Microbial analysis and enrichment of anaerobic phenol and p-cresol degrading consortia with addition of AQDS

Phenol and cresol isomers (ortho-, meta-, and para-cresols) are important petrochemical products and commonly found in wastewaters from coal gasification, petrochemical manufacturing, oil-refining, synthetic chemicals, pesticides, and pharmaceuticals industries. Phenols are also found in municipal landfill leachates, lignin residues in anaerobic agroecosystems, and methanogenic bioreactors treating organic solid wastes, such as slaughter house waste, animal manure, food wastes and crop residues (Levén et al. 2012; Qiao et al. 2013). Therefore, studying anaerobic degradation of phenols is necessary to the industrial wastewater treatment processes and the biogeochemical process of natural phenols.

Thermodynamically, the oxidation of phenol to acetate, carbon dioxide, and hydrogen is unfavorable, and phenol is thought to be metabolized only at a very low hydrogen partial pressure. Under methanogenic conditions, phenol degradation requires the coupling of fermentative bacteria with methanogens to overcome thermodynamics limit. C₆H₆O + 6.5H₂O → 3.5CH₄ + 2.5HCO₃⁻ + 2.5H⁺ (ΔG^0' = −155.3 kJ reaction⁻¹); C₇H₈O + 7.5H₂O → 4.25CH₄ + 2.75HCO₃⁻ + 2.75H⁺ (ΔG^0' = −187.5 kJ reaction⁻¹) (Cervantes et al. 2000a).

The anaerobic degradation of phenols have been reported under methanogenic conditions. Previous studies showed that anaerobic degradation of phenols can be observed at either ambient (26 °C) (Fang et al. 2004), mesophilic (37 °C) (Chen et al. 2008; Franchi et al. 2018; Levén & Schnürer 2010) or thermophilic temperatures (55 °C) (Fang et al. 2006; Chen et al. 2008; Sierra et al. 2018, 2020). Investigations of the methanogenic phenolic compound-degrading microbial community revealed that the majority of functional bacteria belong to Clostridia and subcluster Ih of Desulfotomaculum cluster I within phylum Firmicutes, and Syntrophorhabdaceae and Syntrophus spp. in Deltaproteobacteria (Fang et al. 2006; Chen et al. 2008; Levén et al. 2012). Levén et al. (2012) reviewed that temperature had a strong impact on the degradation efficiency of phenols and phenol-degrading microbes. Desulfotomaculum subcluster Ih was the dominant phenol-degrading population in the thermophilic temperature, while Syntrophorhabdus was the dominant population in the mesophilic temperature (Chen et al. 2008; Levén & Schnürer 2010; Li et al. 2014; Franchi et al. 2018).

Humus is the stable organic matter accumulating in sediments and soils. Lovley et al. (1996) first proposed the concept of humus respiration. They found that Fe(III)-reducing bacteria Geobacter metallireducens and Shewanella alga were able to use humus model AQDS as the sole electron acceptor to oxidize organic compounds or hydrogen, supporting the growth of bacteria. In humus respiration, some quinones act as electron receptors, hence humus respiration is also called quinone respiration (Lovley et al. 1996; Cervantes et al. 2000a). It has been reported that quinones play an active role in the anaerobic transformation of organic acids, poisonous compounds, such as azo dyes (Hong et al. 2007), pentachlorophenol (Zhao et al. 2019), vinyl chloride and dichloroethene (Bradley et al. 1998), polycyclic aromatic hydrocarbons (Ma et al. 2011), benzene (Cervantes et al. 2011) and toluene (Cervantes et al. 2001). Recently, AQDS and AQS were found to serve as the terminal electron acceptor (TEA) for anaerobic oxidation of methane by ANME-2d (Bai et al. 2019). Anaerobic degradation of phenol and p-cresol can be coupled to the reduction of AQDS as TEA. When the humic model compound AQDS was included as an alternative electron acceptor in the cultures inoculated with anaerobic granular sludge and sediment, the oxidation of the phenols was coupled to the reduction of the model humic compound to its corresponding hydroquinone, anthrahydroquinone-2,6-disulfonate. The equations of the theoretical reactions are shown as follows: C₆H₆O + 17H₂O + 14AQDS → 14AH₂QDS + 6HCO₃⁻ + 6H⁺ (ΔG^0′ = −302.0 kJ reaction⁻¹); C₇H₈O + 20H₂O + 17AQDS → 17AH₂QDS + 7HCO₃⁻ + 7H⁺ (ΔG^0′ = −365.7 kJ reaction⁻¹) (Cervantes et al. 2000a). However, the anaerobes responsible for the degradation reaction are unknown.

