Abstract. Ferulic and syringic acids are methoxylated aromatic compounds that often serve as models of the subunits of lignin. Although these compounds have ...
Microb Ecol (1997) 33:206–215 q 1997 Springer-Verlag New York Inc.
Microbial Metabolism of the Plant Phenolic Compounds Ferulic and Syringic Acids under Three Anaerobic Conditions C.D. Phelps, L.Y. Young Rutgers, The State University of New Jersey, Cook College, PO Box 231, New Brunswick, NJ 08903-0231 USA Received: 23 February 1996; Revised: 20 May 1996; Accepted: 24 May 1996
A
B S T R A C T
Ferulic and syringic acids are methoxylated aromatic compounds that often serve as models of the subunits of lignin. Although these compounds have important implications for global carbon cycles, there is limited information on their fate in anoxic environments. Enrichment cultures were established on these two model compounds under methanogenic, sulfidogenic, and denitrifying conditions, using a Raritan River (New Jersey) marsh sediment as the inoculum. All cultures completely degraded ,1.5 mM of both substrates. Methane production in the methanogenic cultures corresponded to the stoichiometric values expected for complete mineralization to CO2 and CH4. Sulfate and nitrate reduction in their respective cultures were both greater than 60% of the amounts predicted for complete mineralization. Aromatic intermediates of ferulic and syringic acid metabolism were identified, and pathways of degradation under sulfidogenic and denitrifying conditions are proposed. Syringic acid is sequentially O-demethylated to gallic acid under both sulfate and nitrate-reducing conditions before ring cleavage occurs. Ferulic acid undergoes propenoate side chain reduction, O-demethylation, removal of an acetate moiety from the side chain, and decarboxylation to form catechol. Catechol is further degraded under sulfidogenic conditions. Under denitrifying conditions, ferulic acid undergoes loss of an acetate moiety, prior to O-demethylation, to form protocatechuic acid, the last product detected before ring cleavage.
Introduction Aromatic compounds constitute a large portion of the organic material available to microorganisms in the natural environment [17, 49]. The largest source of these is lignin,
Correspondence to: L.Y. Young.
a complex three-dimensional polymer of oxyphenylpropane units, which is the second most abundant organic compound on earth following cellulose [12]. Water soluble phenolic compounds are released when lignin is metabolized by fungi and actinomycetes [12, 14]. Similar compounds occur in plant tissues as amino acids, flavonoids, lignin precursors, and tannins [39, 42]. In addition to these natural sources, some agricultural and wood processing wastes such as pulpdebarking effluent (which contains 10–20% monomeric phe-
Anaerobic Metabolism of Plant Phenolics
nols) [35], wine distillery wastewater [5], olive mill wastewater [6, 37], and the accidental and deliberate release of pesticides [24, 42] may also introduce large amounts of phenolics into the environment. Although monoaromatic compounds are readily metabolized under aerobic conditions, the extent of their anaerobic degradation is less understood. In 1979, Healy and Young demonstrated that eleven different monoaromatic lignin derivatives were biodegradable to methane and carbon dioxide [23]. Since that time, a number of studies on phenolic degradation by methanogenic consortia [1, 24, 25, 43] and fermentative bacteria [8, 9, 20, 32, 45] have been undertaken, and these have been thoroughly reviewed [15, 24, 36, 39, 42]. Work has also been done to describe the anaerobic degradation of these methoxylated compounds by photosynthetic bacteria [13, 22, 34, 52] and by an iron-reducing consortium [31]. Despite the number of studies of methoxylated phenolic degradation by acetogenic and fermentative bacteria, there have been very few involving nirate or sulfate respiration. Taylor et al. [46, 47] isolated a denitrifying bacterium on p-OHbenzoate, which was later shown to O-demethylate vanillate, ferulate, and syringate. The acetogen Clostridium thermoaceticum is also capable of using nitrate as an electron acceptor while O-demethylating vanillate [40]. Although several sulfate-reducing bacterial strains, which completely mineralize aromatic compounds, have been described [3, 11, 43], none have been reported to mineralize ferulic or syringic acid. Mixed cultures containing sulfate reducers and acetogens that mineralize syringate, ferulate, vanillate, and trimethoxybenzoate have been described [27, 41]. The acetogens removed the methyl groups; the sulfate reducers mineralized the aromatic ring [41] or the acetate produced by fermentation of the ring [27]. The purpose of this study was to determine how the model plant phenolic compounds, ferulic and syringic acids, are degraded by bacterial consortia enriched from a wetland site under denitrifying, sulfidogenic, and methanogenic conditions. This information can improve our understanding of the transformation and degradation processes of plant residues and pesticides in anoxic environments such as wetlands, marine sediments, or poorly aerated soils.
