Methanogenic degradation of lignin-derived monoaromatic compounds by microbial enrichments from rice paddy field soil

Anaerobic degradation of lignin-derived aromatics is an important metabolism for carbon and nutrient cycles in soil environments. Although there are some studies on degradation of lignin-derived aromatics by nitrate- and sulfate-reducing bacteria, knowledge on their degradation under methanogenic conditions are quite limited. In this study, methanogenic microbial communities were enriched from rice paddy field soil with lignin-derived methoxylated monoaromatics (vanillate and syringate) and their degradation intermediates (protocatechuate, catechol, and gallate) as the sole carbon and energy sources. Archaeal community analysis disclosed that both aceticlastic (Methanosarcina sp.) and hydrogenotrophic (Methanoculleus sp. and Methanocella sp.) methanogens dominated in all of the enrichments. Bacterial community analysis revealed the dominance of acetogenic bacteria (Sporomusa spp.) only in the enrichments on the methoxylated aromatics, suggesting that Sporomusa spp. initially convert vanillate and syringate into protocatechuate and gallate, respectively, with acetogenesis via O-demethylation. As the putative ring-cleavage microbes, bacteria within the phylum Firmicutes were dominantly detected from all of the enrichments, while the dominant phylotypes were not identical between enrichments on vanillate/protocatechuate/catechol (family Peptococcaceae bacteria) and on syringate/gallate (family Ruminococcaceae bacteria). This study demonstrates the importance of cooperation among acetogens, ring-cleaving fermenters/syntrophs and aceticlastic/hydrogenotrophic methanogens for degradation of lignin-derived aromatics under methanogenic conditions.


Results and Discussion
Enrichment of methanogenic microbial communities on lignin-derived aromatics. Metha nogenic microbial communities were enriched from rice paddy field soil using a freshwater basal medium supplemented with different aromatic compounds. Either lignin-derived methoxylated aromatics (5 mM of vanillate or syringate) or a control aromatic compound (5 mM of benzoate) were supplemented as the substrates. After several weeks of cultivation, CH 4 was produced in all of the cultures supplemented with aromatics (Fig. S1). The amount of CH 4 produced reached to 7 to 20 mmol l −1 within 2 month cultivation. On the contrary, only trace amount of CH 4 (less than 0.1 mmol l −1 ) was produced in the cultures without supplementation of the aromatics (Fig. S1), indicating that most of CH 4 produced in the enrichment cultures was derived from degradation of aromatic compounds. Production of CH 4 from the aromatic compounds by respective enrichments (after five successive subcultures) are shown in Fig. 1A-C. It took more than one month to produce stoichiometrically expected CH 4 in all cultures tested. The amounts of CH 4 produced from 5 mM of benzoate, vanillate, and syringate were 17.1 ± 0.5, 18.4 ± 0.3, and 20.1 ± 0.3 mmol l −1 , respectively, and were approximate to the theoretical values (18.75, 20, and 22.5 mmol l −1 , respectively) calculated from the following equations: These results suggest that the each enrichment culture completely degraded the aromatic compounds into CH 4 and CO 2 .
