Sequential anaerobic degradation of 2,4-dichlorophenol in freshwater sediments

2,4-Dichlorophenol (2,4-DCP) was anaerobically degraded in freshwater lake sediments. From observed intermediates in incubated sediment samples and from enrichment cultures, the following sequence of transformations was postulated. 2,4-DCP is dechlorinated to 4-chlorophenol (4-CP), 4-CP is dechlorinated to phenol, phenol is carboxylated to benzoate, and benzoate is degraded via acetate to methane and CO2; at least five different organisms are involved sequentially. The rate-limiting step was the transformation of 4-CP to phenol. Sediment-free enrichment cultures were obtained which catalyzed only the dechlorination of 2,4-DCP, the carboxylation of phenol, and the degradation of benzoate, respectively. Whereas the dechlorination of 2,4-DCP was not inhibited by H2, the dechlorination of 4-CP, and the transformation of phenol and benzoate were. Low concentrations of 4-CP inhibited phenol and benzoate degradation. Transformation rates and maximum concentrations allowing degradation were determined in both freshly collected sediments and in adapted samples: at 31 degrees C, which was the optimal temperature for the dechlorination, the average adaptation time for 2,4-DCP, 4-CP, phenol, and benzoate transformations were 7, 37, 11 and 2 days, respectively. The maximal observed transformation rates for these compounds in acclimated sediments were 300, 78, 2, 130, and 2,080 micromol/liter(-1)/day(-1), respectively. The highest concentrations which still allowed the transformation of the compound in acclimated sediments were 3.1 m/M 2,4-DCP, 3.1 mM 4-CP, 13 mM phenol, and greater than 52 mM benzoate. The corresponding values were lower for sediments which had not been adapted for the transformation steps.     

Sequential anaerobic degradation of 2,4-dichlorophenol in freshwater sediments.

2,4-Dichlorophenol (2,4-DCP) was anaerobically degraded in freshwater lake sediments. From observed intermediates in incubated sediment samples and from enrichment cultures, the following sequence of transformations was postulated. 2,4-DCP is dechlorinated to 4-chlorophenol (4-CP), 4-CP is dechlorinated to phenol, phenol is carboxylated to benzoate, and benzoate is degraded via acetate to methane and CO2; at least five different organisms are involved sequentially. The rate-limiting step was the transformation of 4-CP to phenol. Sediment-free enrichment cultures were obtained which catalyzed only the dechlorination of 2,4-DCP, the carboxylation of phenol, and the degradation of benzoate, respectively. Whereas the dechlorination of 2,4-DCP was not inhibited by H2, the dechlorination of 4-CP, and the transformation of phenol and benzoate were. Low concentrations of 4-CP inhibited phenol and benzoate degradation. Transformation rates and maximum concentrations allowing degradation were determined in both freshly collected sediments and in adapted samples: at 31 degrees C, which was the optimal temperature for the dechlorination, the average adaptation time for 2,4-DCP, 4-CP, phenol, and benzoate transformations were 7, 37, 11 and 2 days, respectively. The maximal observed transformation rates for these compounds in acclimated sediments were 300, 78, 2, 130, and 2,080 micromol/liter(-1)/day(-1), respectively. The highest concentrations which still allowed the transformation of the compound in acclimated sediments were 3.1 m/M 2,4-DCP, 3.1 mM 4-CP, 13 mM phenol, and greater than 52 mM benzoate. The corresponding values were lower for sediments which had not been adapted for the transformation steps.     

Isolation and partial characterization of aClostridium species transforming para-hydroxybenzoate and 3,4-dihydroxybenzoate and producing phenols as the final transformation products

