Anaerobic Toluene Activation by Benzylsuccinate Synthase in a Highly Enriched Methanogenic Culture

Permeabilized cells of a highly enriched, toluene-mineralizing, methanogenic culture catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions. The specific in vitro rate of benzylsuccinate formation was >85% of the specific in vivo rate of toluene consumption. This is the first report of benzylsuccinate synthase activity in a methanogenic culture; the activity has previously been reported to occur in denitrifying, sulfate-reducing, and anoxygenic phototrophic bacteria.     

Biochemical and genetic evidence of benzylsuccinate synthase in toluene-degrading,ferric iron-reducing Geobacter metallireducens

In vitro assays demonstrated that toluene-grown cells of Geobacter metallireducens catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions. The specific in vitro rate of benzylsuccinate formationwas ca. 45% of the specific in vivo rate of toluene consumption. In addition, bssA and bssB, which code for the and subunits of benzylsuccinate synthase (BSS), respectively, were found to have sequences in G. etallireducens similar to the only sequences heretofore available (for three denitrifying strains). This is the first report of the presence of BSS in a ferriciron-reducing bacterium; BSS activity has previously been reported in denitrifying, sulfate-reducing, and anoxygenic phototrophic toluene degraders, as well as in a highly enriched methanogenic, toluene-degrading culture.     

Methanogenic toluene metabolism: community structure and intermediates

Toluene is a model compound used to study the anaerobic biotransformation of aromatic hydrocarbons. Reports indicate that toluene is transformed via fumarate addition to form benzylsuccinate or by unknown mechanisms to form hydroxylated intermediates under methanogenic conditions. We investigated the mechanism(s) of syntrophic toluene metabolism by a newly described methanogenic enrichment from a gas condensate-contaminated aquifer. Pyrosequencing of 16S rDNA revealed that the culture was comprised mainly of Clostridiales. The predominant methanogens affiliated with the Methanomicrobiales. Methane production from toluene ranged from 72% to 79% of that stoichiometrically predicted. Isotope studies using (13)C(7) toluene showed that benzylsuccinate and benzoate transiently accumulated revealing that members of this consortium can catalyse fumarate addition and subsequent reactions. Detection of a BssA gene fragment in this culture further supported fumarate addition as a mechanism of toluene activation. Transient formation of cresols, benzylalcohol, hydroquinone and methylhydroquinone suggested alternative mechanism(s) for toluene metabolism. However, incubations of the consortium with (18)O-H(2)O showed that the hydroxyl group in these metabolites did not originate from water and abiotic control experiments revealed abiotic formation of hydroxylated species due to reactions of toluene with sulfide and oxygen. Our results suggest that toluene is activated by fumarate addition, presumably by the dominant Clostridiales.     

Anaerobic activation of toluene and o-xylene by addition to fumarate in denitrifying strain T.

Anaerobic assays conducted with strain T, a denitrifying bacterium capable of mineralizing toluene to carbon dioxide, demonstrated that toluene-grown, permeabilized cells catalyzed the addition of toluene to fumarate to form benzylsuccinate. This reaction was not dependent on the presence of coenzyme A (CoA) or ATP. In the presence of CoA, formation of E-phenylitaconate from benzylsuccinate was also observed. Kinetic studies demonstrated that the specific rate of benzylsuccinate formation from toluene and fumarate in assays with permeabilized cells was >30% of the specific rate of toluene consumption in whole-cell suspensions with nitrate; this observation suggests that benzylsuccinate formation may be the first reaction in anaerobic toluene degradation by strain T. Use of deuterium-labeled toluene and gas chromatography-mass spectrometry indicated that the H atom abstracted from the toluene methyl group during addition to fumarate was retained in the succinyl moiety of benzylsuccinate. In this study, no evidence was found to support previously proposed reactions of toluene with acetyl-CoA or succinyl-CoA. Toluene-grown, permeabilized cells of strain T also catalyzed the addition of o-xylene to fumarate to form (2-methylbenzyl)succinate. o-Xylene is not a growth substrate for strain T, and its transformation was probably cometabolic. With the exception of specific reaction rates, the observed characteristics of the toluene-fumarate addition reaction (i.e., retention of a methyl H atom and independence from CoA and ATP) also apply to the o-xylene-fumarate addition reaction. Thus, addition to fumarate may be a biochemical strategy to anaerobically activate a range of methylbenzenes.     

