Metabolism of polyethylene glycol by two anaerobic bacteria, Desulfovibrio desulfuricans and a Bacteroides sp.

Two anaerobic bacteria were isolated from polyethylene glycol (PEG)-degrading, methanogenic, enrichment cultures obtained from a municipal sludge digester. One isolate, identified as Desulfovibrio desulfuricans (strain DG2), metabolized oligomers ranging from ethylene glycol (EG) to tetraethylene glycol. The other isolate, identified as a Bacteroides sp. (strain PG1), metabolized diethylene glycol and polymers of PEG up to an average molecular mass of 20,000 g/mol [PEG 20000; HO-(CH2-CH2-O-)nH]. Both strains produced acetaldehyde as an intermediate, with acetate, ethanol, and hydrogen as end products. In coculture with a Methanobacterium sp., the end products were acetate and methane. Polypropylene glycol [HO-(CH2-CH2-CH2-O-)nH] was not metabolized by either bacterium, and methanogenic enrichments could not be obtained on this substrate. Cell extracts of both bacteria dehydrogenated EG, PEGs up to PEG 400 in size, acetaldehyde, and other mono- and dihydroxylated compounds. Extracts of Bacteroides strain PG1 could not dehydrogenate long polymers of PEG (greater than or equal to 1,000 g/mol), but the bacterium grew with PEG 1000 or PEG 20000 as a substrate and therefore possesses a mechanism for PEG depolymerization not present in cell extracts. In contrast, extracts of D. desulfuricans DG2 dehydrogenated long polymers of PEG, but whole cells did not grow with these polymers as substrates. This indicated that the bacterium could not convert PEG to a product suitable for uptake.     

Fermentation of 2,3-butanediol by Pelobacter carbinolicus sp. nov. and Pelobacter propionicus sp. nov., and evidence for propionate formation from C2 compounds

From anaerobic enrichments with 2,3-butanediol as sole substrate pure cultures of new Gram-negative, strictly anaerobic, non-sporeforming bacteria were isolated. Similar isolates were obtained with acetoin as substrate. From marine muds in saltwater medium a short rod (strain Gra Bd 1) was isolated which fermented butanediol, acetoin and ethylene glycol to acetate and ethanol. The DNA base ratio of this strain was 52.3 mol% guanine plus cytosine.From freshwater sediments and sewage sludge, a different type of short rod (strain Ott Bd 1) was isolated in freshwater medium, which fermented butanediol, acetoin, ethanol, lactate and pyruvate stoichiometrically to acetate and propionate. Propanol and butanol were oxidized to the respective fatty acids with concomitant reduction of acetate and bicarbonate to propionate. The DNA base ratio of strain Ott Bd 1 was 57.4 mol% guanine plus cytosine. No other substrates were used by the isolates, and no other products could be detected. In cocultures with Acetobacterium woodii or Methanospirillum hungatei, strain Gra Bd 1 also grew on ethanol, propanol, and butanol by fermenting these alcohols to the respective fatty acids and molecular hydrogen. Cytochromes could not be detected in any of the new isolates. Since both types of bacteria can not be affiliated to any of the existing genera and species, the new species Pelobacter carbinolicus and Pelobacter propionicus are proposed. The mechanism of butanediol degradation and propionate formation from acetate as well as the ecological importance of both processes are discussed.     

Metabolism of polyethylene glycol by two anaerobic bacteria, Desulfovibrio desulfuricans and a Bacteroides sp