To date, two anaerobic phenol-degrading bacteria have been described: Cryptanaerobacter phenolicus belongs to subcluster Ih of Desulfotomaculum cluster I within phylum Firmicutes has been found transform phenol into benzoate via 4-hydroxybenzoate (Juteau et al. 2005); Syntrophorhabdus aromaticivorans belongs to the family Syntrophorhabdaceae in Deltaproteobacteria, an anaerobic microbe that degrades phenol to acetate in syntrophic associations with the hydrogenotrophic methanogen Methanospirillum hungatei (Qiu et al. 2008). The utilization of AQDS by Cryptanaerobacter phenolicus and S. aromaticivorans is unknown.

In this study, we reported the impact of AQDS on phenol and p-cresol degradation by enrichment cultures. The dominant microbial populations were identified by 16S rRNA gene cloning analysis and fluorescence in situ hybridization (FISH). In addition, partial physiological properties of dominant phenol degrading consortia belonging to subcluster Ih of Desulfotomaculum cluster I were characterized.

Methanogenic sludges were taken from two lab-scale, mesophilic (35 °C), upflow anaerobic sludge blanket (UASB) reactors: (i) sludge C treated cresols for one year; (ii) sludge N treated nitro-phenol for three months (Zhang et al. 2005). Two sludges were washed with phosphate buffer (10 mM, pH 7.2) and homogenized briefly for primary enrichment. A total volume of 100 mL sludges were washed with phosphate buffer and then centrifuged at 7,000 × g for 15 min. The pellets were collected and resuspended in 20 mL of fresh medium.

S. aromaticivorans (JCM 13376, DSM 1777) was the first cultured anaerobe capable of degrading phenol to acetate in syntrophic associations with a hydrogenotrophic methanogen (Qiu et al. 2008). Co-culture of S. aromaticivorans with M. hungatei was obtained from our lab and routinely cultured at 37 °C with 2 mM phenol and 0.01% yeast extract (Qiu et al. 2008).

The medium used for cultivation was prepared as described previously (Sekiguchi et al. 2000). All batch experiments were carried out at 37 °C under dark anaerobic conditions, 60 mL serum vials containing 20 mL of medium (pH_{25 °C} 7.0) under an atmosphere of N₂-CO₂ (80:20, v/v) without shaking.

To test the degradability of phenol (2 mM) and cresol isomers (1 mM each) by methanogenic sludges C and N, batch experiments were carried out in 60-mL serum vials with sludges as inoculum at 37 °C without shaking. The experiment lasted 160 days. Concentration of phenol and cresol isomers was measured every 15–20 days.

For enrichment of phenol and p-cresol degrading anaerobes, either phenol (2 mM) or p-cresol (1 mM) was added to the basal medium as the sole energy source with sludges C and N, respectively. Four methanogenic AQDS-free enrichment cultures CP (enriched on phenol medium with sludge C), NP (enriched on phenol with sludge N), CPC (enriched on p-cresol with sludge C) and NPC (enriched on p-cresol with sludge N) were obtained. To evaluate the effect of quinone on phenol and p-cresol degradation, another set of enrichment cultures with addition of 2 mM AQDS were prepared in parallel. The cultures were transferred into fresh medium when approximately 50% of phenol and p-cresol were degraded. For enrichments, large volume of inoculum was always needed (30–50%).

