Anaerobic digestion is a promising strategy for the disposal of diverse categories of waste materials, such as domestic wastewater, agricultural waste, food industry waste, and municipal solid waste, because it has the advantages of low energy consumption, less investment in land and management, nutrient reclamation, and biogas generation (Gallert and Winter, 1997; He et al., 2005; Stabnikova et al., 2008; Chan et al., 2009; Holm-Nielsen et al., 2009; Lytras et al., 2021). Methane (CH₄) is the principal component of biogas produced by anaerobic digestion and can be used as clean, renewable bioenergy that can substitute fossil fuels (Holm-Nielsen et al., 2009; Haberl et al., 2012; Awe et al., 2017; Li et al., 2019). Additionally, many kinds of organic hazards, such as polycyclic aromatic hydrocarbons (PAHs), BTEX, chloramphenicol, polyacrylamide, and antibiotics, can be effectively removed from the initial substrates and converted into CH₄ via anaerobic digestion (Kunapuli et al., 2007; Agarry and Owabor, 2011; Hu et al., 2018; Guo et al., 2019; Bianco et al., 2020). Therefore, how to efficiently harness the anaerobic digestion process for the improvement of CH₄ production and removal the rate of organic pollutants has long been a global challenge.

Co-metabolism is a traditional approach adopted to enhance the degradation of refractory pollutants and CH₄ production in the anaerobic digestion system, which can vigorously promote the growth and activity of indigenous microbes by supplying some popular carbon sources (e.g., glucose, fructose, and sucrose) for microbes to the target system (Li et al., 2016a,b; Xie et al., 2016; Khan et al., 2017; Hadibarata et al., 2018; Feng et al., 2019). Glucose is one of the most favorable carbon (energy) sources for heterotrophic microbes and has been frequently used as a co-metabolic substrate to improve CH₄ production and accelerate the degradation of refractory pollutants in diverse environments (Ambrosoli et al., 2005; Jing et al., 2017; Khan et al., 2017; Shu et al., 2021; Zhou et al., 2021). For example, the addition of glucose was found to significantly enhance the degradation of fluorene, phenanthrene, and pyrene under an anaerobic denitrification system (Ambrosoli et al., 2005), and the addition of glucose effectively alleviated the inhibitory effect of gallic acid on CH₄ production in an anaerobic digestion system (Mousa and Forster, 1999). Therefore, it seems that glucose can be used as a prioritized metabolic substrate to efficiently remove refractory pollutants in anaerobic digestion systems (Li et al., 2016b; Khan et al., 2017).

Polycyclic aromatic hydrocarbons are a group of persistent organic pollutants with intensive toxicity and wide distribution, posing a serious threat to environmental and human health (Xue and Warshawsky, 2005; Couling et al., 2010; Zhou et al., 2018). Previous investigations have reported that co-metabolism triggered by glucose could significantly accelerate the degradation of PAHs under both aerobic and anaerobic digestion environments (Ambrosoli et al., 2005; Ma et al., 2011; Qin et al., 2018; Tripathi et al., 2020; Al Farraj et al., 2021). For example, the addition of glucose was reported to significantly enhance the degradation of PAHs (benzopyrene, anthracene, and pyrene) when treated with some pure bacterial species, such as Cellulosimicrobium, Sphingomonas, and Bacillus (Khanna et al., 2012; Qin et al., 2018; Al Farraj et al., 2021). Additionally, PAH degradation under methanogenesis is thermodynamically feasible and the degradation of PAHs can be coupled with methanogenesis, suggesting that remediation of PAHs pollution via the anaerobic digestion system should be a potentially ideal method marked by the properties of energy conservation and resource reuse (Chang et al., 2005; Dolfing et al., 2009; Berdugo-Clavijo et al., 2012; Wan et al., 2012; Nzila, 2018; Ye et al., 2019; Wang et al., 2021b). Whether methanogens can directly participate in the degradation of PAHs remains unclear, but some potential keystone microbes in this process have been identified. In addition to methanogenic archaea Methanosaeta, Methanoculleus, and Methanosarcina, some syntrophic bacteria, such as Methylibium, Legionella, Clostridiaceae, Citrobacter, and Pseudomonas, have also been found essential for PAH degradation and CH₄ generation (Berdugo-Clavijo et al., 2012; Zhang et al., 2012). Thus, in anaerobic digestion, methanogenesis is the pivotal step that not only can harness biogas (CH₄) production but also can be coupled with PAH degradation. However, the co-metabolic effect induced by glucose on PAH degradation and CH₄ production under methanogenesis has seldom been reported.