Two anaerobic bacteria were isolated from polyethylene glycol (PEG)-degrading, methanogenic, enrichment cultures obtained from a municipal sludge digester. One isolate, identified as Desulfovibrio desulfuricans (strain DG2), metabolized oligomers ranging from ethylene glycol (EG) to tetraethylene glycol. The other isolate, identified as a Bacteroides sp. (strain PG1), metabolized diethylene glycol and polymers of PEG up to an average molecular mass of 20,000 g/mol [PEG 20000; HO-(CH2-CH2-O-)nH]. Both strains produced acetaldehyde as an intermediate, with acetate, ethanol, and hydrogen as end products. In coculture with a Methanobacterium sp., the end products were acetate and methane. Polypropylene glycol [HO-(CH2-CH2-CH2-O-)nH] was not metabolized by either bacterium, and methanogenic enrichments could not be obtained on this substrate. Cell extracts of both bacteria dehydrogenated EG, PEGs up to PEG 400 in size, acetaldehyde, and other mono- and dihydroxylated compounds. Extracts of Bacteroides strain PG1 could not dehydrogenate long polymers of PEG (greater than or equal to 1,000 g/mol), but the bacterium grew with PEG 1000 or PEG 20000 as a substrate and therefore possesses a mechanism for PEG depolymerization not present in cell extracts. In contrast, extracts of D. desulfuricans DG2 dehydrogenated long polymers of PEG, but whole cells did not grow with these polymers as substrates. This indicated that the bacterium could not convert PEG to a product suitable for uptake.     

Degradation of ethylene glycol and polyethylene glycols by methanogenic consortia.

Methanogenic enrichments capable of degrading polyethylene glycol and ethylene glycol were obtained from sewage sludge. Ethanol, acetate, methane, and (in the case of polyethylene glycols) ethylene glycol were detected as products. The sequence of product formation suggested that the ethylene oxide unit [HO-(CH2-CH2-O-)xH] was dismutated to acetate and ethanol; ethanol was subsequently oxidized to acetate by a syntrophic association that produced methane. The rates of degradation for ethylene, diethylene, and polyethylene glycol with molecular weights of 400, 1,000, and 20,000, respectively, were inversely related to the number of ethylene oxide monomers per molecule and ranged from 0.84 to 0.13 mM ethylene oxide units degraded per h. The enrichments were shown to best metabolize glycols close to the molecular weight of the substrate on which they were enriched. The anaerobic degradation of polyethylene glycol (molecular weight, 20,000) may be important in the light of the general resistance of polyethylene glycols to aerobic degradation.     

Microbial Degradation of Polyethylene Glycols

Mono-, di-, tri-, and tetraethylene glycols and polyethylene glycols (PEG) with molecular weight up to 20,000 were degraded by soil microorganisms. A strain of Pseudomonas aeruginosa able to use a PEG of average molecular weight 20,000 was isolated from soil. Washed cells oxidized mono and tetraethylene glycols, but O₂ consumption was not detectable when such cells were incubated for short periods with PEG 20,000. However, the bacteria excreted an enzyme which converted low- and high-molecular-weight PEG to a product utilized by washed P. aeruginosa cells. Gas chromatography of the supernatant of a culture grown on PEG 20,000 revealed the presence of a compound co-chromatographing with diethylene glycol. A metabolite formed from PEG 20,000 by the extracellular enzyme preparation was identified as ethylene glycol by combined gas chromatography-mass spectrometry.     

Ether-cleaving enzyme and diol dehydratase involved in anaerobic polyethylene glycol degradation by a new Acetobacterium sp.

A strictly anaerobic, homoacetogenic bacterium was enriched and isolated from anoxic sewage sludge with polyethylene glycol (PEG) 1000 as sole source of carbon and energy, and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The new isolate fermented ethylene glycol and PEG's with molecular masses of 106 to 1000 to acetate and small amounts of ethanol. The PEG-degrading activity was not destroyed by proteinase K treatment of whole cells. In cell-free extracts, a diol dehydratase and a PEG-degrading (ether-cleaving) enzyme activity were detected which both formed acetaldehyde as reaction product. The diol dehydratase enzyme was oxygen-sensitive and was stimulated 10–14 fold by added adenosylcobalamine. This enzyme was found mainly in the cytoplasmic fraction (65%) and to some extent (35%) in the membrane fraction. The ether-cleaving enzyme activity reacted with PEG's of molecular masses of 106 to more than 20000. The enzyme was measurable optimally in buffers of high ionic strength (4.0), was extremely oxygen-sensitive, and was inhibited by various corrinoids (adenosylcobalamine, cyanocobalamine, hydroxocobalamine, methylcobalamine). This enzyme was found exclusively in the cytoplasmic fraction. It is concluded that PEG is degraded by this bacterium inside the cytoplasm by a hydroxyl shift reaction, analogous to a diol dehydratase reaction, to form an unstable hemiacetal intermediate. The name polyethylene glycol acetaldehyde lyase is suggested for the responsible enzyme.Abbreviations EG ethylene glycol - DiEG diethylene glycol - TriEG triethylene glycol - TeEG tetraethylene glycol - PEG polyethylene glycol (molecular mass indicated)     