Enhanced Anaerobic Biodegradation of Benzene-Toluene-Ethylbenzene-Xylene-Ethanol Mixtures in Bioaugmented Aquifer Columns

Methanogenic flowthrough aquifer columns were used to investigate the potential of bioaugmentation to enhance anaerobic benzene-toluene-ethylbenzene-xylene (BTEX) degradation in groundwater contaminated with ethanol-blended gasoline. Two different methanogenic consortia (enriched with benzene or toluene and o-xylene) were used as inocula. Toluene was the only hydrocarbon degraded within 3 years in columns that were not bioaugmented, although anaerobic toluene degradation was observed after only 2 years of acclimation. Significant benzene biodegradation (up to 88%) was observed only in a column bioaugmented with the benzene-enriched methanogenic consortium, and this removal efficiency was sustained for 1 year with no significant decrease in permeability due to bioaugmentation. Benzene removal was hindered by the presence of toluene, which is a more labile substrate under anaerobic conditions. Real-time quantitative PCR analysis showed that the highest numbers of bssA gene copies (coding for benzylsuccinate synthase) occurred in aquifer samples exhibiting the highest rate of toluene degradation, which suggests that this gene could be a useful biomarker for environmental forensic analysis of anaerobic toluene bioremediation potential. bssA continued to be detected in the columns 1 year after column feeding ceased, indicating the robustness of the added catabolic potential. Overall, these results suggest that anaerobic bioaugmentation might enhance the natural attenuation of BTEX in groundwater contaminated with ethanol-blended gasoline, although field trials would be needed to demonstrate its feasibility. This approach may be especially attractive for removing benzene, which is the most toxic and commonly the most persistent BTEX compound under anaerobic conditions.     

Analysis of the Novel Benzylsuccinate Synthase Reaction for Anaerobic Toluene Activation Based on Structural Studies of the Product

Recent studies of anaerobic toluene catabolism have demonstrated a novel reaction for anaerobic hydrocarbon activation: the addition of the methyl carbon of toluene to fumarate to form benzylsuccinate. In vitro studies of the anaerobic benzylsuccinate synthase reaction indicate that the H atom abstracted from the toluene methyl group during addition to fumarate is retained in the succinyl moiety of benzylsuccinate. Based on structural studies of benzylsuccinate formed during anaerobic, in vitro assays with denitrifying, toluene-mineralizing strain T, we now report the following characteristics of the benzylsuccinate synthase reaction: (i) it is highly stereospecific, resulting in >95% formation of the (+)-benzylsuccinic acid enantiomer [(R)-2-benzyl-3-carboxypropionic acid], and (ii) active benzylsuccinate synthase does not contain an abstracted methyl H atom from toluene at the beginning or at the end of a catalytic cycle.     

Enhanced anaerobic biodegradation of benzene-toluene-ethylbenzene-xylene-ethanol mixtures in bioaugmented aquifer columns

