Fermentative degradation of polyethylene glycol by a strictly anaerobic, gram-negative, nonsporeforming bacterium, Pelobacter venetianus sp. nov.

The synthetic polyether polyethylene glycol (PEG) with a molecular weight of 20,000 was anaerobically degraded in enrichment cultures inoculated with mud of limnic and marine origins. Three strains (Gra PEG 1, Gra PEG 2, and Ko PEG 2) of rod-shaped, gram-negative, nonsporeforming, strictly anaerobic bacteria were isolated in mineral medium with PEG as the sole source of carbon and energy. All strains degraded dimers, oligomers, and polymers of PEG up to a molecular weight of 20,000 completely by fermentation to nearly equal amounts of acetate and ethanol. The monomer ethylene glycol was not degraded. An ethylene glycol-fermenting anaerobe (strain Gra EG 12) isolated from the same enrichments was identified as Acetobacterium woodii. The PEG-fermenting strains did not excrete extracellular depolymerizing enzymes and were inhibited by ethylene glycol, probably owing to a blocking of the cellular uptake system. PEG, some PEG-containing nonionic detergents, 1,2-propanediol, 1,2-butanediol, glycerol, and acetoin were the only growth substrates utilized of a broad variety of sugars, organic acids, and alcohols. The isolates did not reduce sulfate, sulfur, thiosulfate, or nitrate and were independent of growth factors. In coculture with A. woodii or Methanospirillum hungatei, PEGs and ethanol were completely fermented to acetate (and methane). A marine isolate is described as the type strain of a new species, Pelobacter venetianus sp. nov. Its physiology and ecological significance, as well as the importance and possible mechanism of anaerobic polyether degradation, are discussed.     

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.     

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.     

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.     

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.     

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.     

Bacterial Utilization of Ether Glycols

A soil bacterium capable of using oligo- and polyethylene glycols and ether alcohols as sole sources of carbon for aerobic growth was isolated. The effects of substituent groups added to the ether bonds on the acceptability of the compounds as substrates were studied. Mechanisms for the incorporation of two-carbon compounds were demonstrated by the observation that acetate, glyoxylate, ethylene glycol, and a number of the tricarboxylic acid cycle intermediates served as growth substrates in minimal media. The rate of oxidation of the short-chained ethylene glycols by adapted resting cells varied directly with increasing numbers of two-carbon units in the chains from one to four. The amount of oxygen consumed per carbon atom of oligo- and polyethylene glycols was 100% of theoretical, but only 67% of theoretical for ethylene glycol. Resting cells oxidized oligo- and polyethylene glycols with 2 to 600 two-carbon units in the chains. Longer chained polyethylene glycols (up to 6,000) were oxidized at a very slow rate by these cells. Dehydrogenation of triethylene glycol by adapted cells was observed, coupling the reaction with methylene blue reduction.     

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)     

Thermophilic HB8 DNA ligase: effects of polyethylene glycols and polyamines on blunt-end ligation of DNA

NB8 DNA ligase from an extract of Thermus thermophilus HB8 could catalyze blunt-end ligation in the presence of high concentration of polyethylene glycols (PEG) or in the presence of polyamines. In the presence of high molecular weight PEG 20,000, 6,000, or 1,000 (8-28%), the enzyme catalyzed blunt-end intermolecular joining to yield linear oligomers, but no circular DNA forms. But in the presence of low molecular PEG 400, 200 (8-80%), or the monomer, ethylene glycol (16-80%), the circular forms were also detected by intramolecular ligation. In the presence of polyamines, the blunt-end ligation products were linear oligomers and the optimum concentrations were as follows: caldopentamine (0.05 mM), thermine (0.1-0.2 mM), spermine (0.2 mM), thermospermine (0.4 mM), and sperminediol (0.75 mM). Spermidine and putrescine were less capable of producing oligomers. PEG and polyamines elevated the ligation temperature by HB8 DNA ligase. The optimum temperature of blunt-end ligation was about 65 degrees C.     

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.     

Cryoprotection of purified rat kidney transamidinase by polyethylene glycol.

Polyethylene glycol is a water-soluble polymer which is widely used in the pharmaceutical, cosmetic, and chemical industries. In this study, it is shown that polyethylene glycol is an effective cryoprotectant of rat kidney transamidinase purified from both the mitochondria and cytosol. Much of the activity is lost when the purified enzyme is frozen and thawed in sodium-potassium phosphate buffer in the absence of cryoprotectants. Polyethylene glycols with molecular weights of 4000 to 10,000 were effective cryoprotectants. However, polyethylene glycols with a molecular weight of 1000 or lower inhibited the purified enzyme. A concentration of only 0.01% polyethylene glycol 4000, 8000, or 10,000 was required for complete cryoprotection. In addition to polyethylene glycol, 0.5 mM ethylenediaminetetraacetic acid was required in the phosphate buffer for complete cryoprotection. The stabilization of purified transamidinase by polyethylene glycol will facilitate characterization experiments designed to compare the properties of the mitochondrial and cytosolic isozymes.     

Effects of polyethylene glycol on stability of alpha-chymotrypsin in aqueous ethanol solvent

The effects of polyethylene glycol (PEG) of different molecular weights (400, 2000, 6000, 12,000, 20,000, and 35,000) on the conformational stability and catalytic activity of alpha-chymotrypsin in 60% ethanol were studied. The inactivation caused by the organic solvent was not influenced by PEG 400. However, the PEGs with higher molecular weights up to 35,000 increased the stability of the enzyme, but this alpha-chymotrypsin stabilizing effect was molecular weight-independent. With increase of the molecular weight of PEG, a more stable tertiary structure of the enzyme was observed.     