Materials and Methods Establishment of Enrichment Cultures Enrichment cultures were established using a sediment sample from a marsh on the Raritan River (Highland Park, N.J.) as an inoculum.
207 The sediment was actively methanogenic at the time of collection. The water overlying the sediment sample had a pH of 6.5–7.0, a sulfate concentration of 0.3 mM, and no detectable nitrate (,0.02 mM), as determined by ion chromatography. A 10% (vol/vol) sediment slurry was added to defined mineral nitrate [7], sulfate [51], or methanogenic [23] media. The inoculated media was dispensed as 50-ml aliquots into 65-ml serum bottles under an atmosphere of argon (denitrifying medium) or 70% N2/30% CO2 (sulfidogenic and methanogenic media). Bottles were sealed with rubber stoppers and aluminum crimp seals. Strict anaerobic techniques were followed during all procedures. Enrichments were established in triplicate for each substrate, under each of the three reducing conditions, with duplicate sterile (autoclaved three times on consecutive days) and background (no substrate added) controls. Ferulic (3-methoxy-, 4-hydroxy-cinnamic) or syringic (3,5-dimethoxy-,4-hydroxy-benzoic) acid was added as the sole carbon source to all bottles except background controls, to a concentration of ,1.5 mM. All bottles were incubated statically at 308C, in the dark. Enrichments were refed when gas production ceased and there were no longer any detectable aromatic metabolites.
Chemicals Ferulic acid, catechol, and 2-bromoethane sulfonic acid (BESA) were purchased from Sigma (St. Louis, Mo.). Syringic, vanillic, hydrocaffeic, and gallic acids were obtained from Aldrich Chemical Co. (Milwaukee, Wis.). Protocatechuic acid was purchased from Calbiochem (Los Angeles, Calif.), and 5-OH-vanillic acid from Pfaltz and Bauer (Waterbury, Conn.). Hydroferulic acid was prepared from ferulic acid in the laboratory of Dr. Stephen Wilson (Dept. of Chemistry, New York University), and was a gift from Dr. Anne Frazer (Rutgers University). The structures of aromatic compounds referred to in the test are illustrated in Fig. 1.
Analytical Procedures Gas and liquid samples were taken periodically for analysis. The amount of gas produced in each bottle was measured using a 5ml glass syringe (Becton Dickinson, Franklin Lakes, N.J.) as described by Healy and Young [23]. Gas samples were taken by withdrawing a 0.25-ml sample with a gas-tight syringe (Precision Sampling, Baton Rouge, La.) that had been flushed with argon or 70% N2/30% CO2. The percentage of N2, N2O, and CH4 in the headspace gas was determined by analysis with a model 1200 gas partitioner (Fisher Scientific, Pittsburgh, Pa.) equipped with a 3.35-m by 4.76mm column packed with 60/80 mesh 133 molecular sieves (Supelco, Bellefonte, Pa.) in series with a 1.98-m by 3.18-mm column packed with 80/100 mesh Porapak Q (Supelco). Total N2 and CH4 production was calculated from the percentage of each gas and the volume produced. The total N2O production includes the amount dissolved in the medium, which was calculated from the percentage measured in the headspace and Henry’s constant at 258C (moles of N2O in solution 5 [(%N2O 3 0.76)/1.7 3 106] 3 moles of water) [16]. Liquid samples for high performance liquid chromatography (HPLC) and ion chromatography (IC) were withdrawn with sterile syringes that had been flushed with argon or 70% N2/30% CO2 to
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C.D. Phelps, L.Y. Young
Fig. 2. Degradation of ferulic and syringic acid with production of methane in methanogenic enrichment cultures. There was no appreciable loss of substrate in the sterile controls. Both cultures were reamended with substrate on day 84.