Methanogenic flowthrough aquifer columns were used to investigate the potential of bioaugmentation to enhance anaerobic benzene-toluene-ethylbenzene-xylene (BTEX) degradation in groundwater contaminated with ethanol-blended gasoline. Two different methanogenic consortia (enriched with benzene or toluene and o-xylene) were used as inocula. Toluene was the only hydrocarbon degraded within 3 years in columns that were not bioaugmented, although anaerobic toluene degradation was observed after only 2 years of acclimation. Significant benzene biodegradation (up to 88%) was observed only in a column bioaugmented with the benzene-enriched methanogenic consortium, and this removal efficiency was sustained for 1 year with no significant decrease in permeability due to bioaugmentation. Benzene removal was hindered by the presence of toluene, which is a more labile substrate under anaerobic conditions. Real-time quantitative PCR analysis showed that the highest numbers of bssA gene copies (coding for benzylsuccinate synthase) occurred in aquifer samples exhibiting the highest rate of toluene degradation, which suggests that this gene could be a useful biomarker for environmental forensic analysis of anaerobic toluene bioremediation potential. bssA continued to be detected in the columns 1 year after column feeding ceased, indicating the robustness of the added catabolic potential. Overall, these results suggest that anaerobic bioaugmentation might enhance the natural attenuation of BTEX in groundwater contaminated with ethanol-blended gasoline, although field trials would be needed to demonstrate its feasibility. This approach may be especially attractive for removing benzene, which is the most toxic and commonly the most persistent BTEX compound under anaerobic conditions.     

A stable organic free radical in anaerobic benzylsuccinate synthase of Azoarcus sp. strain T

The novel enzyme benzylsuccinate synthase initiates anaerobic toluene metabolism by catalyzing the addition of toluene to fumarate, forming benzylsuccinate. Based primarily on its sequence similarity to the glycyl radical enzymes, pyruvate formate-lyase and anaerobic ribonucleotide reductase, benzylsuccinate synthase was speculated to be a glycyl radical enzyme. In this report we use EPR spectroscopy to demonstrate for the first time that active benzylsuccinate synthase from the denitrifying bacterium Azoarcus sp. strain T harbors an oxygen-sensitive stable organic free radical. The EPR signal of the radical was centered at g = 2.0021 and was characterized by a major 2-fold splitting of about 1.5 millitesla. The strong similarities between the EPR signal of the benzylsuccinate synthase radical and that of the glycyl radicals of pyruvate formate-lyase and anaerobic ribonucleotide reductase provide evidence that the benzylsuccinate synthase radical is located on a glycine residue, presumably glycine 828 in Azoarcus sp. strain T benzylsuccinate synthase.     

Substrate specificities and electron paramagnetic resonance properties of benzylsuccinate synthases in anaerobic toluene and<Emphasis Type="Italic"> m</Emphasis>-xylene metabolism

The anaerobic degradation pathways of toluene and m-xylene are initiated by addition of a fumarate cosubstrate to the methyl group of the hydrocarbon, yielding (R)-benzylsuccinate and (3-methylbenzyl)succinate, respectively, as first intermediates. These reactions are catalyzed by a novel glycyl-radical enzyme, (R)-benzylsuccinate synthase. Substrate specificities of benzylsuccinate synthases were analyzed in Azoarcus sp. strain T and Thauera aromatica strain K172. The enzyme of Azoarcus sp. strain T converts toluene, but also all xylene and cresol isomers, to the corresponding succinate adducts, whereas the enzyme of T. aromatica is active with toluene and all cresols, but not with any xylene isomer. This corresponds to the capabilities of Azoarcus sp. strain T to grow on either toluene or m-xylene, and of T. aromatica to grow on toluene as sole hydrocarbon substrate. Thus, differences in the substrate spectra of the respective benzylsuccinate synthases of the two strains contribute to utilization of different aromatic hydrocarbons, although growth on different substrates also depends on additional determinants. We also provide direct evidence by electron paramagnetic resonance (EPR) spectroscopy that glycyl radical enzymes corresponding to substrate-induced benzylsuccinate synthases are specifically detectable in anoxically prepared extracts of toluene- or m-xylene-grown cells. The presence of the EPR signals and the determined amount of the radical are consistent with the respective benzylsuccinate synthase activities. The properties of the EPR signals are highly similar to those of the prototype glycyl radical enzyme pyruvate formate lyase, but differ slightly from previously reported parameters for partially purified benzylsuccinate synthase.     