Fixation of cells in the enrichment cultures and subsequent whole-cell in situ hybridization were performed as described previously (Sekiguchi et al. 1999). The following 16S rRNA-targeted oligonucleotide probes were used in this study (Table 1): (i) EUB338 mix, specific for Bacteria (Amann et al. 1990); (ii) Ih820, specific for subcluster Ih of Desulfotomaculum cluster I, which was used to detect dominant phenol-degrading bacteria in enrichment culture NPA (enriched on phenol-AQDS with sludge N) (Imachi et al. 2006); (iii) UI178, specific for S. aromaticivorans, which was used to detect dominant phenols degrading bacteria in enrichment cultures CP, NP, CPC and NPC (Qiu et al. 2004); (iv) DEM1164-ar. (5′-CCTTCCTCCGGTTTGTCA-3′), specific for clones CPA01 and CPA02 obtained from phenol-AQDS enrichment culture CPA (enriched on phenol-AQDS with sludge C), which belong to a new undefined subcluster of Desulfotomaculum cluster I. The probe DEM1164-ar. was arranged from Desulfotomaculum cluster I specific probe, DEM1164 (5′-CCTTCCTCCGTTTTGTCA-3′) with a slight modification (Stubner & Meuser 2000) (Figure 3). To test the specificity of probe DEM1164-ar., Sporotomaculum syntrophicum (DSM 14795) and Pelotomaculum terephthalicum (DSM 16121) were used as reference organisms. Hybridization stringency was adjusted by adding formamide to the hybridization buffer (30% [v/v] for Ih820; 15% [v/v] for UI178; 10% [v/v] for EUB338 mix; 0% [v/v] for DEM1164-ar.).

DNA extraction, PCR amplification, cloning, and sequencing procedures for constructing 16S rRNA gene clone libraries were performed as described previously (Sekiguchi et al. 1998) with slight modifications. The following primer sets for PCR amplification of bacterial 16S rRNA genes were used: Bacteria-specific primer EUB8F (5′-AGAGTTTGATCCTGGCTCAG-3′, 8 to 27 in Escherichia coli position) and prokaryote-specific primer UNIVI1492R (5′-TACGGYTACCTTGTTACGACTT-3′; positions 1,492 to 1,513 in E. coli) (Weisburg et al. 1991). The PCR products were purified with a TIAN quick MiDi purification kit (Tiangen Inc., Beijing, China), followed by cloning into plasmids using the TA cloning kit (Promega Inc., Beijing, China). For each enrichment culture, 10–40 clonal rRNA genes were randomly picked and screened by comparing restriction fragment length polymorphism (RFLP) patterns with HaeIII and HhaI restriction endonuclease. Representative clones having different RFLP patterns were then subjected to sequencing.

The phylogenetic tree based on 16S rRNA gene sequences was constructed by the neighbor-joining method (Zhang & Sun 2008) implemented in the MEGA5 computer software program (Tamura et al. 2007). The confidence values of branches in the phylogenetic tree were determined using bootstrap analysis based on 1,000 resamplings (Felsenstein 1985).

To test whether AQDS could be used as electron acceptor, batch experiments were conducted with enrichment cultures CPA, NPA and syntrophic co-culture of S. aromaticivorans with M. hungatei. In this test, 5 mM 2-bromoethane-sulfonate (2-BES) was added to inactivate the methanogenesis in enrichment cultures. The concentrations of AQDS were set at 5 and 30 mM respectively, according to the theoretic chemical equation C₆H₆O + 17H₂O + 14AQDS → 14AH₂QDS + 6HCO₃⁻ + 6H⁺ (ΔG^0′ = −302.0 kJ reaction⁻¹) and previous studies (Cervantes et al.2000a, 2000b). In total, six sets of assays were run as follows: (i) phenol; (ii) phenol-BES; (iii) phenol-AQDS (5 mM); (iv) phenol-AQDS (5 mM)-BES; (v) phenol-AQDS (30 mM); (vi) phenol-AQDS (30 mM)-BES (Table 2). Sterilized controls were also included to evaluate the chemical transformation. For enrichment cultures CPA & NPA (more than 20 successive transfers over three years), a total volume of 200 mL exponential phase cultures in the exponential phase were collected by centrifugation at 7,000 × g for 15 min at 35 °C, and then used for inoculation (inoculum size, 30%) (Qiu et al. 2004). All the experiments were performed in duplicate at 37 °C and pH 7.0 for four months.