Detection of degradation intermediates from the enrichment cultures. Among  study by the high performance liquid chromatography (HPLC) analysis. Acetate accumulated in the enrichment cultures to 8 to 12 mM during methanogenesis, followed by the complete consumption of the accumulated acetate ( Fig. 1A-C), indicating that acetate is one of the important intermediates of methanogenic degradation of the aromatic compounds. In order to determine the degradation pathway of vanillate and syringate in the enrichment cultures, intermediate metabolites produced during cultivation were analyzed by HPLC coupled with an electrospray ionization-mass spectrometry (ESI-MS). It should be noted that CoA-derivatives of aromatic compounds could not be detected by our analytical protocol. In the vanillate enrichment cultures, two peaks (compound I, II) appeared at day 10, followed by an appearance of another peak (compound III) at day 24 ( Fig. 2A). Based on the comparison of retention time, UV/VIS spectrum, and negative ESI-MS spectrum with authentic compounds, compound I, II, and III were identified as PCA, catechol, and  3-hydroxybenzoate (3-HB), respectively (Figs S2 and S3). Generation of PCA at day 10 suggests that O-demethylation of vanillate is the first step of vanillate degradation. Catechol and 3-HB were appeared to be produced by the decaroboxylation and dehydroxylation of PCA, respectively, probably as intermediates of methanogenic degradation pathway of PCA (discussed below). In the degradation of syringate, two metabolites, compound IV and V, were found and identified as 3-MGA and gallate, respectively ( Fig. 2B, S2, and S3). These observations indicate that syringate is first converted to gallate with successive O-demethylation reactions via 3-MGA. In the benzoate enrichment cultures, no aromatic compound other than benzoate were detected (data not shown), suggesting that benzoate is firstly converted to benzoyl-CoA and then the ring structure is reductively cleaved via benzoyl-CoA pathway as reported 25,26 . Enrichment cultures on the degradation intermediates. In order to further elucidate the mechanisms for degradation of the lignin-derived aromatics, the enrichment cultures on vanillate and syringate were subjected to further enrichments with respective degradation intermediates. Enrichment cultures on PCA and gallate were constructed with vanillate-and syringate-enrichments as the inoculum, respectively, and generated considerable amounts of CH 4 within 40 days (Fig. 3A). Although enrichment cultures on catechol was also constructed using the vanillate-enrichments as the inoculum, the cultures supplemented with 5 mM catechol produced only little amounts of CH 4 with a long lag time (> 50 days, data not shown). Since cytotoxicity of relatively high concentration of catechol was considered as the reason for the low methanogenic activity, another set of enrichment cultures was constructed with The amount of CH 4 produced from PCA (5 mM), gallate (5 mM), and catechol (1 mM) were 15.5 ± 0.5, 14.4 ± 0.5, and 2.8 ± 0.3 mmol l −1 , respectively, and were approximate to the theoretical values (16.25, 15, and 3.25 mmol l −1 , respectively) calculated from the following equations: These results suggest that methanogenic microbial communities that completely degrade the respective intermediates of vanillate and syringate were successfully enriched.

Dominant microorganisms in the enrichment cultures.
Microbial community structures of the enrichments were assessed based on 16S rRNA gene clone library analysis to elucidate the microorganisms participating in methanogenic degradation of the lignin-derived aromatics. The summarized features of the bacterial and archaeal community analyses are presented in Fig. 4. All phylotypes detected from each enrichment culture are listed in Tables S1 and S2.
Only 4 archaeal phylotypes were recovered from the enrichment cultures ( Fig. 4A and Table S1). The phylotype ArA-01 (98.9% identity to Methanosarcina vacuolata), which dominated in all enrichment cultures, was the only phylotype classified as an aceticlastic methanogen. The phylotypes classified as hydrogenotrophic methanogens, namely the phylotype ArA-02 (97.9% identity to Methanoculleus palmolei) and ArA-03 (97.5% identity to Methanocella arvoryzae), were also detected from all enrichment cultures. These results suggest that both aceticlastic and hydrogenotrophic methanogenesis contribute to methanogenic degradation of the lignin-derived aromatics.
The predominant bacterial phylotype in the benzoate-enrichments was the phylotype ArB-25 (Fig. 4B  and Table S2), which is classified into the class Deltaproteobacteria and has 99.0% identity to Syntrophus aciditrophicus. S. aciditrophicus has ability to degrade benzoate via benzoyl-CoA pathway in syntrophic association with methanogens 27,28 . In the benzoate-enrichment cultures, Syntrophus sp. appears to decompose benzoate into acetate and H 2 /CO 2 , which is further converted into CH 4 by aceticlastic and hydrogenotrophic methanogens.