Fermentation of polyethylene glycol via acetaldehyde in Pelobacter venetianus

Summary Pelobacter venetianus, a strictly anaerobic bacterium recently isolated with polyethylene glycol (PEG) as substrate, ferments PEG's with molecular masses of 106–40000, as well as acetoin, ethanolamine, choline, and ethoxyethanol, to acetate and ethanol. Ethylene glycol (EG) and acetaldehyde were fermented in the same manner at limiting concentrations in continuous culture. Growth with glycolaldehyde led to acetate as sole fermentation product. Acetaldehyde appeared as byproduct of PEG fermentation, and accumulated to high concentrations during degradation of PEG 4000 and PEG 6000. Utilization of PEG's was constitutive, whereas acetoin degradation was inducible. Acetaldehyde was shown to be the primary product of EG degradation, and inhibited utilization of other substrates. Enzymes involved in the fermentation of PEG, EG, acetoin, and glycolaldehyde were demonstrated in cell-free extracts, except for the PEG degrading enzyme and EG dehydrase. These results demonstrate that acetaldehyde plays a central role in the metabolism of Pelobacter venetianus. A scheme of intermediary metabolism and PEG degradation is discussed.Abbreviations EG ethylene glycol - Di-EG diethylene glycol - PEG (20 000) polyethylene glycol (molecular weight 20 000)     

Propionigenium modestum gen. nov. sp. nov. a new strictly anaerobic,nonsporing bacterium growing on succinate

From marine and freshwater mud samples and from human saliva new strictly anaerobic, Gram-negative, nonsporeforming bacteria were isolated growing with succinate as sole source of carbon and energy. All strains grew in defined mineral media containing at least 1% sodium chloride. Succinate was stoichiometrically transformed to propionate und carbon dioxide; the growth yield varied between 2.1 and 2.4 g cell dry weight per mol of succinate fermented. In addition to succinate, only fumarate, l-aspartate, l-malate, oxaloacetate and pyruvate, were utilized and were stoichiometrically fermented to propionate and acetate. Yeast extract was not fermented but enhanced growth rates and yields. Neither sulfate, sulfur, nor nitrate were reduced. The DNA base ratio was 33.9±0.3 mol % guanine plus cytosine. A marine isolate, strain Gra Succ 2, is described as type strain of a new species, Propionigenium modestum gen. nov. sp. nov., in the family Bacteroidaceae.     

Oxidation of primary aliphatic alcohols by Acetobacterium carbinolicum sp. nov., a homoacetogenic anaerobe

Four strains of new homoacetogenic bacteria were enriched and isolated from freshwater sediments and sludge with ethanol, propanol, 1,2-propanediol, or 1,2-butanediol as substrates. All strains were Gram-positive nonsporeforming rods and grew well in carbonate-buffered defined media under obligately anaerobic conditions. Optimal growth occurred at 27° C around pH 7.0. H₂/CO₂, primary aliphatic alcohols C₃–C₅, glucose, fructose, lactate, pyruvate, ethylene glycol, 1,2-propanediol, 2,3-butanediol, acetoin, glycerol, and methyl groups of methoxylated benzoate derivates and betaine were fermented to acetate or, in case of primary alcohols C₃–C₅ and 1,2-propanediol, to acetate and the respective fatty acid. In coculture with methanogens methane was formed, probably due to interspecies hydrogen transfer. Strain WoProp 1 is described as a new species, Acetobacterium carbinolicum. It had a DNA base composition of 38.5±1.0% guanine plus cytosine, and contained murein of crosslinkage type B similar to A. woodii.     