In this study, we enriched the phenanthrene-degrading microbial consortium under methanogenic conditions from a long-term rice paddy, which was used to investigate the co-metabolic effect of glucose on CH₄ production and phenanthrene degradation during methanogenesis. The response of the microbial community and functional composition to the bio-stimulation of glucose in the enriched microbial consortium was further analyzed based on the results of metagenomic sequencing. This study aimed to examine whether the co-metabolic substrate (glucose) could enhance CH₄ production and phenanthrene degradation under the methanogenesis system and explore the community and functional responses of the phenanthrene-degrading microbial consortium to the bio-stimulation of glucose, providing useful information for the enhancement of CH₄ production and PAHs elimination during the anaerobic digestion of waste containing PAHs.

In this study, the CH₄ production rate of the enriched microbial consortium was significantly improved by the addition of glucose (Figure 3A), which was consistent with findings in the literature that glucose could enhance CH₄ production in anaerobic digestion systems (Bucha et al., 2018; Maletic et al., 2018; Vu and Min, 2019; Li et al., 2021). Compared with phenanthrene, glucose can be more quickly and easily utilized and subsequently converted into carbon dioxide (CO₂), hydrogen (H₂), and acetate by the microbes in the enriched consortium under anaerobic conditions, supplying feasible substrates for methanogens to synthesize CH₄ (Khan et al., 2017; Zhang et al., 2019). However, our results indicated that the addition of glucose did not promote the degradation of phenanthrene (Figure 3B). Several studies have reported the enhancement of PAH degradation caused by glucose addition or other co-metabolic substrates under anaerobic conditions (Yuan and Chang, 2007; Ma et al., 2011; Li et al., 2016a). For example, Li et al. (2016a) reported that glucose addition enhanced the degradation rate of petroleum hydrocarbons by 200%, while the promotion of petroleum hydrocarbons degradation was mainly attributed to the stimulation of the biocurrent triggered by glucose. Maletic et al. (2018) found that glucose addition improved CH₄ production but had no effect on the degradation of PAHs, which is consistent with our current results and should be explained by the following reasons. The utilization of phenanthrene was delayed by the addition of glucose, which should start after the complete consumption of glucose, and then the metabolism of glucose resulted in the improvement of CH₄ production. However, the glucose addition might neither promote microbial activity to degrade phenanthrene nor induce the microbial consortium to produce phenanthrene-degrading enzymes during methanogenesis.

This study revealed that the addition of glucose decreased the α-diversity of the enriched consortium (Supplementary Figure 1), which is consistent with reports that glucose addition decreased the microbial α-diversity in some enriched consortia (Li et al., 2016a; Goldford et al., 2018; Zhang et al., 2019; Shu et al., 2021), indicating the selectivity of a single carbon source on the microbial community. Additionally, glucose addition had no significant influence on the microbial community of the enriched phenanthrene-degrading consortium (Figure 4A). The addition of co-metabolic substrates has usually been shown to trigger shifts in the microbial community structure in anaerobic digestion systems (Li et al., 2016a,b; Zhang et al., 2019). In these studies, the wastes treated in their systems contained inartificial microbes originating from soil or wastewater, which should be complex, unstable, and fictile, and then susceptible to external carbon sources such as glucose. By contrast, the microbial consortium in our study had undergone an enrichment period of 10 rounds supplied with the sole carbon source (phenanthrene) and nutrient components, which should be a stable ecosystem insensitive to the external addition of glucose. Sundh et al. (2003) also reported that glucose addition significantly enhanced CH₄ yield, but only slightly affected the microbial community in an anaerobic digestion system fed with a synthetic substrate. Furthermore, our result showed that MPR and PDR were significantly correlated with the microbial community composition of the enriched consortium (Table 1). Generally accepted is that the microbial community structure determines its function (de Souza and Santos, 2018; Zhou et al., 2019). The close linkage between CH₄ production and microbial communities in diverse ecosystems has been confirmed (Shrestha et al., 2011; Edwards et al., 2015; Wang et al., 2020; Watanabe et al., 2020). However, the strong selectivity of PAHs to environmental microbes has led to a strong relationship between PAH degradation and microbial community structure (Zhou et al., 2017a; Spini et al., 2018; Wang et al., 2021a). Moreover, our results revealed that MPR explained a greater percentage of the variance in the microbial community than PDR (Figure 4B), indicating that the former might be more dependent on the microbial community structure than the latter, which might be due to the different microbial mechanisms of CH₄ synthesis and phenanthrene degradation. In detail, the CH₄ synthesis process might involve a series of steps driven by diverse microbes, including the primary metabolism of carbon sources, the transformation of the intermediates, and the generation of substrates (CO₂, H₂, and acetate) for methanogens (Maletic et al., 2018), whereas the degradation rate of phenanthrene might be determined by limited microbes responsible for the cleavage of the benzene ring (Meckenstock and Mouttaki, 2011). Therefore, MPR should be more important in explaining the variance of the microbial community than PDR in the phenanthrene-degrading system during methanogenesis.