By contrast, almost no Deltaproteobacteria were recovered from the enrichment cultures with other aromatic compounds. Instead, the phylotypes classified into phylum Firmicutes dominated in the enrichments supplemented with the lignin-derived aromatics and their degradation intermediates (Fig. 4B). Among the Firmicutes phylotypes, the phylotype ArB-01 (99.6% identity to Sporomusa sphaeroides) and ArB-02 (89.8% identity to Sporomusa paucivorans) were abundantly detected from vanillate-and syringate-enrichments, but not from enrichments with their degradation intermediates. Sporomusa spp. were reported to have the ability to acetogenetically grow on diverse array of methoxylated aromatic compounds, such as vanillate, ferulate, and 3,4-dimethoxybenzoate, via respective O-demethylation reactions 29,30 . These findings suggested that Sporomusa spp. contribute to the initial step of anaerobic degradation of the lignin-derived aromatics in the enrichments, namely the conversion of vanillate and syringate into PCA and gallate, respectively.
In the enrichments on vanillate and its degradation intermediates (PCA and catechol), the phylotypes classified into family Peptococcaceae were dominant, while the dominant phylotypes varied among the supplemented aromatic compounds (Fig. 4B and Table S2). The phylogenetic tree based on 16S rRNA gene sequences of representative Peptococcaceae isolates and the Peptococcaceae phylotypes detected in this study is shown in Fig. 5A. The dominant phylotypes ArB-04 (96.8% identity to Pelotomaculum schinkii) and ArB-06 (98.4% identity to Cryptanaerobacter phenolicus) were clustered into the Desulfotomaculum subcluster Ih 31 . Microorganisms in the Desulfotomaculum subcluster Ih were characterized by their ability to anaerobically metabolize various organic substrates in syntrophic association with hydrogenotrophic methanogens 32 . Furthermore, Desulfotomaculum subcluster Ih includes some strains that syntrophically degrade a range of aromatic compounds 33,34 and was frequently detected as the dominant bacteria in microbial communities that anaerobically degrade diverse aromatic compounds [35][36][37][38] . The other dominant phylotype ArB-05 (97.9% identity to Desulfosporosinus auripigmenti) was separately clustered from ArB-04 and -06 and affiliated with genus Desulfosporosinus (Fig. 5A). Desulfosporosinus spp. were generally characterized as sulfate-reducing bacteria and its syntrophic metabolism with methanogens has not been tested 39 . However, all crucial domains for syntrophic metabolisms were found in the genome of Desulfosporosinus meridiei, suggesting their possible ability to grow in syntrophic association with methanogens 40 . While there have been no reports on degradation of aromatic compounds by Desulfosporosinus spp., sequences related to Desulfosporosinus were frequently recovered from various aromatics-degrading microbial communities [41][42][43] . Taken together, it is very likely that the Peptococcaceae phylotypes mainly contributed to the ring-cleavage of the vanillate and/or its degradation intermediates via syntrophic interaction with hydrogenotrophic methanogens.
The phylotypes classified into family Ruminococcaceae were dominant in the enrichments with syringate and gallate ( Fig. 4B and Table S2). The phylogenetic tree for family Ruminococcaceae is shown in Fig. 5B. One of the phylotypes, ArB-08, was not clustered with known Ruminococcaceae isolates and has only quite low 16S rRNA gene sequence identity to isolated microorganisms (92.0% identity to Acetivibrio sp. [6][7][8][9][10][11][12][13]. The other dominant phylotype ArB-09 (97.1% identity to Intestinimonas butyriciproducens) was placed within the "Clostridium cluster IV 44,45 ". Although the closest relative of ArB-09 (I. butyriciproducens) has not been reported to degrade aromatic compounds 46 , some strains in the Clostridium cluster IV were reported to fermentatively decompose diverse aromatic compounds. For example, Flavonifractor plautii (formerly Clostridium orbiscindens) has the ability to degrade flavonoids via phloroglucinol (1,3,5-trihydroxybenzene) as the intermediate 47 . Most notably, Sporobacter termitidis grows exclusively on a limited range of methoxylated aromatic compounds, including syringate and vanillate, through their O-demethylation and ring cleavage 17 . These reports support the assumption that the Ruminococcaceae phylotypes dominant in the syringate-and gallate-enrichments mainly contributed to their ring cleavage.