external standards using Chrome-Jet integrators (Spectra-Physics, San Jose, Calif.). The identity of catechol was confirmed by mass spectroscopy performed by Wei Fang in the laboratory of Dr. Arthur Greenberg (Rutgers University).
Results Fig. 1. Structures of aromatic compounds.
Degradation of Substrates
remove oxygen. The samples (0.5 ml) were transferred to Spinprep vials with 0.22-mm pore-size nylon filters (VWR Scientific, West Chester, Pa.) containing 20 ml of 1 N HCl (to inhibit chemical oxidation of aromatic compounds) and centrifuged immediately to remove all suspended solids. The disappearance of substrate was monitored using a Beckman (Fullerton, Calif.) System Gold HPLC equipped with a C18 reverse phase column (250 mm by 4.6 mm; particle size, 5 mm), and a Gilson (Middleton, Wis.) autosampler. Aromatic compounds were detected by measuring absorbance at 280 nm. The mobile phase was 64% 5 mM formic acid in water/30% methanol/6% acetonitrile at a flow rate of 1 ml min21. Intermediate metabolites were tentatively identified by coelution with authentic compounds on this system. Nitrate, nitrite, and sulfate were measured using a Dionex (Sunnyvale, Calif.) model 100 ion chromatograph equipped with an IonPac AS9 column (Dionex), a conductivity detector, and an autosampler. The eluent was 2 mM Na2CO3, 0.75 mM NaHCO3, at a flow rate of 2 ml min21. All compounds measured on the gas partitioner, HPLC, and IC were quantitated by comparison with
Ferulic and syringic acids were readily degraded by the enrichments generated under all three reducing conditions. Under methanogenic (Fig. 2) and sulfidogenic (Fig. 3) conditions, ,1.5 mM of each substrate had disappeared within 10 days of the original feeding. Under denitrifying conditions (Fig. 4), ferulic acid was utilized within 10 days, but syringic acid persisted for 12 days before degradation began, and was not completely removed until after 40 days. After the second feeding, both substrates disappeared rapidly under all three conditions, indicating that an enrichment had been established. No substrate loss was detected in sterile controls. The production of methane and the reduction of nitrate and sulfate were followed during degradation of the two substrates. The cumulative amount of methane formed is shown in Fig. 2. These values have been adjusted by subtracting the amount of methane formed in the unamended background controls. The extent and rate of methane production increased after the second addition of substrate. Sulfate reduction (Fig. 3) was not significant until after both compounds had disap-
Anaerobic Metabolism of Plant Phenolics
Fig. 3. Degradation of ferulic and syringic acid with loss of sulfate in sulfidogenic enrichment cultures. There was no appreciable loss of substrate in the sterile controls. Both cultures were reamended with substrate on day 36.