Organisms present in methanogenic freshwater lake sediments from the vicinity of Athens, Georgia, were adapted to mineralize 2,4-dichlorophenol. Repeated addition of 0.5 to 2.7 mmol/liter of phenol, and later of 0.5–6.2 mmol/liter p-hydroxybenzoate (p-OHB), to such enrichments led to the conversion of p-OHB to phenol at a rate of up to 100 mmol p-OHB per liter per day. Subsequently, a spore-forming, obligately anaerobic bacterium, strain JW/Z-1, was isolated which transformed p-OHB to phenol and 3,4-dihydroxybenzoate (3,4-OHB) to catechol (1,2-dihydroxybenzene) stoichiometrically without further metabolism of the phenols. The strain did not transform benzoate, 4-chlorophenol, 2,4-dichlorophenol, 4-chlorobenzoate, o- and m-hydroxybenzoate, 2,4- and 3,5-dihydroxybenzoate, 2,3,4- and 3,4,5-trihydroxybenzoate, or 4-aminobenzoate. Yeast extract was required for growth of strain JW/Z-1 and only high concentrations of casein hydrolysate or tryptone could substitute it, to some extent. Except for sodium acetate, and some amino acids together with a 20-fold increased concentration of vitamins, no single carbohydrate or defined organic compound has been found to support growth of this strain in the presence (or in the absence) of 0.2 to 0.5% (w/v) yeast extract. The fermentation products during growth on yeast extract indicated that the metabolism of amino acid degradation was the major source for growth. The decarboxylating activity was inducible by p-OHB for the decarboxylation of p-OHB, and at a lower rate for 3,4-OHB, and by 3,4-OHB only for 3,4-OHB, suggesting that two different enzyme systems exist. The addition of the aromatic amino acids phenol or benzoate did not induce the decarboxylation activity in cultures growing with yeast extract. Growth was observed at temperatures ranging from 12–41°C (T_opt, 33–34°C) and at pH-values ranging from 6.0–10.0 (pH_opt, 7.2–8.2). The shortest doubling time observed for strain JW/Z-1 was 3.2 hours.     

Degradation of phenol under meso- and thermophilic, anaerobic conditions

Based on the results of preliminary studies on phenol degradation under mesophilic conditions with a mixed methanogenic culture, we proposed a degradation pathway in which phenol is fermented to acetate: Part of the phenol is reductively transformed to benzoate while the rest is oxidised, forming acetate as end product. According to our calculations, this should result in three moles of phenol being converted to two moles of benzoate and three moles of acetate (3 phenol + 2 CO2 + 3 H2O --> 3 acetate + 2 benzoate): To assess the validity of our hypothesis concerning the metabolic pathway, we studied the transformation of phenol under mesophilic and thermophilic conditions in relation to the availability of hydrogen. Hence, methanogenic meso- and thermophilic cultures amended with phenol were run with or without an added over-pressure of hydrogen under methanogenic and non-methanogenic conditions. Bromoethanesulfonic acid (BES) was used to inhibit methanogenic activity. In the mesophilic treatments amended with only BES, about 70% of the carbon in the products found was benzoate. During the course of phenol transformation in these BES-amended cultures, the formation pattern of the degradation products changed: Initially nearly 90% of the carbon from phenol degradation was recovered as benzoate, whereas later in the incubation, in addition to benzoate formation, the aromatic nucleus degraded completely to acetate. Thus, the initial reduction of phenol to benzoate resulted in a lowering of H2 levels, giving rise to conditions allowing the degradation of phenol to acetate as the end product. Product formation in bottles amended with BES and phenol occurred in accordance with the hypothesised pathway; however, the overall results indicate that the degradation of phenol in this system is more complex. During phenol transformation under thermophilic conditions, no benzoate was observed and no phenol was transformed in the BES-amended cultures. This suggests that the sensitivity of phenol transformation to an elevated partial pressure of H2 is higher under thermophilic conditions than under mesophilic ones. The lack of benzoate formation could have been due to a high turnover of benzoate or to a difference in the phenol degradation pathway between the thermophilic and mesophilic cultures.     

Fluorophenols and 3-fluorobenzoate in phenol-degrading methanogenic cultures

3-Fluorobenzoate and all three isomers of fluorophenol were used as analogues and inhibitors of phenol degradation in a methanogenic consortium. 3-Fluorobenzoate was not transformed by phenol-degrading cultures, but it facilitated the detection of the formation of 4-hydroxybenzoate and benzoate from phenol. The effects of the fluorophenols depended on their concentration in the cultures. When added at 0.90 mM, all fluorophenols prevented phenol transformation. At concentrations of 0.45 to 1.8 mM, 2-fluorophenol was transformed to 3-fluoro-4-hydroxybenzoate which accumulated in the medium. When both 2-fluorophenol and phenol were added to cultures at concentrations of 1 mM each, 3-fluoro-4-hydroxybenzoate, 4-hydroxybenzoate, 3-fluorobenzoate and benzoate were detected. 4-Fluorophenol was never transformed, and when it was present at 0.22 mM, it had no effect on phenol degradation. At concentrations 0.09 mM, 2-fluorophenol was mineralized by the phenol-degrading cultures to methane, carbon dioxide, and fluoride. The release of fluoride was also observed from 3-fluorophenol when it was initially present at 0.09 mM. These results support the proposed pathway for phenol degradation involving an initial para-carboxylation to 4-hydroxybenzoate followed by dehydroxylation to benzoate and further metabolism to carbon dioxide and methane. They also demonstrate defluorination of 2- and 3-fluorophenols under methanogenic conditions.     

