Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system

Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B₁ and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.     

Microbial dehalogenation of trichloroacetic acid

A pure bacterial culture and a two-membered mixed culture were isolated that degraded trichloroacetic acid if a second, readily metabolizable substrate was present in the growth medium. Previous doubts over the microbial dehalogenation of trichloroacetic acid (TCA) may be due to its inability to act as a sole carbon and energy source. TCA dehalogenation was associated with conventional 2-haloalkanoic acid dehalogenases but oxalate, the putative dehalogenase product, was not detected. CO₂ was produced rapidly and concomitantly with Cl^– ion release during dehalogenation of TCA. An alternative mechanism is suggested for TCA dehalogenation via an initial decarboxylation reaction. This mechanism predicts that carbon monoxide is a product of TCA decarboxylation and it was significant that one of the organisms isolated,Pseudomonas carboxydohydrogens, was a carboxytroph and a second was an unidentified facultative methylotroph.     

Dichloromethane utilized by an anaerobic mixed culture: acetogenesis and methanogenesis

Dichloromethane (8.9 mg/l) was eliminated from industrially polluted, anaerobic groundwater in a fixed-bed reactor (43 m³) which was packed with activated charcoal and operated continuously for over three years. The elimination of dichloromethane over this period was some ten-fold in excess of the sorptive capacity of the charcoal, and the elimination (3.7 mg/h·[kg of charcoal]: residence time, 49 h) was tentatively attributed to dehalogenative microorganisms immobilized on the charcoal. Anaerobic enrichment cultures, with dichloromethane as the sole added source of carbon and energy, were inoculated with material from the reactor. Reproducibly complete substrate disappearance in subcultures was observed when traces of groundwater (1%) or yeast extract (0.01%) were supplied. Fed-batch experiments under an atmosphere of CO₂ plus N₂ led to the conversion in 11 days of 11 mM dichloromethane to 3 mM acetate and 2 mM methane, with a growth yield of 0.4 g of protein/mol of dichloromethane; insignificant amounts (<1 M) of chloromethane accumulated. Methanogenesis could be inhibited by 50 mM 2-bromoethane sulfonate without any effect on the dehalogenation rate. The maximum dehalogenation rate was 0.13 mmol dichloromethane/h·l (2.6 mkat/kg of protein).Abbreviation DCM dichloromethane     

Conversion of the reactive sulfhydryl groups of proteins to the S-trifluoroethyl derivatives: Application to human hemoglobin

The S-2,2,2-trifluoroethyl residue (-SCH₂CF₃) has been incorporated into human hemoglobin, Hb₄(SH)₂, as an extrinsic probe at Cys-β93 through formation of a disulfide bond. The thiol group was activated by reaction with 5,5′-dithiobis(2-nitrobenzoic acid), Hb₄(SSCH₂CF₃)₂ then being obtained by reaction with 2,2,2-trifluoroethanethiol. Both disulfide interchange reactions proceed to completion with a modest excess of reagents using dilute solutions of hemoglobin (0.005 m heme). The stoichiometry of each disulfide interchange reaction is readily determined by measurement of 5-thio-2-nitrobenzoic acid, a product of each reaction. The functional properties of Hb₄ (SSCH₂CF₃)₂ were found to be similar to those of Hb₄(SH)₂. At pH 7.0 and 20°C the partial pressure of oxygen required for half saturation was 0.45 mm Hg in 0.050 m 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilo-triethanol, 4.1 mm Hg in 0.050 m potassium phosphate, and 16.5 mm Hg in 0.010 m inositol hexaphosphate. The n values of the Hill plots were 1.45, 1.80, and 2.3, respectively. The equilibrium constant for the tetramer-dimer dissociation reaction, K_4,2, of the carbon monoxide derivative was 2.1 × 10^?7m. The time course for combination with carbon monoxide was homogeneous at 432 nm. In the presence of inositol hexaphosphate the time course of combination with carbon monoxide was wavelength dependent.     