Glycosidases in organic solvents: II. Transgalactosylation catalysed by polyethylene glycol-modified β-galactosidase

β- -Galactoside galactohydrolase (E.C. 3.2.1.23) was chemically modified with 1,1′-carbonyldiimidazole-activated polyethylene glycols (MW 2,000, 8,000, and 20,000). The modified β-galactosidases had over 50% of amino groups coupled to polyethylene glycol but retained over 50% of the original activity. The hydrophobically modified enzymes were soluble in chlorinated organic solvents in which transferase activity has been demonstrated. The transferase activity, its dependency on water content, and the thermostability of all three modified enzymes were compared.     

Microbial Degradation of Polyethylene Glycols

Three bacterial strains have been isolated that differ in their ability to degrade polyethylene glycols (PEGs). Strains R and O showed a marked preference for growth on the low and high molecular weight PEGs, respectively, while strain Z utilized mono-ethylene glycol only. The partial degradation of PEG 200 by strains R and O was studied in some detail and the results suggested that those components of the mixture that were not utilized were converted into acidic derivatives which accumulated in the medium.     

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)     

Hemoglobin precipitation by polyethylene glycols leads to underestimation of membrane pore sizes

The size of pores formed in the plasma membrane by various substances is frequently determined using polyethylene glycols as osmotic protectants. In this work, we have found that the size of pores formed by saponin in the red blood cell membrane determined by hemolysis versus molecular weight of polyethylene glycol was different to that estimated by light dispersion of cell suspensions. After complete swelling of cells induced by saponin in semiisotonic salt media containing 150 mOsm PEG-4000 or PEG-3000, a significant increase in the light absorbance at 640 nm was developed resulting from the formation of hemoglobin precipitates. Easily sedimenting aggregates were also formed when the supernatant of lysed cells was added to the equiosmotic solutions of polyethylene glycols with molecular weight higher than 1000. We suggest that the real size of large pores could be underestimated due to the phenomenon of hemoglobin precipitation by polyethylene glycols.     

Mechanism of poly(ethylene glycol) interaction with proteins

Poly(ethylene glycol) (PEG) is one of the most useful protein salting-out agents. In this study, it has been shown that the salting-out effectiveness of PEG can be explained by the large unfavorable free energy of its interaction with proteins. Preferential interaction measurements of beta-lactoglobulin with poly(ethylene glycols) with molecular weights between 200 and 1000 showed preferential hydration of the protein for those with Mr greater than or equal to 400, the degree of hydration increasing with the increase in poly(ethylene glycol) molecular weight. The preferential interaction parameter had a strong cosolvent concentration dependence, with poly(ethylene glycol) 1000 having the sharpest decrease with an increase in concentration. The preferential hydration extrapolated to zero cosolvent concentration increased almost linearly with increasing size of the additive, suggesting steric exclusion as the major factor responsible for the preferential hydration. The poly(ethylene glycol) concentration dependence of the preferential interactions could be explained in terms of the nonideality of poly(ethylene glycol) solutions. All the poly(ethylene glycols) studied, when used at levels of 10-30%, decreased the thermal stability of beta-lactoglobulin, suggesting that caution must be exercised in the use of this additive at extreme conditions such as high temperature.     

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.     

Decrease in intestinal permeability to polyethylene glycol 1000 during development in the pig

Changes in intestinal permeability during postnatal development in the pig were investigated by using different-sized polyethylene glycols in the Mr 766-1338 range (polyethylene glycol 1000) as permeability probes. Pigs of varying age, newborn (Oh), 36-45 h old and 22-28 days old, were gavage fed polyethylene glycol 1000 together with the macromolecular markers bovine serum albumin, ovalbumin or FITC-labelled dextran 70,000. The 4-h blood serum concentrations of the different markers were determined and taken as an estimate of their intestinal transmission. In the newborn pigs, high serum levels of polyethylene glycols were obtained, concomitant with high serum levels of bovine serum albumin and FITC-dextran. After intestinal macromolecular closure in the 36-45 h-old pigs, lower serum polyethylene glycol levels were found, especially of those with a Mr greater than 1100 Da. In the 22-28 days-old pigs, polyethylene glycol levels were reduced to one-tenth or less of those in the 36-45 h-old pigs, with the levels decreasing markedly with increasing molecular size. These results show that there is a correlation between the intestinal permeability of polyethylene glycols, especially those larger than 1100, and macromolecules in the newborn pig around intestinal closure, suggesting that such polyethylene glycols traverse the gut by the macromolecular route. During later development, further intestinal maturation results in a markedly reduced permeability to polyethylene glycol 1000.     

Precipitation of collagens by polyethylene glycols

Types I, II, and III collagens are readily precipitated at neutral pH by polyethylene glycols (PEG). As the molecular weight fraction of the polyethylene glycols increases, they become more effective as precipitants on a weight basis. The amount of PEG required for precipitation depends on the pH, the ionic strength, and the nature of the buffer or salts present. In tissue culture media, low concentrations of collagens and procollagens may be quantitatively precipitated and readily collected by low-speed centrifugation. Polyethylene glycol precipitation can be used to obtain collagens and procollagens from tissue culture media at either analytical or preparative scale, and since the polyethylene glycols do not bind to collagens, the precipitates may be further analyzed directly by chromatographic or electrophoretic methods.