Effect of fluorinated analogues of phenol and hydroxybenzoates on the anaerobic transformation of phenol to benzoate

The effects of fluorinated analogues on the anaerobic transformation of phenol to benzoate were examined. At 250 M 2- or 3-fluorophenol, phenol transformation was delayed. 2-Fluorophenol had no apparent effect on subsequent degradation of benzoate, but benzoate accumulated in the presence of 250 M 3-fluorophenol. In contrast, 4-fluorophenol at 2 mM had no effect on either phenol transformation or benzoate degradation. Phenol and 2-, or 3-fluorophenol were transformed simultaneously, but phenol was transformed more rapidly than either fluorophenol. Thus, fluorinated analogues of phenol did not prevent anaerobic transformation of phenol to benzoate. 2-Fluorophenol was converted to 3-fluorobenzoate, and phenol enhanced the rate and extent of its transformation. 3-Fluorophenol was transformed to 2-fluorobenzoate to a limited extent (3%) when phenol was present. 4-Fluorophenol was not transformed regardless of the presence of phenol. 3-Fluoro-4-hydroxybenzoate, a potential fluorinated intermediate product of para-carboxylation, was transformed rapidly to 2-fluorophenol and 3-fluorobenzoate, irrespective of the presence of phenol, indicating that both dehydroxylation and decarboxylation occurred. Initially, 2-fluorophenol and 3-fluorobenzoate were rapidly formed in an approximate molar ratio of 2 : 1. Once 3-fluoro-4-hydroxybenzoate was completely removed, the 2-fluorophenol, initially formed, was converted to 3-fluorobenzoate at a slower rate. Thus, phenol enhanced transformation of the fluorinated analogues, and the products of transformation suggested para-carboxylation. 3-Fluoro-2-hydroxybenzoate was not transformed in either the presence or absence of phenol, indicating that ortho-carboxylation did not occur.Abbreviations 3F4HB 3-fluoro-4-hydroxybenzoate - 3F2HB 3-fluoro-2-hydroxybenzoate (3-fluorosalicylate) Contribution No. 692, Environmental Research Laboratory, U.S. EPA, Gulf Breeze, FL. 32561, USA     

Absorbance signals from resting frog skeletal muscle fibers injected with the pH indicator dye, phenol red

Singly dissected twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with the pH indicator dye, phenol red. Dye-related absorbances in myoplasm, denoted by A0(lambda) and A90(lambda), were estimated as a function of wavelength lambda (450 nm less than or equal to lambda less than or equal to 640 nm) with light polarized parallel (0 degrees) and perpendicular (90 degrees) to the fiber axis respectively. At all lambda, A0(lambda) was slightly greater than A90(lambda), indicating that some of the phenol red molecules were bound to oriented structures accessible to myoplasm. The phenol red "isotropic" signal, [A0(lambda) + 2A90(lambda)]/3, a quantity equal to the average absorbance of all the dye molecules independent of their orientation, had a spectral shape that was red-shifted by approximately 10 nm in comparison with in vitro dye calibration curves measured in 140 mM KCl. The red-shifted spectrum also indicates that some phenol red molecules were bound in myoplasm. A quantitative estimate of indicator binding was obtained from measurements of the dye's apparent diffusion constant in myoplasm, denoted by Dapp. The small value of Dapp, 0.37 x 10(-6) cm2 s-1 (at 16 degrees C), can be explained if approximately 80% of the dye was bound to myoplasmic sites of low mobility. To estimate the apparent myoplasmic pH, denoted by pHapp, the isotropic absorbance of phenol red was fitted by in vitro calibration spectra. pHapp was found to be independent of dye concentration (0.2-2 mM), but varied widely (range, 6.8-7.5; mean value, 7.17) among fibers judged from functional characteristics to be normal. When fibers were subjected to acid or alkaline loads by exposure to Ringer's solution containing, respectively, dissolved CO2 or NH3, the changes in pHapp were in agreement with those expected from pH micro-electrode studies. It is concluded that in spite of the several indications for the presence of bound phenol red inside muscle cells, the pHapp signal from the indicator is useful for monitoring changes in myoplasmic pH in response to physiological and pharmacological manipulations.     