Reductive Desulfurization of Dibenzyldisulfide

Dibenzyldisulfide was reductively degraded by a methanogenic mixed culture derived from a sewage digestor. Toluene was produced with benzyl mercaptan as an intermediate in sulfur-limited medium. Toluene production was strictly associated with biological activity; however, the reducing agent for the culture medium, Ti(III), was partially responsible for production of benzyl mercaptan. Sulfide was not detected. Additions of sodium sulfide did not inhibit toluene production. Additions of 2-bromoethane sulfonic acid prevented methanogenesis but did not adversely affect toluene yields.     

Identification of Desulfosporosinus as toluene-assimilating microorganisms from a methanogenic consortium

To understand the potential for toluene removal under electron acceptor depleted conditions, stable isotope probing (SIP) was applied to a methanogenic toluene degrading culture to identify the microorganisms responsible for toluene assimilation. Both bacterial and archaeal communities were investigated. The approach involved addition of labeled and unlabeled toluene to microcosms, DNA extraction, ultracentrifugation, and analysis of the generated fractions, as well as the total genomic DNA. Three genes were investigated in the fractions, including the 16S rRNA gene, bssA (encoding for benzylsuccinate synthase α-subunit) and bamA (encoding for 6-oxocylcohex-1-ene-1-carbonyl-CoA hydrolase). Analysis of the total genomic 16S rRNA gene clone library indicated the microbial community was reasonably diverse, containing microorganisms from six phyla (Proteobacteria, Firmicutes, Acidobacteria, Actinobacteria, Deferribacteres, Bacteroidetes). In contrast, only four phylotypes were found in the heavy fraction 16S rRNA gene clone library (from three phyla: Firmicutes, Acidobacteria, Actinobacteria). When these data were correlated with the TRFLP fragments enriched in the heavy fractions, three phylotypes were identified. Specifically, a Desulfosporosinus phylotype was highly enriched in the heavy fractions and was therefore the key consumer of the labeled carbon from toluene. Two other phylotypes, Peptostreptococcaceae and Pseudonocardia were presumed to consume daughter products and produce methane precursors, which in turn were likely utilized by Methanomicrobia to produce methane. Further, the SIP results suggested that the enzymes encoding by functional genes (bssA and bamA) were likely to be harbored by the Desulfosporosinus phylotype.     

Operon structure and expression of the genes for benzylsuccinate synthase in<Emphasis Type="Italic"> Thauera aromatica</Emphasis> strain K172

The first step in anaerobic toluene degradation is the addition of a fumarate cosubstrate to the methyl group of toluene, as catalyzed by the glycyl radical enzyme benzylsuccinate synthase. The bssDCAB genes code for the subunits of benzylsuccinate synthase (BssA, B and C) and an additional enzyme implicated in activating the enzyme by introducing the glycyl radical (BssD). Quantitation of the amounts of benzylsuccinate synthase and activating enzyme showed that both proteins are only synthesized in toluene-grown cells, and that the activating enzyme is present in about 14-fold lower amounts. Two mRNA species of the bss gene cluster were identified, one beginning in front of bssD, and a second in front of bssC. Only the first mRNA 5'-end correlates with a toluene-induced promoter, which is similar to that preceding the bbs operon coding for the further enzymes of toluene catabolism of the same strain. The second mapped 5'-end appears to be generated by endonucleolytic processing. The mRNA segment containing the bssD gene is very short-lived, while that containing the bssCAB genes is more stable. The RNA stability data are consistent with the observed amounts of encoded gene products. Furthermore, the previously known bssDCAB genes are apparently cotranscribed with a fifth gene ( bssE) whose product may function as a putative ATP-dependent chaperone for assembly and/or activation of benzylsuccinate synthase.     