It is widely recognized that 4-hydroxybenzoate and benzoate are intermediates during anaerobic phenol degradation (Juteau et al. 2005; Qiu et al. 2008; Levén et al. 2012). To test the degradation of phenol, o, m, p-cresols, 4-hydroxybenzoate and benzoate by enrichment cultures CPA and NPA, batch experiments were performed with highly enriched dense cell suspensions (more than three years successive transfers). The degradability was evaluated by percent substrate degradation in 1–4 months' incubation. For the other possible aromatic substrates such as catechol, 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, hydroquione, 2-hydroxybenzoate, 3-hydroxybenzoate, 3-phenylpropionate, and phthalate isomers, the utilization was evaluated by turbidity of cultures and methane production over four months' incubation.

An Olympus microscope equipped for epifluorescence was used for studies of cell morphology and epifluorescence (Olympus BX50F). Concentrations of phenol, o-, m-, p-cresols, benzoate and 4-hydroxybenzoate were analyzed by high-performance liquid chromatography (HPLC) with an UV detector as described previously (Qiao et al. 2013). Short-chain fatty acids, methane, and hydrogen were measured as described previously (Yuan et al. 2011). Concentrations of AH₂QDS were determined spectrophotometrically by monitoring the absorbance at 450 nm in an anaerobic chamber as described previously (Cervantes et al. 2000a; Li et al. 2019).

The GenBank/EMBL/DDBJ accession numbers of the 16S rRNA gene sequences of phenol-degrading clones are AB853905 to AB853925.

To assess the biodegradability of two sludges C and N, batch experiments were performed with either phenol or o-, m-, p-cresols as sole carbon source. Both sludges were able to degrade phenol and three cresol isomers within 150 days of incubation (Figure 1), suggesting functional phenol and cresol isomers degrading organisms existed in the sludges. Phenols are toxic to microorganisms, that cannot be degraded easily under anaerobic conditions. Therefore, in most cases, long lag period is often required. Phenol and p-cresol were completely degraded by both sludges within three months. o-Cresol was completely degraded by sludge C, while partially (25%) degraded by sludge N within five months. Only 20% m-cresol was degraded by both sludges within five months of incubation. Phenol and p-cresol were easy to degrade, whereas o- and m-cresols were more difficult to degrade. Our result was consistent with the previous study (Veeresh et al. 2005).

To enrich phenol and p-cresol degrading microbes, primary enrichment was made with each phenol (2 mM) and p-cresol (1 mM) as the sole carbon and energy source with two sludges C and N as inoculum. Four methanogenic AQDS-free enrichment cultures CP, NP, CPC and NPC were obtained. To evaluate the effect of quinone on phenol and p-cresol degradation, another set of enrichment cultures with addition of 2 mM AQDS was prepared in parallel. 2 mM AQDS completely inhibited p-cresol degradation, thus, two phenol-AQDS enrichment cultures CPA and NPA were obtained.

In methanogenic AQDS-free enrichment cultures, the growth of microbes and phenol or p-cresol degradation accompanied by methane production were observed after 1–2 months of incubation. In total, four successful methanogenic enrichment cultures were obtained: (i) enrichment CP with phenol from sludge C; (ii) enrichment NP with phenol from sludge N; (iii) enrichment CPC with p-cresol from sludge C; (iv) enrichment NPC with p-cresol from sludge N. After two years of successive transfers, short rods (later identified as S. aromaticivorans by FISH) were the major morphotypes in all four enrichment cultures by microscopic observation. Methanospirillum-like F₄₂₀-autofluorescent rods and Methanosaeta-like thick rods in some cases were also detected.

