Anaerolineaceae, Dechloromonas, Bacteroidetes_vadin HA17 and Geobacter were key participants in the biodegradation of PAHs. The diversity analysis of functional bacteria based on the bamA gene and microcosm experiments further demonstrated that nitrate was the primary electron acceptor for PAH biodegradation. These findings provide a new perspective on the mechanism of PAH biodegradation in the TGR and knowledge that can be used to develop strategies for environmental management.

Introduction

Polycyclic aromatic hydrocarbons (PAHs), which are carcinogenic, teratogenic, mutagenic, and harmful to human health and the ecological environment, are ubiquitous persistent organic pollutants (POPs) (Deyerling et al., 2014; Zhang et al., 2019a). PAHs in the environment may originate from natural sources such as volcanoes and forest fires but are derived mainly from anthropogenic sources such as the incomplete combustion of biomass and fossil fuels and oil spills (Wang et al., 2017; Zhang et al., 2019a). Owing to their high hydrophobicity and low water solubility, PAHs are readily adsorbed onto soil particles and difficult to degrade. Hence, they can remain in the soil for decades (Ping et al., 2007; Wang et al., 2017; Zhang et al., 2019a). Studies have shown that soil is the most important sink for PAHs and is estimated to hold nearly 90% of the total PAHs in the environment (Wang et al., 2015; Zhang et al., 2019a). Further, PAHs in the soil may be emitted into water and the atmosphere through evaporation, leading to secondary contamination (Sarria-Villa et al., 2016). Hence, soil plays a vital role in controlling PAH contamination and in understanding the human exposure risk to these toxic pollutants (Hu et al., 2017a).

The water-level-fluctuation zone (WLFZ) is a special habitat with alternating flood and dry cycles caused by periodic submergence and exposure due to water level changes in rivers and reservoirs (Zhang et al., 2012). To achieve the multiple purposes of power generation, shipping and flood control, the water level of the Three Gorges Reservoir (TGR), China, is kept between 175 m from Oct to Mar and 145 m from Mar to Oct. Hence, the WLFZ is a special sink for pollutants because it suffers from the effects of both aquatic and terrestrial ecosystems (Ye et al., 2011). Currently, severe pollution, including that involving organics, heavy metals, nitrogen and phosphorus, has been found in the WLFZ (Hu et al., 2017a; Xia et al., 2018; Ye et al., 2019). Among them, PAHs have been suggested to be a priority organic pollutant and have drawn broad attention in the WLFZ soil of the TGR (Floehr, 2015; Hu et al., 2017a). Under the low-water-level (LWL) conditions, the PAHs in the WLFZ generally originate from domestic sewage and industrial wastewater as well as atmospheric precipitation. During the high-water-level (HWL) period, PAHs from shipping and pollutants seep into the water in the upper reaches and can accumulate in the WLFZ during flooding (Hu et al., 2017a). The ΣPAH content in WLFZ soil has previously been reported to range from 18.4 to 463.1 ng/g, and the highest PAH occurrence was detected in the Changshou district of the Chongqing municipality (Hu et al., 2017a).

Biodegradation is a unique economic and sustainable method for removing PAHs, and this metabolic activity is readily effective in the presence of molecular oxygen (Toyama et al., 2011; Xu et al., 2014; Luo et al., 2019). In this process, the aromatic nucleus is activated by inserting molecular oxygen and forming cis-dihydrodiol through a multicomponent aromatic ring-hydroxylating-dioxygenase (RHD) enzyme system (Kauppi et al., 1998). The genes that encode the enzymes are widely found in gram-negative (GN) and some gram-positive (GP) bacteria (Habe and Omori, 2003). However, in polluted sites, such as soil and sediment, oxygen is usually depleted by the degradation of easily degradable organics and cellular respiration (Lovley, 1997). Under anaerobic conditions, sulphate, carbon dioxide, ferrihydrite and nitrate are common electron acceptors for the mineralization of PAHs (Meckenstock et al., 2016; Nzila, 2018; Peng et al., 2017; Vogt et al., 2011; Yang et al., 2020). In this process, organisms can transform aromatic compounds to benzoyl-CoA prior to ring cleavage (Boll, 2005). The enzyme 6-oxocyclohex-1-ene-1-carbonyl-CoA hydrolase, encoded by the bamA gene, is a key enzyme in the process of reductive benzene ring cleavage (Porter and Young, 2014; Ruan et al., 2016; Wischgoll et al., 2005). Hence, the RHD and bamA genes are ideal biomarkers for PAH-degrading bacteria in aerobic and anaerobic pathways, respectively (Kuntze et al., 2011; Porter and Young, 2013; Staats et al., 2011). In the environment, in situ microbial interaction analyses and microcosm experiments are effective tools for exploring the biodegradable mechanism of PAHs. Xu et al. (2015) reported that nitrate plays a vital role in aromatic hydrocarbon biodegradation in river sediment based on a functional genes analysis. Moreover, nitrate has been proven to be an important electron acceptor for PAH biodegradation in river estuary sediment based on an in situ microbiota and microcosmic experiment (Ribeiro et al., 2018). In addition, a network analysis based on co-occurrence patterns provides a new way of identifying keystone species and illustrating the relationship between geochemical properties (including PAHs) and microbial communities in lakes, rivers, soil, coastal sediment and activated sludge bacteria (Barberan et al., 2012; Hu et al., 2017b; Ju and Zhang, 2015; Yan et al., 2019). Recent investigations showed that iron and nitrate reduction were key factors promoting PAH degradation in two different riverine sediments (Yan et al., 2019; Yang et al., 2020). Hence, the combination of qPCR, co-occurrence network analyses and microcosmic experiments can be used as operational tools to identify the PAH degradation pathway and key functional bacteria to develop sustainable bioremediation strategies for PAH-contaminated environments (Atashgahi et al., 2018).

However, nearly all of the previous research has focused on occurrence, source and health risk assessments (Deyerling et al., 2014;Floehr, 2015; Hu et al., 2017a; Tang et al., 2017), and few studies have focused on the degradation of PAHs driven by bacteria in the WLFZ of the TGR. Therefore, it is still unclear whether the natural attenuation of PAHs is an obstacle when taking appropriate measures to promote the removal of PAHs in the WLFZ. In this study, we proved that the concentration of PAHs in urban sections was significantly higher than that in the rural sections in the WLFZ of the TGR. Further, the results of the network analyses and microcosmic experiments suggested that Anaerolineaceae, Dechloromonas, Bacteroidetes and Geobacter were key species, and anaerobic pathways with nitrate as the electron acceptor play a key role in the biodegradation of PAHs. These findings provide crucial data for developing effective strategies to promote the remediation of PAH contamination in the WLFZ of the TGR.