Methanogenic Microbial Community Composition of Oily Sludge and Its Enrichment Amended with Alkanes Incubated for Over 500 Days

Methanogenic microbial community is responsive to the availability of hydrocarbons and such information is critical for the assessment of hydrocarbon degradation in remediation and also in biologically enhanced recovery of energy from non-producing oil reserves. In this study, methanogenic enrichment cultures from oily sludge amended with n-alkanes (C₁₅-C₂₀) showed a development of active methanogenic alkanes-degrading consortium for over a total of 1000 days of incubation at 37°C. Total genomic DNAs were extracted from three types of samples, the original oily sludge (OS), the sludge after incubation for 500 days under methanogenic condition without any external carbon addition (EC), and the enrichment culture from the EC amended with n-alkanes (ET) incubated for another 500 days. The phylogenetic diversities of microbial communities of the three samples were analyzed by PCR amplification of partial 16S rRNA genes. The catabolic genes encoding benzylsuccinate synthase (bssA) and alkylsuccinate synthase (assA) were also examined by PCR amplification. These results provide important evidence in that microbial populations in an oily sludge shifted from methanogenic aromatic compounds degrading communities to potential methanogenic alkane-degrading communities when the enrichment was supplemented with n-alkanes and incubated under anaerobic conditions.     

Substrate induction and metabolite accumulation during anaerobic toluene utilization by the denitrifying strain T1.

The denitrifying strain T1 utilizes toluene anaerobically. We now report that anaerobic toluene degradation is inducible in strain T1. Fluoracetate treatment of cell suspensions inhibited both the rate of toluene metabolism and the formation of the toluene dead-end products benzylsuccinate and benzylfumarate, which is consistent with the pathway proposed by Evans et al. (Appl. Environ. Microbiol. 58:496-501, 1992). In addition, when either nitrate was limiting or fluoroacetate was added, benzoate was detected during toluene metabolism.     

Initial reactions of anaerobic metabolism of alkylbenzenes in denitrifying and sulfate-reducing bacteria

The initial activation reactions of anaerobic oxidation of the aromatic hydrocarbons toluene and ethylbenzene were investigated in cell extracts of a toluene-degrading, sulfate-reducing bacterium, Desulfobacula toluolica, and in cell extracts of strain EbN1, a denitrifying bacterium capable of degrading toluene and ethylbenzene. Extracts of toluene-grown cells of both species catalysed the addition of fumarate to the methyl group of [phenyl-¹⁴C]-toluene and formed [¹⁴C]-labeled benzylsuccinate. Extracts of ethylbenzene-grown cells of strain EbN1 did not catalyse this reaction, but catalysed the formation of 1-phenylethanol and acetophenone from [methylene-¹⁴C]-ethylbenzene. Toluene-grown cells of D. toluolica and strain EbN1 synthesised highly induced polypeptides corresponding to the large subunits of benzylsuccinate synthase from Thauera aromatica. These polypeptides were absent in strain EbN1 after growth on ethylbenzene, although a number of different polypeptides were highly induced. Thus, formation of benzylsuccinate from toluene and fumarate appears to be the general initiating step in anaerobic toluene degradation by bacteria affiliated with the phylogenetically distinct β-subclass (strain EbN1 and T. aromatica) and δ-subclass (D. toluolica) of the Proteobacteria. Anaerobic ethylbenzene oxidation proceeds via a different pathway involving a two-step oxidation of the methylene group to an alcohol and an oxo group; these steps are most probably followed by a biotin-independent carboxylation reaction and thiolytic cleavage. Received: 16 March 1998 / Accepted: 27 June 1998     

Acidophilic degradation of methanol by a methanogenic enrichment culture

Abstract An acidophilic methanogenic enrichment culture was obtained in a continuous up-flow anaerobic sludge blanket reactor operated at pH 4.2 with methanol as the sole carbon source. The specific methylotrophic methanogenic activity of the enriched reactor sludge at pH 5 was 3.57 g COD g⁻¹ volatile suspended solids day⁻¹ and the apparent doubling time of the biomass was 15.8 h. Acidic conditions were obligatory, since the enrichment culture was not able to produce methane or to grow at pH 7. Based on morphological characteristics, the dominant methanogenic species in the enrichment culture was a Methanosarcina .