Anaerobic oxidation of fatty acids by Clostridium bryantii sp. nov., a sporeforming,obligately syntrophic bacterium

From marine and freshwater mud samples strictly anaerobic, Gram-positive, sporeforming bacteria were isolated which oxidized fatty acids in obligately syntrophic association with H₂-utilizing bacteria. Even-numbered fatty acids with up to 10 carbon atoms were degraded to acetate and H₂, odd-numbered fatty acids with up to 11 carbon atoms including 2-methylbutyrate were degraded to acetate, propionate and H₂. Neither fumarate, sulfate, thiosulfate, sullur, nor nitrate were reduced. A marine isolate, strain CuCal, is described as type strain of a new species, Clostridium bryantii sp. nov.     

Fermentation of trihydroxybenzenes by Pelobacter acidigallici gen. nov. sp. nov., a new strictly anaerobic,non-sporeforming bacterium

Five strains of rod-shaped, Gram-negative, non-sporing, strictly anaerobic bacteria were isolated from limnic and marine mud samples with gallic acid or phloroglucinol as sole substrate. All strains grew in defined mineral media without any growth factors; marine isolates required salt concentrations higher than 1% for growth, two freshwater strains only thrived in freshwater medium. Gallic acid, pyrogallol, 2,4,6-trihydroxybenzoic acid, and phloroglucinol were the only substrates utilized and were fermented stoichiometrically to 3 mol acetate (and 1 mol CO₂) per mol with a growth yield of 10g cell dry weight per mol of substrate. Neither sulfate, sulfur, nor nitrate were reduced. The DNA base ratio was 51.8% guanine plus cytosine. A marine isolate, Ma Gal 2, is described as type strain of a new genus and species, Pelobacter acidigallici gen. nov. sp. nov., in the family Bacteroidaceae. In coculture with Acetobacterium woodii, the new isolates converted also syringic acid completely to acetate. Cocultures with Methanosarcina barkeri converted the respective substrates completely to methane and carbon dioxide.     

New types of acetate-oxidizing,sulfate-reducing Desulfobacter species,D. hydrogenophilus sp. nov., D. latus sp. nov., and D. curvatus sp. nov.

Sulfate-reducing bacteria with oval to rod-shaped cells (strains AcRS1, AcRS2) and vibrio-shaped cells (strains AcRM3, AcRM4, AcRM5) differing by size were isolated from anaerobic marine sediment with acetate as the only electron donor. A vibrio-shaped type (strain AcKo) was also isolated from freshwater sediment. Two strains (AcRS1, AcRM3) used ethanol and pyruvate in addition to acetate, and one strain (AcRS1) grew autotrophically with H₂, sulfate and CO₂. Higher fatty acids or lactate were never utilized. All isolates were able to grow in ammonia-free medium in the presence of N₂. Nitrogenase activity under such conditions was demonstrated by the acetylene reduction test. The facultatively lithoautotrophic strain (AcRS1), a strain (AcRS2) with unusually large cells (2×5 m), and a vibrio-shaped strain (AcRM3) are described as new Desulfobacter species, D. hydrogenophilus, D. latus, and D. curvatus, respectively.     

Fermentation of acetylene by an obligate anaerobe,Pelobacter acetylenicus sp. nov.

Four strains of strictly anaerobic Gram-negative rod-shaped non-sporeforming bacteria were enriched and isolated from marine and freshwater sediments with acetylene (ethine) as sole source of carbon and energy. Acetylene, acetoin, ethanolamine, choline, 1,2-propanediol, and glycerol were the only substrates utilized for growth, the latter two only in the presence of small amounts of acetate. Substrates were fermented by disproportionation to acetate and ethanol or the respective higher acids and alcohols. No cytochromes were detectable; the guanine plus cytosine content of the DNA was 57.1±0.2 mol%. Alcohol dehydrogenase, aldehyde dehydrogenase, phosphate acetyltransferase, and acetate kinase were found in high activities in cell-free extracts of acetylene-grown cells indicating that acetylene was metabolized via hydration to acetaldehyde. Ethanol was oxidized to acetate in syntrophic coculture with hydrogen-scavenging anaerobes. The new isolates are described as a new species in the genusPelobacter, P. acetylenicus.Dedicated to Professor Dr. Norbert Pfennig on occasion of his 60th birthday     