The addition of 2 mM AQDS diminished phenol degradation and completely inhibited p-cresol degradation. The growth rate of phenol-AQDS cultures was approximately 1.5–2 times slower than that of AQDS-free phenol enrichment cultures, and phenol was degraded after 3–4 months. Methane production was detected along with phenol degradation no matter with or without AQDS addition. In total, two stable phenol-AQDS enrichment cultures were obtained: (i) enrichment CPA with phenol-AQDS from sludge C; (ii) enrichment NPA with phenol-AQDS from sludge N. Microscopic observation revealed that major morphotypes in two phenol-AQDS enrichment cultures CPA and NPA were slightly different. In enrichment culture CPA, short rods (later identified as C. phenolicus resembling bacterium with 87% sequence similarity, and Syntrophus aciditrophicus with 99% similarity) were observed as major morphotypes. In enrichment culture NPA, at least two major bacterial cell morphotypes were observed: one was short rods (later identified as C. phenolicus resembling bacterium with 96% similarity), the other was sporulating rods (later identified as Sporotomaculum syntrophicum with 100% similarity). Methanospirillum-like F₄₂₀-autofluorescent rods and Methanosaeta-like thick rods were presented in both CPA and NPA enrichment cultures.

After two years of successive transfers, short rods S. aromaticivorans like microbes were major morphotypes in all four AQDS-free enrichment cultures CP, NP, CPC and NPC by microscopic observation. To confirm whether S. aromaticivorans actually represented the predominant bacterial populations in four enrichments, FISH with S. aromaticivorans-specific16S rRNA-targeted oligonucleotide probe UI178 (Qiu et al. 2004) was done (Figure 2). Bacteria-specific probe EUB338 mix was also applied. At least 10 microscopic fields were randomly acquired. FISH analysis indicated that short rods in all of the four cultures reacted with the probe UI178, and represented approximately 80–90% of the total bacteria detected by probe EUB338 mix.

To identify bacterial populations in two phenol-AQDS enrichments CPA and NPA, 16S rRNA gene clone libraries were constructed as described above. RFLP analysis indicated that three RFLP patterns were found in enrichment culture CPA. Phylogenetic analysis indicated that clones CPA01 (3/9 clones) and CPA02 (3/9 clones) were affiliated with phenol degrading bacterium C. phenolicus (87% similarity), clone CPA06 (3/9 clones) was affiliated with syntrophic benzoate degrading bacterium Syntrophus aciditrophicus (99% similarity) (Jackson et al. 1999) (Figure 3 and Table S1 in Supplementary Information). For enrichment culture NPA, eight RFLP patterns were found. Phylogenetic analysis indicated that clones NPA01 (4/41 clones) and NPA02 (2/41 clones) were affiliated with phenol degrading bacterium C. phenolicus (96% similarity) and benzoate-degrading bacterium Sporotomaculum syntrophicum (100% similarity) (Qiu et al. 2003), respectively.

Phylogenetic analysis indicated that dominant microbes in cultures NPA and CPA were affiliated with two different groups in Desulfotomaculum cluster I of Firmicutes: clone NPA01 belonged to subcluster Ih, whereas clones CPA01 and CPA02 represented an undefined subcluster in Desulfotomaculum cluster I (Figure 3). To detect dominant microbes in enrichment culture NPA, FISH with Desulfotomaculum subcluster Ih-specific probe Ih820 was used (Imachi et al. 2006) (Table 1). To detect clones CPA01 and CPA02, an arranged probe DEM1164-ar was applied to enrichment culture CPA (Stubner & Meuser 2000). Bacteria-specific probe EUB338 mix and S. aromaticivorans-specific probe UI178 were also applied to the enrichment cultures. In enrichment culture NPA, non-spore forming short rodshaped microbes reacted with probe Ih820, indicating that bacteria resembling C. phenolicus in Desulfotomaculum subcluster Ih were represented (Figure 2). In enrichment culture CPA, short rod-shaped bacteria reacted with probe DEM1164-ar, but did not react with probe Ih820 (data not shown), confirming that the dominant populations belonged to a new sucluster in Desulfotomaculum cluster I (Figure 2). Both enrichment cultures CPA and NPA did not react with S. aromaticivorans-specific probe UI178, indicating that S. aromaticivorans were not present in the two phenol-AQDS cultures (data not shown).