peared, suggesting that aromatic intermediates were being formed during the initial phase of degradation. Stoichiometry and Nitrogen Balance In order to link degradation of the substrates to reduction of the respective electron acceptors, a comparison of the amount of nitrate and sulfate reduced during degradation
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to the amount predicted by the theoretical stoichiometry is shown in Table 1. The measured amount of electron acceptor loss was determined by comparing the concentration of these ions after all of the substrate from two feedings had been metabolized (as judged by the absence of any detectable intermediates and cessation of gas production), with the concentration on the starting day. The amount of nitrate or sulfate loss in the background controls was substracted from the final value. As noted in Table 1, the amount of sulfate reduced in the sulfidogenic cultures was greater than 60% of that predicted for both compounds, and nitrate reduction was more than 70% in the denitrifying cultures. The predicted amount of nitrate or sulfate loss does not account for the possibility of incomplete reduction in N2O in the case of nitrate, or incorporation of carbon into cell mass. Also shown in Table 1 is the stoichiometric balance for methane production. The value for measured methane is the sum of the amounts produced on each sampling date minus the amount produced in the background controls. The degradation of both substrates resulted in nearly stoichiometric production of methane (ferulic acid, 99%; syringic acid, 106%). Nitrogen balance data was also obtained for a set of subcultures propagated from the original denitrifying ferulate enrichments. After the enrichments had completely degraded two additions of substrate, they were diluted 1 to 10 (vol/ vol) into fresh media and ,3 mM ferulate was added. Three active bottles were sampled on the starting day and set aside for analysis of nitrate loss and N2 and N2O formation at the end of the incubation. Of the 0.90 mmoles of nitrate lost, 0.84 mmoles (94%) was recovered as N2 (0.36 mmoles N) and N2O (0.48 mmoles N). Sulfate Dependence
Fig. 4. Degradation of ferulic and syringic acid with loss of nitrate in denitrifying enrichment cultures. There was no appreciable loss of substrate in the sterile controls. Both cultures were reamended with substrate on day 84.
The dependence of the sulfidogenic enrichment cultures on sulfate as the sole electron acceptor was tested by establishing a series of subcultures diluted 1 to 10 (vol/vol) for the original enrichments. These subcultures were divided into four sets of duplicate bottles for each carbon source. One set served as the active controls and contained the normal medium with no additions. The second set was amended with 0.1 mM BESA (a specific inhibitor of methanogenesis). The third contained 20 mM molybdate (MoO422) (a specific inhibitor of sulfate reduction), and the last contained no sulfate. All replicates were fed either ,2 mM ferulic or syringic acid and sampled periodically to measure metabolite production and sulfate loss.
210 Table 1.
C.D. Phelps, L.Y. Young Stiochiometry of enrichment culturesa Electron acceptor loss
Substrate Nitrate Ferulic acid Syringic acid Sulfate Ferulic acid Syringic acid
Substrate metabolized
Predicted
Measured
% of expected
2.61 mM 2.77 mM
21.95 mM 19.91 mM
15.28 mM 15.50 mM
69.6 6 4.9 77.9 6 0.3
2.86 mM 2.98 mM
15.00 mM 13.40 mM
10.20 mM 8.23 mM
68.0 6 6.62 61.5 6 1.27
Methane produced
Ferulic acid Syringic acid a
2.55 mM 2.57 mM
Predicted
Measured
0.67 mmoles 0.58 mmoles
0.66 mmoles 0.61 mmoles
98.8 6 10.5 106.2 6 28.3
Stoichiometry was calculated using the formulas: Ferulic Acid C10H10O4 1 5.25 SO224 1 10.5 H1 r 10 CO2 1 5 H2O 1 5.25 H2S C10H10O4 1 8.4 NO23 1 8.4 H1 r 10 CO2 1 9.2 H2O 1 4.2 N2 C10H10O4 1 5.5 H2O r 5.25 CH4 1 4.75 CO2
All of the ferulic acid–fed replicates were able to transform ferulate to hydrocaffeic acid. However, those that lacked sulfate, or where sulfate reduction was inhibited by molybdate, were unable to catalyze the remaining steps of degradation, including ring cleavage, leading to an accumulation of aromatic metabolites (Fig. 7a). In cultures containing sulfate and not inhibited by molybdate, substrate utilization was complete; no aromatic metabolites accumulated, indicating aromatic rings were cleaved. Syringic acid disappeared rapidly in all replicates with no dependence on the presence of sulfate.