Alcohol and acid formation during the anaerobic decomposition of propylene glycol under methanogenic conditions

Intermediates formed during the anaerobic decomposition of propylene glycol under methanogenic conditions were studied using a serum bottle technique. The pathway is similar to the anaerobic decomposition of ethylene glycol as previously reported. For both compounds, the decomposition is believed to proceed via an initial disproportionation of the glycol to form equal molar amounts of the volatile fatty acid and normal alcohol of the same chain length. In the case of ethylene glycol, disproportionation results in the formation of acetate and ethanol, while disproportionation of propylene glycol produces propionate and n-propanol. Following disproportionation, the alcohols produced from glycol fermentation are oxidized to their corresponding volatile fatty acid with the reduction of protons to form hydrogen. Ethanol and propionate oxidation to acetate proceeds via a well-established syntrophic pathway that is favorable only under low hydrogen partial pressures. Subsequent degradation of acetate proceeds via acetoclastic methanogenesis with the production of carbon dioxide and methane. Despite the production of hydrogen in the initial steps of glycol degradation, both compounds are completely degradable under the methanogenic conditions tested in this study.     

Fermentation of methoxyacetate to glycolate and acetate by newly isolated strains of Acetobacterium sp.

Three strains of new mesophilic homoacetogenic bacteria were enriched and isolated from sewage sludge and from marine sediment samples with methoxyacetate as sole organic substrate in a carbonate-buffered medium under anoxic conditions. Two freshwater isolates were motile, Gram-positive, non-sporeforming rods. The marine strain was an immotile, Gram-positive rod with a slime capsula. All strains utilized only the methyl residue of methoxyacetate and released glycolic acid. They also fermented methyl groups of methoxylated aromatic compounds and of betaine to acetate with growth yields of 6–10 g dry matter per mol methyl group. H₂/CO₂, formate, methanol, hexamethylene tetramine, as well as fructose, numerous organic acids, glycerol, ethylene glycol, and glycol ethers were fermented to acetate as well. High activities of carbon monoxide dehydrogenase (0.4–2.2 U x mg protein^–1) were detected in all three isolates. The guanine-plus-cytosine-content of the DNA of the freshwater isolates was 42.7 and 44.4 mol %, with the marine isolate it was 47.7 mol %. The freshwater strains were assigned to the genus Acetobacterium as new strains of the species A. carbinolicum. One freshwater isolate, strain KoMac1, was deposited with the Deutsche Sammlung von Mikroorganismen GmbH, Braunschweig, under the number DSM 5193.     

Fermentative degradation of nonionic surfactants and polyethylene glycol by enrichment cultures and by pure cultures of homoacetogenic and propionate-forming bacteria.

Linear alkyl ethoxylates (polyethylene glycol alkyl ethers) were fermented completely to methane and CO2 in enrichment cultures inoculated with anoxic sewage sludge. Long-chain fatty acids were released as intermediates. No degradation was found with polypropylene glycol and polypropylene glycol-containing surfactants. Two types of primary ethoxylate-degrading bacteria were isolated and characterized. Both degraded polyethylene glycols with molecular weights of 1,000 completely. Strain KoB35 fermented polyethylene glycol, ethoxyethanol, and lactate to acetate and propionate and was assigned to the described species Pelobacter propionicus. Strain KoB58 converted polyethylene glycol and many other substrates to acetate only and was assigned to the genus Acetobacterium. The pathways of anaerobic degradation of nonionic surfactants are discussed with respect to their limitations and the various groups of bacteria involved.     

Bacterial oxidation of polyethylene glycol.