Inhibition of ethinyloestradiol and tolbutamide metabolism by quinoline derivatives in vitro

The effects of the quinoline derivatives amodiaquine (AQ), chloroquine (CQ), mefloquine (MQ), primaquine (PQ), quinine (Q) and quinidine (QD) on in vitro hepatic metabolism has been studied using as substrates ethinyloestradiol (EE2) and tolbutamide (TOL). The 2-hydroxylation of EE2 and the hydroxylation of TOL were determined in the presence of variable concentrations of each compound. MQ, PQ, AQ and Q significantly inhibited EE2 metabolism at each of the concentrations studied (0.1, 0.2 and 0.5 mM) as shown by an increase in the percentage of unmetabolised EE2. QD significantly inhibited metabolism at 0.2 and 0.5 mM but CQ was without effect. In terms of recovery of 2-OHEE2, PQ was the most potent inhibitor. At an inhibitor concentration of 0.5 mM the order of potency was PQ greater than or equal to MQ greater than or equal to Q greater than or equal to QD greater than or equal to AQ greater than or equal to CQ. TOL hydroxylase activity in control microsomes was 1.52 +/- 0.33 nmol. min-1 X mg protein-1. The order of potency of the inhibitors (0.5 mM) was PQ greater than or equal to MQ greater than or equal to Q greater than or equal to QD greater than or equal to AQ greater than or equal to CQ. These data provide further evidence of the inhibitory potential of some of the quinoline derivatives. PQ, MQ, and to a lesser extent Q produce the most marked inhibitory effects. QD and AQ are of intermediate potency and CQ is essentially non-inhibitory.     

Separation of a phenol carboxylating organism from a two-member, strict anaerobic co-culture

In a culture converting phenol to benzoic acid under anaerobic conditions and previously described as being constituted of only a Clostridium-like strain 6, another bacterium (strain 7) was observed. Each organism was enriched by centrifugation on a Percoll gradient. Strain 6 was purified by dilution and plating. Strain 7 did not grow on solid media, but a strain 7 culture, cleared of strain 6, was obtained by subculturing in the presence of ampicillin and by dilution. In fresh medium, phenol was transformed by the reconstituted co-culture but not by each strain alone. In a supernatant from a co-culture or from a strain 6 culture, strain 7 alone transformed phenol but not strain 6. Maintenance of an active strain 7 in fresh medium instead of co-culture supernatant became possible when phenol was replaced by 4-hydroxybenzoate (4-OHB), which is decarboxylated to phenol before being transformed to benzoate. Even with 4-OHB, the use of co-culture (or strain 6 culture) supernatant resulted in faster transformation activity and growth rate. A phylogenetic analysis placed strain 7 in a cluster of uncultivated or nonisolated bacteria (92-96% homology). Strain 7 is also related to Desulfotomaculum, Desulfitobacterium, Desulfosporosinus, Moorella, and Sporotomaculum genera (87-92% homology).     

Brain distribution of 6-mercaptopurine is regulated by the efflux transport system in the blood-brain barrier