Proposed model for methanogenic degradation of vanillate and syringate. The proposed models for methanogenic degradation of the lignin-derived aromatics are shown in Fig. 6. Analysis on degradation intermediates and microbial communities in the enrichments suggested that vanillate is firstly converted into PCA by Sporomusa spp. with acetate generation via O-demethylation reactions (Fig. 6A). Peptococcaceae bacteria (Desulfotomaculum subcluster Ih and/or Desulfosporosinus spp.) are the plausible candidates for decomposing PCA into acetate and H 2 /CO 2 , which are the substrates for methanogenesis by aceticlastic methanogens (Methanosarcina spp.) and hydrogenotrophic methanogens (Methanoculleus and Methanocella spp.), respectively. Anaerobic degradation of PCA has been investigated for some nitrate-and sulfate-reducing bacteria 20,21 , by which PCA is firstly activated to protocatechuyl-CoA and reductively dehydroxylated to 3-HB-CoA, followed by ring cleavage via the (3-hydroxy)benzoyl-CoA pathway. However, we could not confirm the existence of this degradation pathway in the enrichment cultures, because CoA-derivatives of aromatic compounds could not be detected by our analytical protocol. Instead, catechol and 3-HB were detected as the possible intermediate compounds through degradation of PCA ( Fig. 2A). Although there have been no reports on microbial enzymes that dehydroxylate PCA into 3-HB under anoxic conditions, it might be possible that certain bacteria utilize such reaction followed by CoA activation and ring cleavage via (3-hydroxy)benzoyl-CoA pathway. While enzymatic activities to generate catechol by decarboxylation of PCA were reported for some anaerobic microorganisms 48,49 , further catechol degradation routes have not been identified. Some nitrate-and sulfate-reducing bacteria were reported to decompose catechol 20,50 . These bacteria initially convert catechol into PCA by carboxylation, followed by activation with CoA-ligation, dehydroxylation and reductive ring cleavage via the (3-hydroxy)benzoyl-CoA pathway. Although there are some reports on anaerobic degradation of catechol under methanogenic conditions 51-53 , the responsible microorganisms and the degradation pathway were not identified.
As in the case of vanillate, the first step of syringate degradation appears to be acetogenic O-demethylation catalyzed by Sporomusa spp. (Fig. 6B). The demethoxylated product, gallate, seems to be further decomposed into acetate and H 2 /CO 2 mainly by Ruminococcaceae bacteria. Anaerobic degradation of gallate were reported for some fermenting bacteria, including E. oxidoreducens, which is classified into Firmicutes and distantly related to the Ruminococcaceae phylotypes detected in this study 22,23 . E. oxidoreducens initially converts gallate into trihydroxybenzene by decarboxylation reaction, followed by ring cleavage via the phloroglucinol pathway 22 . Although the decarboxylated products of gallate were not detected in this study, it is highly possible that the trihydroxybenzene degradation is much faster than its generation.
Concluding remarks. In this study, we successfully enriched methanogenic microbial communities that decompose the lignin-derived monoaromatics, namely syringate and vanillate. This study is the first to demonstrate that cooperation of microorganisms with a diverse range of trophic groups are required for methanogenic degradation of the lignin-derived aromatics. The initial step appears to be catalyzed by Sporomusa spp. that generate acetate via O-demethylation of the methoxylated aromatics. The resultant demethoxylated aromatics were decomposed into acetate and H 2 /CO 2 by Firmicutes bacteria, while the bacterial groups responsible for PCA (family Peptococcaceae) and gallate (family Ruminococcaceae) were not identical. Finally, both aceticlastic and hydrogenotrophic methanogens generate methane from acetate and H 2 /CO 2 , respectively. Although the details in the pathway of aromatic ring cleavage have not been characterized, isolation of aromatics-degrading Peptococcaceae and Ruminococcaceae strains identified in this study followed by genomic and enzymatic studies will shed light on novel aspects of methanogenic degradation of lignin-derived aromatics in anaerobic environments.