The metabolism of polyethylene glycol (PEG) was investigated with a synergistic, mixed culture of Flavobacterium and Pseudomonas species, which are individually unable to utilize PEGs. The PEG dehydrogenase linked with 2,6-dichlorophenolindophenol was found in the particulate fraction of sonic extracts and catalyzed the formation of a 2,4-dinitrophenylhydrazine-positive compound, possibly an an aldehyde. The enzyme has a wide substrate specificity towards PEGs: from diethylene glycol to PEG 20,000 Km values for tetraethylene glycol (TEG), PEG 400, and PEG 6,000 were 11, 1.7, and 15 mM, respectively. The metabolic products formed from TEG by intact cells were isolated and identified by combined gas chromatography-mass spectrometry as triethylene glycol and TEG-monocarboxylic acid plus small amounts of TEG-dicarboxylic acid, diethylene glycol, and ethylene glycol. From these enzymatic and analytical data, the following metabolic pathway was proposed for PEG: HO(CH2CH2O)nCH2CH2OH leads to HO(CH2CH2O)nCH2CHO leads to HO(CH2CH2O)nCH2COOH leads to HO(CH2CH2O)n-1CH2CH2OH.     

A new 3-hydroxybutyrate fermenting anaerobe,Ilyobacter polytropus,gen. nov. sp. nov., possessing various fermentation pathways

From marine anoxic mud, a new strictly anaerobic, Gram-negative, non-sporeforming bacterium was isolated with 3-hydroxybutyrate as substrate. 3-Hydroxybutyrate and crotonate were fermented to acetate and butyrate. Glycerol was fermented to 1,3-propanediol and 3-hydroxypropionate. Acetate and formate were the only products of pyruvate or citrate fermentation. Glucose and fructose were fermented to acetate, formate and ethanol. Malate and fumarate were fermented to acetate, formate and propionate. Neither sulfate, sulfur, nor nitrate was reduced. The DNA base ratio was 32.2±0.5 mol% guanine plus cytosine. Strain CuHbu1 is described as type strain of a new genus and species, Ilyobacter polytropus gen. nov. sp. nov., in the family Bacteroidaceae.     

Fermentative degradation of nonionic surfactants and polyethylene glycol by enrichment cultures and by pure cultures of homoacetogenic and propionate-forming bacteria

Linear alkyl ethoxylates (polyethylene glycol alkyl ethers) were fermented completely to methane and CO2 in enrichment cultures inoculated with anoxic sewage sludge. Long-chain fatty acids were released as intermediates. No degradation was found with polypropylene glycol and polypropylene glycol-containing surfactants. Two types of primary ethoxylate-degrading bacteria were isolated and characterized. Both degraded polyethylene glycols with molecular weights of 1,000 completely. Strain KoB35 fermented polyethylene glycol, ethoxyethanol, and lactate to acetate and propionate and was assigned to the described species Pelobacter propionicus. Strain KoB58 converted polyethylene glycol and many other substrates to acetate only and was assigned to the genus Acetobacterium. The pathways of anaerobic degradation of nonionic surfactants are discussed with respect to their limitations and the various groups of bacteria involved.     

Transformation of tetrachloromethane to dichloromethane and carbon dioxide by Acetobacterium woodii.

Five anaerobic bacteria were tested for their abilities to transform tetrachloromethane so that information about enzymes involved in reductive dehalogenations of polychloromethanes could be obtained. Cultures of the sulfate reducer Desulfobacterium autotrophicum transformed some 80 microM tetrachloromethane to trichloromethane and a small amount of dichloromethane in 18 days under conditions of heterotrophic growth. The acetogens Acetobacterium woodii and Clostridium thermoaceticum in fructose-salts and glucose-salts media, respectively, degraded some 80 microM tetrachloromethane completely within 3 days. Trichloromethane accumulated as a transient intermediate, but the only chlorinated methanes recovered at the end of the incubation were 8 microM dichloromethane and traces of chloromethane. Desulfobacter hydrogenophilus and an autotrophic, nitrate-reducing bacterium were unable to transform tetrachloromethane. Reduction of chlorinated methanes was thus observed only in the organisms with the acetyl-coenzyme A pathway. Experiments with [14C]tetrachloromethane were done to determine the fate of this compound in the acetogen A. woodii. Radioactivity in an 11-day heterotrophic culture was largely (67%) recovered in CO2, acetate, pyruvate, and cell material. In experiments with cell suspensions to which [14C]tetrachloromethane was added, 14CO2 appeared within 20 s as the major transformation product. A. woodii thus catalyzes reductive dechlorinations and transforms tetrachloromethane to CO2 by a series of unknown reactions.