6-mercaptopurine (6-MP) has been used clinically for 40 years to maintain remission in patients with acute lymphoblastic leukemia (ALL). However, central nervous system (CNS) relapses frequently occur in patients with ALL who continuously receive anticancer drugs, including 6-MP, during remission maintenance therapy. The cause of such CNS relapse is not well understood. One possible reason may involve the restricted distribution of 6-MP in the brain. This study, therefore, investigates the blood-brain barrier (BBB) transport which largely regulates 6-MP distribution in the brain using a quantitative microdialysis technique and centers on the efflux transport of 6-MP across the BBB. The brain tissue, cerebrospinal fluid (CSF), or hippocampal interstitial fluid (ISF) concentration of 6-MP was very low compared with the unbound plasma concentration, suggesting that 6-MP distribution in the brain is highly restricted. Kinetic analyses of this BBB transport showed that the efflux clearance from brain ISF to plasma across the BBB (CLout) is approximately 20-times greater than the influx clearance from plasma to brain (CLin). The CLout was significantly reduced by 1mM N-ethylmaleimide (NEM), a sulfhydryl-modifying agent, suggesting the participation of transport protein in the efflux of 6-MP across the BBB. In addition, efflux transport was inhibited by an intracerebral infusion of probenecid (1.5 mM), p-aminohippuric acid (PAH, 3.0 mM), benzoate (3.6 mM), or salicylate (3.7 mM) administered through a microdialysis probe, but neither choline (0.8 mM) nor tetraethylammonium (TEA, 0.7 mM) had any effect. These data suggest that the restricted 6-MP brain distribution may be ascribed to efficient efflux from the brain, possibly via both the organic anion transport system, shared with probenecid and PAH, and the monocarboxylic acid transport system, shared with benzoate and salicylate.     

The effects of cyclopropane carboxylate on hepatic pyruvate metabolism

The effects of cyclopropane carboxylate on gluconeogenesis and pyruvate decarboxylation from [1-14C]-labeled pyruvate and lactate were investigated in perfused livers from fasted rats. With high concentrations of pyruvate (greater than or equal to 0.5 mM) in the perfusion medium, infusion of cyclopropane carboxylate inhibited pyruvate decarboxylation and gluconeogenesis by 30 and 40%, respectively. With low, more physiological concentrations of pyruvate (50 microM) or with lactate (1 mM), cyclopropane carboxylate, at a concentration which elicits maximal inhibition of pyruvate decarboxylation from pyruvate (greater than or equal to 0.5 mM), did not affect either pyruvate decarboxylation or gluconeogenesis. Evidence is presented for the rapid formation of the coenzyme-A ester of cyclopropane carboxylate in perfused livers. Infusion of l-(-)carnitine (20 mM) prevented the inhibitory effects of cyclopropane carboxylate on pyruvate decarboxylation and gluconeogenesis from pyruvate (greater than or equal to 0.5 mM). Interestingly, no decrease in the tissue level of cyclopropanecarboxyl-CoA occurs under these conditions. The present study suggests that cyclopropane carboxylate, through a presently ill-defined mediator, inhibits pyruvate decarboxylation and gluconeogenesis by interfering with the pyruvate----oxalacetate----phosphoenolpyruvate----pyruvate cycle when pyruvate (greater than or equal to 0.5mM) supports gluconeogenesis.     

Phenylacetylene reversibly inhibits the phenol hydroxylase of Pseudomonas sp. CF600 at high concentrations but is oxidized at lower concentrations

Alkynes are mechanism-based inhibitors of several bacterial monooxygenases, including the soluble methane monooxygenase (sMMO) of Methylococcus capsulatus and the toluene o-monooxygenase (TOM) of Burkholderia cepacia G4. In this paper, we investigated the inhibition of the phenol hydroxylase of Pseudomonas sp. CF600 by the alkyne phenylacetylene. Growth of CF600 on phenol and phenol hydroxylase activity were inhibited by phenylacetylene concentrations greater than 1.0 mM. Unlike other alkynes, which irreversibly inhibit a number of monooxygenases, inhibition of phenol hydroxylase by phenylacetylene was reversible, as demonstrated by the ability of washed cells to regain phenol hydroxylase activity. Additionally, phenylacetylene was metabolized by phenol-grown cells, yielding a yellow meta-ring fission product which absorbed light maximally at 412 nm. Phenol-grown CF600 transformed phenylacetylene to hydroxyphenylacetylene and 2-hydroxy-6-oxo-octa-2,4-dien-7-ynoic acid as detected by gas chromatography–mass spectroscopy and high-performance liquid chromatography (HPLC), respectively, while neither a derivative of CF600 with a non-functional phenol hydroxylase nor wild-type CF600 grown on acetate transformed phenylacetylene. These results demonstrate that the phenol hydroxylase of CF600 has broader substrate specificity than previously reported. They also suggest that phenylacetylene acts as a competitive inhibitor rather than as a mechanism-based inhibitor of this phenol hydroxylase.     