16S rRNA gene cloning analysis and FISH results revealed the predominant populations were affiliated with C. phenolicus (similarity, 87% and 96%, respectively) in CPA and NPA enrichments, and syntrophic phenol degrading bacterium S. aromaticivorans were detected in four methanogenic AQDS-free enrichments. To test whether AQDS could be used as an electron acceptor to oxidize phenol by three cultures, batch experiments were conducted. Results revealed that AQDS reduction was not found by three phenol-degrading cultures after four months of incubation, suggesting that neither C. phenolicus resembling microbes nor S. aromaticivorans could use AQDS (5 mM or 30 mM) as an electron acceptor to oxidize phenol (Table 2). For enrichment cultures CPA and NPA, phenol was completely converted to methane in the absence of AQDS and BES, while phenol was completely converted to benzoate and acetate in the presence of BES. For enrichment culture CPA, the presence of 5 mM AQDS showed significant inhibitory effect on phenol degrading consortia, with only tiny phenol (17–20%) was degraded into benzoate, methane or acetate, however, the addition of 30 mM AQDS completely inhibited the phenol degradation. For enrichment culture NPA, phenol degradation was not observed in the presence of 5 mM or 30 mM AQDS, indicating that AQDS had a strong inhibition to phenol degrading consortia in culture NPA. For S. aromaticivorans co-culture, phenol was completely converted to methane and acetate only in the absence of AQDS and BES. However, the degradation of phenol was not observed either with AQDS (5 mM or 30 mM) or BES, indicating that AQDS also had a strong inhibition to S. aromaticivorans.

Previous investigations suggest that high concentrations of AQDS (more than 5 mM) have a high redox potential, thus causing the inhibition of methanogens due to the unfavorable environment (Cervantes et al. 2000a, 2000b, 2008; Yang et al. 2012). Our results provide further support for this suggestion. The AQDS utilization assays showed that S. aromaticivorans was more sensitive to AQDS than C. phenolicus resembling bacteria of Desulfotomaculum cluster I. This may be related to different cell wall structures of these two groups. S. aromaticivorans is a gram negative bacterium, whereas C. phenolicus resembling microbes are gram positive and heat-resistant bacteria (although spore was not observed) (Juteau et al. 2005; Qiu et al. 2008). It has been proved that the cell wall of gram-positive bacteria is (20–80 nm) thicker than that of negative bacteria, which provides greater strength and rigidity by multiple cross linked peptidoglycan layers (Yang et al. 2020). C. phenolicus resembling microbes may be more tolerant to adverse environments. It can be inferred that different tolerance of Desulfotomaculum cluster I and Syntrophorhabdaceae to AQDS occurring conditions resulted in different distribution of these two clusters in enrichment cultures.

Phylogenetic analysis results suggested that dominant phenol degrading bacterium in enrichments CPA and NPA was related to phenol degrading Cryptanaerobacter phenolicus (similarity, 87% and 96%, respectively), representing new species. Aromatic compounds utilization by two cultures CPA and NPA were studied.

Both enrichment cultures CPA and NPA could degrade phenol, 4-hydroxybenzoate and benzoate completely to methane after one month of incubation, but could not use three cresol isomers (Figure 4, Table 3). Both enrichment cultures CPA and NPA could convert 4-hydroxybenzoate to phenol, and phenol was further degraded to methane. During phenol degradation, 0.28 mM benzoate and 0.01 mM 4-hydroxybenzoate were detected in culture CPA, and 0.26 mM benzoate was detected in culture NPA, indicating that phenol was degraded via 4-hydroxybenzoate and benzoate for two cultures.

Utilization of other aromatic substrates by enrichment cultures CPA and NPA were also tested (Table 3). According to the methane production over four months' incubation, enrichment culture CPA could metabolize 3-hydroxybenzoate, but not catechol, 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 2-hydroxybenzoate, 3-phenylpropionate, hydroquione and phthalate isomers. Enrichment culture NPA could metabolize catechol, 2,5-dihydroxybenzoate, 3-hydroxybenzoate and 3-phenylpropionate, but not 2,4-dihydroxybenzoate, 2-hydroxybenzoate, hydroquione and phthalate isomers.