Microbial CO Conversions with Applications in Synthesis Gas Purification and Bio-Desulfurization

ABSTRACT

Recent advances in the field of microbial physiology demonstrate that carbon monoxide is a readily used substrate by a wide variety of anaerobic micro-organisms, and may be employed in novel biotechnological processes for production of bulk and fine chemicals or in biological treatment of waste streams. Synthesis gas produced from fossil fuels or biomass is rich in hydrogen and carbon monoxide. Conversion of carbon monoxide to hydrogen allows use of synthesis gas in existing hydrogen utilizing processes and is interesting in view of a transition from hydrogen production from fossil fuels to sustainable (CO₂-neutral) biomass. The conversion of CO with H₂O to CO₂ and H₂ is catalyzed by a rapidly increasing group of micro-organisms. Hydrogen is a preferred electron donor in biotechnological desulfurization of wastewaters and flue gases. Additionally, CO is a good alternative electron donor considering the recent isolation of a CO oxidizing, sulfate reducing bacterium. Here we review CO utilization by various anaerobic micro-organisms and their possible role in biotechnological processes, with a focus on hydrogen production and bio-desulfurization.     

Degradation of 3-Chlorobenzoate under Low-Oxygen Conditions in Pure and Mixed Cultures of the Anoxygenic Photoheterotroph Rhodopseudomonas palustris DCP3 and an Aerobic Alcaligenes Species

The presence or absence of molecular oxygen has been shown to play a crucial role in the degradability of haloaromatic compounds. In the present study, it was shown that anaerobic phototrophic 3-chlorobenzoate (3CBA) metabolism by Rhodopseudomonas palustris DCP3 is oxygen tolerant up to a concentration of 3 μM O₂. Simultaneous oxidation of an additional carbon source permitted light-dependent anaerobic 3CBA degradation at oxygen input levels which, in the absence of such an additional compound, would result in inhibition of light-dependent dehalogenation. Experiments under the same experimental conditions with strain DCP3 in coculture with an aerobic 3CBA-utilizing heterotroph, Alcaligenes sp. strain L6, revealed that light-dependent dehalogenation of 3CBA did not occur. Under both oxygen limitation (O₂ < 0.1 μM) and low oxygen concentrations (3 μM O₂), all the 3CBA was metabolized by the aerobic heterotroph. These data suggest that biodegradation of (halo)aromatics by photoheterotrophic bacteria such as R. palustris DCP3 may be restricted to anoxic photic environments.     

Mass-spectrometric kinetic studies of the nitrogenase and hydrogenase activities in in-vivo cultures of Azospirillum brasilense Sp. 7

Acetylene reduction, deuterium uptake and hydrogen evolution were followed in in-vivo cultures of Azospirillum brasilense, strain Sp 7, by a direct mass-spectrometric kinetic method. Although oxygen was needed for nitrogenase functioning, the enzyme was inactivated by a fairly low oxygen concentration in the culture and an equilibrium had to be found between the rate of oxygen diffusion and bacterial respiration. A nitrogenase-mediated hydrogen evolution was observed only in the presence of carbon monoxide inhibiting the uptake hydrogenase activity which normally recycles all the hydrogen produced. However, under anaerobic conditions and in the presence of deuterium, a bidirectional hydrogenase activity was observed, consisting in D₂ uptake and in H₂ and HD evolution. In contrast to the nitrogenase-mediated H₂ production, this anaerobic H₂ and HD evolution was insensitive to the presence of acetylene and was partly inhibited by carbon monoxide. It was moreover relatively unaffected by the deuterium partial pressure. These results suggest that the anaerobic H₂ and HD evolution can be ascribed to a reverse hydrogenase activity under conditions where D₂ is saturating the uptake process and scavenging the electron acceptors. Although the activities of both nitrogenase and hydrogenase were thus clearly differentiated, a close relationship was found between their respective functioning conditions.     

Reductive Dehalogenations of Halobenzoates by Anaerobic Lake Sediment Microorganisms

Methane-producing freshwater lake sediment was found to dehalogenate chloro-, bromo-, and iodobenzoates by a reductive reaction in which the halogen was replaced by a hydrogen atom. The identity of the dehalogenated products was confirmed by mass spectrometry, nuclear magnetic resonance, or cochromatography. Removal of the halogens to produce benzoate was necessary before mineralization to CH₄ + CO₂ could occur. The dehalogenation occurred after a lag period which lasted from 1 week to more than 6 months, depending on the chemical. Dehalogenation was not observed in the absence of CH₄ production, and it was inhibited by the addition of 20% O₂. Once sediment was acclimated to halobenzoate dehalogenation, new additions of the halobenzoate were degraded without lag. Acclimation was observed regardless of whether the parent substrates were eventually mineralized to CH₄ + CO₂. Sediment acclimated to bromo- and chlorobenzoate degradation generally metabolized bromo- and chlorobenzoates, but sediment acclimated to iodobenzoate degradation only metabolized iodobenzoate. Prior acclimation of sediment to benzoate decomposition did not alter the pattern of dehalogenation, and sediment acclimated to dehalogenation was not concurrently acclimated to benzoate degradation. The presence of this apparent specificity, the lag period, and subsequent acclimation, together with our findings of the absence of dehalogenation in sterile sediments and by sediments previously incubated at ≥39°C, suggests that this reaction was biologically catalyzed. Apparently, a pathway for the reductive dehalogenation of aryl halides is present in anaerobic microorganisms of this methanogenic sediment.     