In this study, the PCoA profile based on the composition of functional genes indicated that the macro-function of the enriched consortium was not affected by the addition of glucose, which was consistent with the response of the microbial community structure to glucose (Figure 6A; Supplementary Figure 2). Additionally, the significant correlation between the functional composition and MPR or PDR (Table 1), and the result of VPART analysis based on the functional composition (Figure 6B), were similar to the results based on the microbial community structure, further confirming the uniformity between the microbial community and its functions. In this study, the genes involved in the degradation of some refractory compounds and CH₄ biosynthesis were selected from the functional pool of KEGG for further analysis. The relative abundance of those degrading genes showed a decreasing tendency due to the addition of glucose (Figure 7A), indicating that the metabolic characteristic of the microbial community also followed the principle of “glucose repression.” Glucose (monosaccharide) will hamper the Escherichia coli from utilizing lactose (disaccharide), known as “glucose repression,” suggesting that an easy-utilized carbon source will be preferentially metabolized by microbes and thus hinder the utilization of the complex one (Rose et al., 1991). Nevertheless, the relative abundance values of degrading genes in the PME and PMEG treatments were of the same order of magnitude (Figure 7A), which the following reasons might explain. First, the DNA samples were collected at the end of the incubation (day 21) when the glucose had been exhausted; second, the metagenomic sequencing in this study was based on the DNA level, which could not reflect the actual transcription level of those degrading genes. The relative abundance of the genes related to CH₄ synthesis pathways was not significantly different between the PME and PMEG treatments, and the pathway of CH₄ synthesis via acetate was dominant in this enriched consortium (Figure 7B). The acetate pathway has been reported to be the key CH₄ synthesis pathway in paddy soils (Conrad, 1999; Glissmann and Conrad, 2002; Masuda et al., 2018). Acetate is also an essential intermediate or terminal product in the degradation of crude oil (a major source of PAHs) or some PAHs (naphthalene, phenanthrene, anthracene, and pyrene) during methanogenesis (Dolfing et al., 2008, 2009; Jones et al., 2008). Therefore, CH₄ synthesis via the acetate pathway should be dominant in the anaerobic phenanthrene digestion system during methanogenesis.

In this study, we examined the co-metabolic effect of glucose on CH₄ production and phenanthrene degradation in a phenanthrene-degrading consortium under methanogenesis. We found that glucose addition significantly enhanced the CH₄ production rate of the enriched consortium but had no influence on its ability to degrade phenanthrene. Glucose addition significantly decreased the α-diversity of the enriched microbial consortium but showed no significant effect on either the microbial community or the macro-function of the enriched consortium. Glucose addition only slightly decreased the relative abundance of the genes involved in the degradation of aromatic compounds, and the acetate pathway was dominant for CH₄ synthesis in this anaerobic phenanthrene digestion system under methanogenesis. These results indicate that it is difficult to remarkably enhance PAH degradation in a single-stage anaerobic digestion system under methanogenesis via the addition of co-metabolic substrates. Therefore, we propose that a multistage and combined pattern comprising methanogenesis and other more efficient anaerobic digestion pathways of PAH (sulfate-reducing or denitrification) should be a promising strategy to enhance the CH₄ production and PAH degradation rate in the anaerobic digestion of PAH-containing wastes.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/, PRJNA744876.

ZiZ and YW conducted the experiment, analyzed the data, and wrote the original draft. MW arranged the investigation consortium and contributed to the phenanthrene and methane detection. ZhZ designed the experiment and contributed to the edit, data analysis, and review of the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (41771501).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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