Syringic Acid C9H10O5 1 4.5 SO224 1 9 H1 r 9 CO2 1 5 H2O 1 4.5 H2S C9H10O5 1 7.2 NO23 1 7.2 H1 r 9 CO2 1 8.6 H2O 1 3.6 N2 C9H10O5 1 4 H2O r 4.5 CH4 1 4.5 CO2
followed by protocatechuic acid and catechol. The catechol persisted for ,30 days before being degraded.
Discussion Degradation Under Three Anaerobic Conditions Both ferulic and syringic acid were completely degraded by the bacterial consortia enriched under all three reducing
Metabolites Detected During Degradation Transient metabolites were observed during syringate metabolism under both sulfidogenic and denitrifying conditions. These were identified as 5-OH-vanillic acid and gallic acid. The 5-OH-vanillate appeared first, followed by gallate (Fig. 5). The pattern was the same under both reducing conditions. Metabolites of ferulate degradation were also identified under both conditions. The intermediates detected under denitrifying conditions are shown in figure 6a. Vanillic acid accumulated to nearly stoichiometric amounts and did not decrease until the ferulic acid had completely disappeared. Subsequent to this, protocatechuic acid was detected and was rapidly degraded. No further aromatic compounds were observed. The order of appearance of intermediates under sulfidogenic conditions is illustrated in figure 6b. Hydroferulic acid (not shown in the figure) appeared first, then hydrocaffeic acid,
Fig. 5. Appearance and disappearance of aromatic metabolites during degradation of syringic acid under denitrifying conditions.
210 Table 1.
C.D. Phelps, L.Y. Young Stiochiometry of enrichment culturesa Electron acceptor loss
Substrate Nitrate Ferulic acid Syringic acid Sulfate Ferulic acid Syringic acid
Substrate metabolized
Predicted
Measured
% of expected
2.61 mM 2.77 mM
21.95 mM 19.91 mM
15.28 mM 15.50 mM
69.6 6 4.9 77.9 6 0.3
2.86 mM 2.98 mM
15.00 mM 13.40 mM
10.20 mM 8.23 mM
68.0 6 6.62 61.5 6 1.27
Methane produced
Ferulic acid Syringic acid a
2.55 mM 2.57 mM
Predicted
Measured
0.67 mmoles 0.58 mmoles
0.66 mmoles 0.61 mmoles
98.8 6 10.5 106.2 6 28.3
Stoichiometry was calculated using the formulas: Ferulic Acid C10H10O4 1 5.25 SO224 1 10.5 H1 r 10 CO2 1 5 H2O 1 5.25 H2S C10H10O4 1 8.4 NO23 1 8.4 H1 r 10 CO2 1 9.2 H2O 1 4.2 N2 C10H10O4 1 5.5 H2O r 5.25 CH4 1 4.75 CO2
All of the ferulic acid–fed replicates were able to transform ferulate to hydrocaffeic acid. However, those that lacked sulfate, or where sulfate reduction was inhibited by molybdate, were unable to catalyze the remaining steps of degradation, including ring cleavage, leading to an accumulation of aromatic metabolites (Fig. 7a). In cultures containing sulfate and not inhibited by molybdate, substrate utilization was complete; no aromatic metabolites accumulated, indicating aromatic rings were cleaved. Syringic acid disappeared rapidly in all replicates with no dependence on the presence of sulfate.
Syringic Acid C9H10O5 1 4.5 SO224 1 9 H1 r 9 CO2 1 5 H2O 1 4.5 H2S C9H10O5 1 7.2 NO23 1 7.2 H1 r 9 CO2 1 8.6 H2O 1 3.6 N2 C9H10O5 1 4 H2O r 4.5 CH4 1 4.5 CO2
followed by protocatechuic acid and catechol. The catechol persisted for ,30 days before being degraded.