Metabolism of homoacetogens

Homoacetogenic bacteria are strictly anaerobic microorganisms that catalyze the formation of acetate from C₁ units in their energy metabolism. Most of these organisms are able to grow at the expense of hydrogen plus CO₂ as the sole energy source. Hydrogen then serves as the electron donor for CO₂ reduction to acetate. The methyl group of acetate is formed from CO₂ via formate and reduced C₁ intermediates bound to tetrahydrofolate. The carboxyl group is derived from carbon monoxide, which is synthesized from CO₂ by carbon monoxide dehydrogenase. The latter enzyme also catalyzes the formation of acetyl-CoA from the methyl group plus CO. Acetyl-CoA is then converted either to acetate in the catabolism or to cell carbon in the anabolism of the bacteria. The homoacetogens are very versatile anaerobes, which convert a variety of different substrates to acetate as the major end product.     

Biodegradation of atrazine under denitrifying conditions

Anaerobic biodegradation of atrazine by the bacterial isolate M91-3 was characterized with respect to mineralization, metabolite formation, and denitrification. The ability of the isolate to enhance atrazine biodegradation in anaerobic sediment slurries was also investigated. The organism utilized atrazine as its sole source of carbon and nitrogen under anoxic conditions in fixed-film (glass beads) batch column systems. Results of HPLC and TLC radiochromatography suggested that anaerobic biotransformation of atrazine by microbial isolate M91-3 involved hydroxyatrazine formation. Ring cleavage was demonstrated by ¹⁴CO₂ evolution. Denitrification was confirmed by detection of ¹⁵N₂ in headspace samples of K¹⁵NO₃-amended anaerobic liquid cultures. In aquatic sediments, mineralization of uniformly ring-labeled [¹⁴C]atrazine occurred in both M91-3-inoculated and uninoculated sediment. Inoculation of sediments with M91-3 did not significantly enhance anaerobic mineralization of atrazine as compared to uninoculated sediment, which suggests the presence of indigenous organisms capable of anaerobic atrazine biodegradation. Results of this study suggest that the use of M91-3 in a fixed-film bioreactor may have applications in the anaerobic removal of atrazine and nitrate from aqueous media. Received: 3 September 1997 / Received revision: 4 December 1997 / Accepted: 2 January 1998     

Nutritional growth requirements forButyribacterium methylotrophicum on single carbon substrates and glucose

Butyribacterium methylotrophicum, an anaerobic acetogen, obligately required pantothenate for growth on either glucose, CH₃OH?CO₂, H₂?CO₂, or carbon monoxide. Growth on glucose but not single carbon substrates was stimulated by lipoate and biotin. Sulfide but not sulfate served as the sole sulfur source for growth. This study established thatB. methylotrophicum was both a true autotroph when grown on H₂?CO₂ and a unicarbonotroph on CO as the sole carbon and energy source. In addition, the vitamin requirements of this species further suggest its distinctiveness fromEubacterium limosum (Butyribacterium rettgeri).     