Methods
Enrichment cultures of methanogenic microbial communities. Methanogenic microbial communities were enriched in serum bottles (68 ml in capacity) filled with 20 ml of a bicarbonate-and HEPES-buffered freshwater basal medium (pH 7.0) 54 supplemented with aromatic compounds (benzoate, vanillate, or syringate) as a substrate. The cultures without supplementation of organic substrates were also prepared as a no-amendment control. Approximately 50 mg (wet weight) of rice paddy field soil was inoculated as a microbial source. The cultures were incubated at 30 °C under an atmosphere of N 2 / CO 2 [80:20 (v/v)] without shaking. When methanogenesis reached a plateau, 0.5 ml of culture solution was transferred to the fresh media. Enrichment cultures on PCA/catechol and gallate were constructed in the same medium with the vanillate-and syringate-enrichments as the inoculum, respectively. After more than three passages, the enrichment cultures were subjected to chemical and phylogenetic analyses. All culture experiments were conducted in triplicate.
Chemical analyses. The partial pressure of CH 4 was determined using a gas chromatograph (GC-2014, Shimadzu) as described previously 55 . The concentrations of organic acids were determined using a HPLC (D-2000 LaChrom Elite HPLC system, HITACHI) as described previously 56 . To identify metabolic intermediates, the authentic compounds and supernatant of enrichment cultures were analyzed by HPLC (ACQUITY UPLC system, Waters) coupled with ESI-MS (ACQUITY TQ detector, Waters) using a TSKgel ODS-140HTP column (2.1 by 100 mm, Tosoh) as described previously 57 . In the HPLC analysis, the mobile phase was a 90:10 (v/v) water:acetonitrile at a flow rate of 0.3 ml/min. Formic acid (0.1%) was added to the mobile phase solvent as a means of increasing ionization efficiency for ESI-MS. Degradation products of vanillate and syringate were detected at the wavelength of 220 nm and 270 nm, respectively. Wavelength for the detection and the retention times of authentic compounds were described in Figs S2 and S3. In the ESI-MS analysis, mass spectra were obtained by negative and positive modes with the following settings: capillary voltage, 3.0 kv; cone voltage, 10 to 40 V; source temperature, 120 °C; desolvation temperature, 350 °C; desolvation gas flow rate, 650 liter h −1 ; and cone gas flow rate, 50 liter h −1 .
Scientific RepoRts | 5:14295 | DOi: 10.1038/srep14295 Phylogenetic analyses. Microbial DNA was extracted with the FAST DNA Spin Kit for soil (MP Biomedicals) according to the manufacturer's instructions. PCR amplification of 16S rRNA gene fragments for clone library analyses was conducted as described previously 58 , with primer sets of U515f and U1492r for bacteria and A25f and A958r for archaea 59 . The sequences obtained were assigned to each phylotype using BLASTClust program 60 with a cut-off value of 97% sequence identity. Classification of phylotypes was performed using the Classifier program in the Ribosomal Database Project database 61 . The sequence of each phylotype was compared to those stored in the GenBank nucleotide sequence database using the BLAST program 60 to infer the closest relatives. Phylogenetic trees were constructed by the neighbor-joining method 62 using program MEGA 63 . The bootstrap resampling method 64 was used with 1000 replicates to evaluate the robustness of the phylogenetic trees.
Nucleotide sequence accession numbers. The nucleotide sequence data reported here have been submitted to GenBank under Accession No. LC036665-LC036702.