Nowdays, two anaerobic phenol-degrading bacteria have been isolated, pure culture of Cryptanaerobacter phenolicus belongs to phylum Firmicutes (Juteau et al. 2005) and co-culture of S. aromaticivorans belongs to Deltaproteobacteria (Qiu et al. 2008). Physiological traits and metabolic pathways of the two isolates are obviously different: S. aromaticivorans transforms phenol, p-cresol, 4-hydroxybenzoate, isophthalate and benzoate directly to acetate and methane in syntrophic co-culture with hydrogenotrophic methanogens (Qiu et al. 2008); however, Cryptanaerobacter phenolicus pure culture can only transform phenol to benzoate via 4-hydroxybenzoate (Juteau et al. 2005).

Microbes resembling C. phenolicus were likely responsible for the conversion of phenol to benzoate, which was further degraded by benzoate-degraders of Syntrophus aciditrophicus (Jackson et al. 1999) or Sporotomaculum syntrophicum (Qiu et al. 2003) (Figure 5). In this study, C. phenolicus affinities was dominant in two phenol-AQDS enrichments CPA and NPA, representing new species (similarity, 87% and 96%, respectively) (Figure 3). Phylogenetic analysis and further degradation experiments suggested that C. phenolicus related microbes in enrichments CPA and NPA were responsible for the conversion of phenol to benzoate. Biodegradation experiment indicated that both enrichment cultures CPA and NPA could degrade phenol completely to methane, benzoate was detected as intermediate (Figure 4, Table 3). Phylogenetic analysis indicated, syntrophic benzoate degrading bacterium Syntrophus aciditrophicus (99% similarity) represented 33.3% (3/9 clones) in CPA enrichments, and another type benzoate-degrading bacterium Sporotomaculum syntrophicum (100% similarity) represented 4.9% (2/41 clones) in NPA enrichments (Jackson et al. 1999; Qiu et al. 2003).

The dominant phenol-degrading bacteria in enrichment cultures CPA and NPA show similar metabolic pathways with the cultured C. phenolicus (Juteau et al. 2005), however, their substrates range for growth are different. Pure culture of C. phenolicus can utilize phenol and 4-hydroxybenzoate, while enrichment cultures CPA and NPA could utilize phenol, 4-hydroxybenzoate as well as several other aromatic compounds. Enrichment culture CPA could utilize 3-hydroxybenzoate, and enrichment culture NPA could utilize catechol, 2,5-dihydroxybenzoate, 3-hydroxybenzoate and 3-phenylpropionate. Considering the microbial complexity of enrichment cultures, further isolation attempts of NPA and CPA are in progress to verify the substrate utilization of bacteria in Desulfotomaculum cluster I.

The degradation test using phenol and p-cresol as substrate were observed significantly faster than o-, and m-cresols. 2 mM AQDS mitigated anaerobic phenol degradation and completely inhibited p-cresol degradation. 16S rRNA gene analysis revealed that S. aromaticivorans (similarity, 100%) was dominant in four AQDS-free enrichments, whereas C. phenolicus (similarity, 87% and 96%) was dominant in two phenol-AQDS enrichments. Neither S. aromaticivorans nor C. phenolicus resembling microbes could metabolize phenol using AQDS as the terminal electron acceptor. The phenol-degrading bacteria in enrichment cultures CPA and NPA show similar metabolic pathways with C. phenolicus, however, their substrates range are different.

We thank Dr Shuizhou Ke in Hunan University for providing sludges. This work was supported by the National Natural Science Foundation of China (grant number 51878363); Natural Science Foundation of Shandong (grant numbers ZR2019MEE028, ZR2019YQ18); Key R & D plan of Ningxia Hui Autonomous Region (grant number 2020BBF03008) and the Science and Technology Support Plan for Youth Innovation of Colleges in Shandong Province (grant number DC2000000961).

All relevant data are included in the paper or its Supplementary Information.