Anaerobic degradation of chlorophenol by an enrichment culture

Summary An anaerobic mixed culture from sewage sludge was enriched in a yeast extract and peptone-containing medium; it was able to degrade 2-cholorophenol completely to methane and CO₂. Degradation rates of 2-chlorophenol of up to 0.18 g/l per day were observed in suspended cultures without biomass retention and of 0.375 g/l per day in cultures immobilized on Liapor clay beads. Attempts to isolate the dechlorinating organism failed. The mixed culture was reduced to three morphologically distinctive microorganisms using a medium with limited amounts of yeast extract and peptone and n-butyrate as a co-substrate. Under these conditions the phenol-degrading bacterium was lost and phenol accumulated in the medium. No growth and no dehalogenation of 2-chlorophenol was obtained when yeast extract and peptone were omitted completely. Besides serving as a source of supplementary components, yeast extract and peptone were apparently required as the main source of carbon, wereas reducing equivalents for reductive dehalogenation were obtained by oxidation of n-butyrate. A spirochaete-like organism was presumably the dechlorinating bacterium. The mixed culture lost its dehalogenation capability if this organism was lost. n-Butyrate could be replaced by n-valerate, hexanoate, heptanoate, octanoate, pelargonic acid, n-decanoic acid or palmitate as co-substrates for dehalogenation of either 2-chlorophenol, 2-bromophenol or complete dechlorination of 2,6-dichlorophenol, whereas from 2,4-dichlorophenol only the substituent in the ortho-position could be eliminated.Dedicated to Professor O. Kandler on the occassion of his 70th birthdayOffprint requests to: J. Winter     

Genomic insights into the metabolic potential of the polycyclic aromatic hydrocarbon degrading sulfate‐reducing Deltaproteobacterium N47

Anaerobic degradation of polycyclic aromatic hydrocarbons (PAHs) is an important process during natural attenuation of aromatic hydrocarbon spills. However, knowledge about metabolic potential and physiology of organisms involved in anaerobic degradation of PAHs is scarce. Therefore, we introduce the first genome of the sulfate‐reducing Deltaproteobacterium N47 able to catabolize naphthalene, 2‐methylnaphthalene, or 2‐naphthoic acid as sole carbon source. Based on proteomics, we analysed metabolic pathways during growth on PAHs to gain physiological insights on anaerobic PAH degradation. The genomic assembly and taxonomic binning resulted in 17 contigs covering most of the sulfate reducer N47 genome according to general cluster of orthologous groups (COGs) analyses. According to the genes present, the Deltaproteobacterium N47 can potentially grow with the following sugars including d ‐mannose, d ‐fructose, d ‐galactose, α‐d ‐glucose‐1P, starch, glycogen, peptidoglycan and possesses the prerequisites for butanoic acid fermentation. Despite the inability for culture N47 to utilize NO₃^‐ as terminal electron acceptor, genes for nitrate ammonification are present. Furthermore, it is the first sequenced genome containing a complete TCA cycle along with the carbon monoxide dehydrogenase pathway. The genome contained a significant percentage of repetitive sequences and transposase‐related protein domains enhancing the ability of genome evolution. Likewise, the sulfate reducer N47 genome contained many unique putative genes with unknown function, which are candidates for yet‐unknown metabolic pathways.     

Microbial carbon oxidation rates and pathways in sediments of two Tanzanian mangrove forests

Temporal and spatial variations in benthic metabolism and anaerobic carbon oxidation pathways were assessed in an anthropogenically impacted (Mtoni) and a pristine (Ras Dege) mangrove forest in Tanzania. The objectives were (1) to evaluate how benthic metabolism is affected by organic carbon availability; (2) to determine the validity of diffusive release of CO₂ as a measure benthic carbon oxidation; and (3) to assess the partitioning of anaerobic carbon pathways and factors controlling the availability of electron acceptors (e.g. oxidized iron). Microbial carbon oxidation measured as diffusive exchange of O₂ and CO₂ (32?C67 and 28?C115 mmol m^?2 day^?1, respectively) showed no specific temporal patterns. Low intertidal sediments at Mtoni fed by labile algal carbon of anthropogenic origin had higher diffusive CO₂ release than high intertidal sediments that primarily received less reactive mangrove detritus. Diffusive release of CO₂ apparently underestimated total sediment carbon oxidation due to CO₂ loss from deep sediments via emission through biogenic structures (i.e. crab burrows and pneumatophores) and porewater seepage into creeks. We propose that diffusive fluxes in the present mangrove sediments are roughly equivalent to depth-integrated reactions occurring in the upper 12 cm. Anaerobic carbon oxidation was dominated by FeR irrespective of anthropogenic influence in sediments where the oxidizing effects of biogenic structures increased the Fe(III) level. More than 80% of the anaerobic carbon oxidation in Mtoni and Ras Dege sediments was due to FeR when reactive Fe(III) exceeded 30 ??mol cm^?3. The anthropogenic influence at Mtoni was primarily noted as up to one order of magnitude higher denitrification than at Ras Dege, but this process always accounted for less than 1% of total carbon oxidation. It is noteworthy that organic and nutrient enrichment of anthropogenic origin in Mtoni has no measurable effect on microbial processes, other than carbon oxidation in the low intertidal area and denitrification throughout the forest, and indicates a strong resilience of mangrove environments towards disturbances.     