Discussion Degradation Under Three Anaerobic Conditions Both ferulic and syringic acid were completely degraded by the bacterial consortia enriched under all three reducing
Metabolites Detected During Degradation Transient metabolites were observed during syringate metabolism under both sulfidogenic and denitrifying conditions. These were identified as 5-OH-vanillic acid and gallic acid. The 5-OH-vanillate appeared first, followed by gallate (Fig. 5). The pattern was the same under both reducing conditions. Metabolites of ferulate degradation were also identified under both conditions. The intermediates detected under denitrifying conditions are shown in figure 6a. Vanillic acid accumulated to nearly stoichiometric amounts and did not decrease until the ferulic acid had completely disappeared. Subsequent to this, protocatechuic acid was detected and was rapidly degraded. No further aromatic compounds were observed. The order of appearance of intermediates under sulfidogenic conditions is illustrated in figure 6b. Hydroferulic acid (not shown in the figure) appeared first, then hydrocaffeic acid,
Fig. 5. Appearance and disappearance of aromatic metabolites during degradation of syringic acid under denitrifying conditions.
Anaerobic Metabolism of Plant Phenolics
Fig. 6. Appearance and disappearance of aromatic metabolites during degradation of ferulic acid under (a) denitrifying and (b) sulfidogenic conditions.
conditions. The lack of a perceptible lag period before degradation began suggests that the sediment contained populations of bacteria capable of initiating metabolism. Syringic acid fed–cultures did exhibit a short lag after the initial feeding under denitrifying conditions, but not after refeeding, indicating that the community became acclimated. The methanogenic enrichments exhibited relatively slow methane generation after the initial feeding (see Fig. 2). The response was much faster after refeeding. This acclimation can be explained either by an increase in the population of methanogens, and/or by an increase in the rate that intermediate metabolites were fermented to substrates the methanogens can metabolize. The stoichiometric balance of methane produced by these cultures indicated these substrates were completely mineralized to methane and carbon dioxide. No aromatic intermediates were detected during degradation. This is not surprising, since other researchers have had to use metabolic inhibitors to promote intermediate accumulation (53). Earlier studies using sediments as an inoculum have also reported rapid degradation without a lag [1, 24, 25]. Although the amount of sulfate reduced was lower than the predicted amount for both ferulic (68%) and syringic
211
acid (62%) enrichments, these values are higher than could be accounted for by metabolism of side chain constituents alone. Since only 40% of the carbon in ferulate, and 30% in syringate is accessible without ring fission, we conclude that the aromatic ring was utilized. Some of the remaining reducing power not used in sulfate reduction may have been assimilated into cell mass or accumulated as an incompletely oxidized metabolite such as acetate. Because sulfate-reducing bacteria generally use fermentation products rather than complex organic molecules, it is possible that the bacterial populations responsible for ferulate or syringate disappearance were not sulfidogens. However, some sulfate reducers are able to metabolize aromatic compounds such as phenol, benzoate, and protocatechuate [3, 11, 43]. In selectively inhibited sulfidogenic enrichments, the first two steps of ferulate metabolism do not require sulfate reduction (Fig. 7a). These steps, saturation of the propenoate side chain and O-demethylation, are common reactions carried out by other groups of organisms, such as acetogenic bacteria [18]. The next transformation, conversion of hydrocaffeic acid to protocatechuic acid by removal of two carbons from the side chain, appears to be dependent on sulfate-reducing bacteria (either directly or indirectly), since it does not occur in the absence of sulfate or when molybdate is present. The degradation of syringic acid proceeded in the absence of sulfate, indicating that the transformations leading to ring cleavage are carried out by other anaerobic organisms such as fermentors and/or acetogens. The stoichiometric results indicate, however, that over 60% of the carbon is being used to reduce sulfate by the enriched cosortium (Table 1). The products of ring cleavage are apparently being mineralized by sulfidogens as in the defined mixed culture reported by Kreikenbohm and Pfennig [27]. In the denitrifying enrichments, the amount of nitrate reduced during degradation (Table 1) and the nitrogen balance results (Table 2) indicate that degradation and ring cleavage were dependent on denitrifying bacteria. The stoichiometric equation used to predict the amount of nitrate loss does not include accumulation of cell mass. Inclusion of cell production would increase the estimated percentage of expected nitrate reduction (Table 1). The degradation of similar compounds under denitrifying conditions has been reported previously [4, 46, 47]. Pathways of Degradation Based on the order of appearance of the various intermediate compounds during the course of degradation, pathways are proposed for ferulic and syringic acid under sulfidogenic
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C.D. Phelps, L.Y. Young
Fig. 7. Proposed degradation pathways: (a) ferulic acid, (b) syringic acid.