Factors influencing the production of hydrogen by the purple non‐sulphur phototrophic bacterium Rhodopseudomonas acidophila KU001

Rhodopseudomonas acidophila KU001 was isolated from leather industry effluents and the effect of different cultural conditions on hydrogen production was studied. Anaerobic light induced more hydrogen production than anaerobic dark conditions. Growing cells produced more amounts of hydrogen between 96 and 144 h of incubation. Resting and growing cells preferred a pH of 6.0 ± 0.24 for hydrogen production. Succinate was the most preferred carbon source for the production of hydrogen while citrate was a poor source of carbon. Acetate and malate were also good carbon sources for hydrogen production under anaerobic light. Among the nitrogen sources, R. acidophila preferred ammonium chloride followed by urea for production of hydrogen_. L‐tyrosine was the least preferred nitrogen source by both growing and resting cells.     

Transport,anoxia and end-product accumulation control carbon dioxide and methane production and release in peat soils

Anaerobic respiration and methanogenesis have been found to slow-down in water saturated peat soils with accumulation of metabolic end-products, i.e. dissolved inorganic carbon (DIC) and methane (CH₄), due to a lack of solute and gas transport. So far it is not well understood how solute and gas transport may control this effect. We conducted a column experiment with homogenized ombrotrophic peat over a period of 300 days at 20 °C. We specifically evaluated the effects of diffusive flux as control, downward advective water flux, intensified ebullition by conduit gas transport and diffusive oxygen supply on controlling anaerobic decomposition rates and carbon (C) turnover. To simulate advective flux, water and solutes were recirculated downward through the column after stripping of dissolved gases. We analyzed DIC and CH₄ concentrations, production rates and fluxes, gas filled porosity, oxygen profiles (O₂) and microbial C biomass over time. DIC residence time thereby served as proxy to characterize transport. A slowdown of anaerobic respiration and methanogenesis evolved with the accumulation of the end-products DIC and CH₄ and set in after 150 days. This slow-down was accompanied by a decrease in the distribution of microbial biomass C with depths. Anaerobic DIC and CH₄ production rates were fastest close to the water table and sharply slowed with depth. Accumulation of DIC and CH₄ in the homogeneous peat material throughout the column decreased decomposition constants from about 10^?5 near the surface to 10^?9 year^?1 deeper in the profile. Advective water transport extended the zone of active methanogenesis compared to a diffusive system; experimental enhancement of ebullition had little or no effect as well as strictly anoxic conditions. DIC residence time was negatively correlated to anaerobic respiration suggesting this parameter to be a predictor of anaerobic peat decomposition in peatlands. Overall, this study suggests that burial of peat and accumulation of metabolic end-products effectively slows decomposition and that this effect needs to be considered to explain peat accumulation and the response of peat mineralization rates to changes in environmental conditions.     

Pseudomonas sp. Strain 273, an Aerobic α,ω-DichloroalkaneDegrading Bacterium

A gram-negative, aerobic bacterium was isolated from soil; this bacterium grew in 50% (vol/vol) suspensions of 1,10-dichlorodecane (1,10-DCD) as the sole source of carbon and energy. Phenotypic and small-subunit ribosomal RNA characterizations identified the organism, designated strain 273, as a member of the genus Pseudomonas. After induction with 1,10-DCD, Pseudomonas sp. strain 273 released stoichiometric amounts of chloride from C₅ to C₁₂ α,ω-dichloroalkanes in the presence of oxygen. No dehalogenation occurred under anaerobic conditions. The best substrates for dehalogenation and growth were C₉ to C₁₂ chloroalkanes. The isolate also grew with nonhalogenated aliphatic compounds, and decane-grown cells dechlorinated 1,10-DCD without a lag phase. In addition, cells grown on decane dechlorinated 1,10-DCD in the presence of chloramphenicol, indicating that the 1,10-DCD-dechlorinating enzyme system was also induced by decane. Other known alkane-degrading Pseudomonas species did not grow with 1,10-DCD as a carbon source. Dechlorination of 1,10-DCD was demonstrated in cell extracts of Pseudomonas sp. strain 273. Cell-free activity was strictly oxygen dependent, and NADH stimulated dechlorination, whereas EDTA had an inhibitory effect.     