and denitrifying conditions (see Fig. 7). Although many of the transformations in these pathways are similar to those that have been previously reported [53], this is the first time that these compounds have been systematically examined under denitrifying, sulfate-reducing, and methanogenic conditions.
Table 2.
The degradation of syringic acid appears to follow the same pathways under both denitrifying and sulfidogenic conditions (Fig. 7b). The parent compound is sequentially Odemethylated through 5-OH-vanillate to gallate. This has been reported to be the pathway used by many acetogenic cultures [2, 19, 30, 32, 41], a methylotroph [10], a fermentor
Nitrogen balance of propagated denitrifying cultures
Ferulate metabolized
Nitrate reduced
Nitrous oxide produced
Dinitrogen produced
% of Nitrogen recovered
2.87 mM
0.90 mmoles
0.24 mmoles
0.18 mmoles
93.68 6 2.7
Anaerobic Metabolism of Plant Phenolics
[28], a methanogenic consortium [25], a sulfide methylating homoacetogen [26] and the denitrifyer PN-1 [46]. Although no metabolites of gallic acid were detected in this study, the degradation pathway used by fermentative bacteria has been reported to occur through decarboxylation to form pyrogallol, which is then rearranged to phloroglucinol, then reduced to dihydro-phloroglucinol. This nonaromatic metabolite then undergoes hydrolytic cleavage [8, 9, 21, 29]. Under denitrifying conditions, ferulate was degraded via removal of two carbons from the side chain to vanillate and then O-demethylated to form protocatechuate. Alternatively, under sulfidogenic conditions, ferulate is first converted to hydroferulate, then O-demethylated to hydrocaffeate with loss of an acetate moiety from the side chain to form protocatechuate, which is then decarboxylated to catechol (Fig. 7a). In the sulfidogenic cultures, the propenoate side chain of ferulate was reduced prior to O-demethylation (Fig. 7a). This reaction was not dependent on sulfate reduction. Enoate reduction is used as an electron-accepting reaction by acetogenic bacteria [2, 18, 48] and it has been previously reported to occur on ferulate-related compounds [2, 19, 28, 33, 38, 45, 48, 50]. The decarboxylation of protocatechuate to form catechol, which occurred in the sulfidogenic enrichments, has been reported to occur in rat intestinal microbiota [38] and in methanogenic enrichments [24]. Although we did not detect downstream aromatic metabolites after catechol, Szewzyk et al. reported that it can be dehydroxylated to phenol [44]. The results of this study demonstrate that the plant phenolic compounds ferulic and syringic acid are readily degraded by consortia of bacteria from this site under methanogenic, sulfidogenic, and denitrifying conditions. Because aromatic compounds constitute a large portion of the carbon available to microorganisms in the environment, anaerobic mineralization of these and related compounds can have a significant impact on global carbon cycling, particularly in environments such as wetlands and marine sediments that are dominated by anoxic conditions. In addition, these degradation processes may also be useful for developing treatment methods for some agricultural wastes, as well as determining the fate of aromatic pesticides in the environment.
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New Jersey, The New Jersey Commission on Science and Technology, and ARPA-URI. Thanks to Drs. Peter Coschigano, Anne Frazer, Max Ha¨ggblom, and Junko Kazumi for many helpful discussions, and to Maria Rivera for technical assistance.
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