Isolation and characterization of Dehalobacterium formicoaceticum gen. nov. sp. nov., a strictly anaerobic bacterium utilizing dichloromethane as source of carbon and energy

A strictly anaerobic, dichloromethane-utilizing bacterium was isolated from a previously described dichloromethane-fermenting, two-component mixed culture. In a mineral medium with vitamins, the organism converted 5 mM dichloromethane within 7 days to formate plus acetate in a molar ratio of 2:1 and to biomass and traces of pyruvate. Of 50 potential substrates and combinations of substrates tested, only dichloromethane supported growth. The organism had a DNA G+C content of 42.7 mol%. From its phylogenetic position deduced from 16S rDNA analysis and from its unique substrate range, we conclude that the organism represents a new genus and a new species within the phylum of the gram-positive bacteria for which we propose the name Dehalobacterium formicoaceticum. Cell extracts were found to contain carbon monoxide dehydrogenase, methylene tetrahydrofolate dehydrogenase, formyl tetrahydrofolate synthetase, and hydrogenase activities, whereas activities of methenyl tetrahydrofolate cyclohydrolase and methylene tetrahydrofolate reductase were not detectable. Activity for dehalogenation of dichloromethane was lost on preparation of cell extracts, but was maintained in cell suspensions. Oxygen and reagents that react with thiol groups caused irreversible inhibition, and propyl iodide caused reversible inhibition of dehalogenation. Our observations suggest: 1) conversion of dichloromethane to methylene tetrahydrofolate, which gives rise to both formate and the methyl group of acetate, or 2) conversion of two molecules of dichloromethane to methylene tetrahydrofolate (which is oxidized to formate) and parallel reductive dehalogenation of one dichloromethane to the methyl group of the corrinoid-protein involved in acetate formation. Received: 11 March 1996 / Accepted 3 May 1996     

Anaerobic degradation of acetone by Desulfococcus biacutus spec. nov.

From anaerobic digestor sludge of a waste water treatment plant, a gram-negative, strictly anaerobic sulfate-reducing bacterium was isolated with acetone as sole organic substrate. The bacterium was characterized as a new species, Desulfococcus biacutus. The strain grew with acetone with doubling times of 72 h to 120 h; the growth yield was 12.0 (±2.1) g · [mol acetone]^-1. Acetone was oxidized completely, and no isopropanol was formed. In labelling studies with ¹⁴CO₂, cell lipids (including approx. 50% PHB) of acetone-grown cells became labelled 7 times as high as those of 3-hydroxy-buyrate-grown cells. Enzyme studies indicated that acetone was degraded via acetoacetyl-CoA, and that acetone was channeled into the intermediary metabolism after condensation with carbon dioxide to a C₄-compound, possibly free acetoacetate. Acetoacetyl-CoA is cleaved by a thiolase reaction to acetyl-CoA which is completely oxidized through the carbon monoxide dehydrogenase pathway. Strain KMRActS was deposited with the Deutsche Sammlung von Mikroorganismen, Braunschweig, under the number DSM 5651.     

Aromatic and Volatile Acid Intermediates Observed during Anaerobic Metabolism of Lignin-Derived Oligomers

Anaerobic enrichment cultures acclimated for 2 years to use a ¹⁴C-labeled, lignin-derived substrate with a molecular weight of 600 as a sole source of carbon were characterized by capillary and packed column gas chromatography. After acclimation, several of the active methanogenic consortia were inhibited with 2-bromoethanesulfonic acid, which suppressed methane formation and enhanced accumulation of a series of metabolic intermediates. Volatile fatty acids levels in 2-bromoethanesulfonic acid-amended cultures were 10 times greater than those in the uninhibited, methane-forming consortia with acetate as the predominant component. Furthermore, in the 2-bromoethanesulfonic acid-amended consortia, almost half of the original substrate carbon was metabolized to 10 monoaromatic compounds, with the most appreciable quantities accumulated as cinnamic, benzoic, caffeic, vanillic, and ferulic acids. 2-Bromoethanesulfonic acid seemed to effectively block CH₄ formation in the anaerobic food chain, resulting in the observed buildup of volatile fatty acids and monoaromatic intermediates. Neither fatty acids nor aromatic compounds were detected in the oligolignol substrate before its metabolism, suggesting that these anaerobic consortia have the ability to mediate the cleavage of the β-aryl-ether bond, the most common intermonomeric linkage in lignin, with the subsequent release of the observed constituent aromatic monomers.