Detection of intermediate metabolites of benzene biodegradation under microaerophilic conditions

The intermediate metabolites of benzene transformation by a microaerophilic bacterial consortium, adapted to degrade gasoline and benzene at low concentrations of dissolved oxygen (<1 mg l^-1), were identified. The examined range of initial DO concentration, 0.05 to 1 mg l^-1, was considerably lower than the previously reported values believed to be necessary to initiate benzene biodegradation. An extensive transformation of benzene, higher than the theoretical predictions for its aerobic oxidation, was observed. Phenol was identified as the most stable and the major intermediate metabolite which was subsequently transformed into catechol and benzoate. The use of ¹³C-labeled compounds identified benzene as the source of phenol, and phenol as the source of catechol and benzoate, suggesting the involvement of a monooxygenase enzymatic system in biodegradation of benzene at low DO concentrations. A metabolic sequence was proposed to describe the simultaneous detection of catechol and benzoate during the microaerophilic transformation of benzene. The results of this work demonstrate that it is possible to transform benzene, a highly carcinogenic hydrocarbon and a major contaminant of groundwater, to more easily biodegradable compounds in the presence of very small amounts of oxygen.     

Anaerobic transformation of phenol to benzoate via para-carboxylation: use of fluorinated analogues to elucidate the mechanism of transformation

Isomeric fluorophenols were used as phenol analogues to investigate the transformation of phenol to benzoate by an anaerobic, phenol-degrading consortium derived from freshwater sediment. Transformation of 2-fluorophenol and 3-fluorophenol led to the accumulation of fluorobenzoic acids. 2-Fluorophenol was transformed in the presence or absence of phenol, while 3-fluorophenol transformation was only observed in the presence of phenol. Identification of the resulting fluorobenzoate products as 3-fluorobenzoate and 2-fluorobenzoate isomers, respectively, together with the nontransformation of 4-fluorophenol indicated that the carboxyl group was introduced para to the phenolic hydroxyl group.     

Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6-bisphosphate in yeast

Phosphofructokinase 2 from Saccharomyces cerevisiae was purified 8500-fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP-agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [gamma-32P]ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase from beef heart; there was a parallel incorporation of 32P into a 105-kDa peptide and also, but only faintly, into a 162-kDa subunit. A low-Km (0.1 microM) fructose-2,6-bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6-[2-32P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300-fold, had an apparent molecular mass of 110 kDa and was made of two 56-kDa subunits. It was inhibited by fructose 6-phosphate (Ki = 5 microM) and stimulated 2-3-fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low-Km fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-Km (approximately equal to 100 microM), apparently specific, fructose 2,6-bisphosphatase was separated by anion-exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6-bisphosphate, which it converts to fructose 6-phosphate and Pi, because it is not inhibited by fructose 6-phosphate, glucose 6-phosphate or Pi. Several other phosphatases able to hydrolyze fructose 2,6-bisphosphate into a mixture of fructose 2-phosphate, fructose 6-phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6-bisphosphate (Km greater than 50 microM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.     

Effect of the ionic strength of solutions on the size of ganglioside mycelia

Gangliosides in aqueous media of low ionic strength (2-5 mM NaCl) and in concentrations over the critical ones (10(-5) M) form micellas which do not differ from liposomes as regards the chromatographic behavior on Sepharose 4R, with a molecular weight of greater than or equal to 10(7) dalton. In aqueous media of a higher ionic strength (greater than or equal to 20 mM NaCl), gangliosides form micellas which are eluted during chromatography in far later fractions than liposomes 70-80 nm in diameter, with a molecular weight of (1-5) X 10(5) dalton. It is assumed that the conclusions about ganglioside incorporation into the liposomal membrane made on the basis of their peaks coincidence are correct, provided that ganglioside-containing liposomes are obtained and chromatographed under high ionic strength (greater than or equal to 20 mM NaCl).     

Effect of Added Heavy Metal Ions on Biotransformation and Biodegradation of 2-Chlorophenol and 3-Chlorobenzoate in Anaerobic Bacterial Consortia

The effect of added Cd(II), Cu(II), Cr(VI), or Hg(II) at 0.01 to 100 ppm on metabolism in anaerobic bacterial consortia which degrade 2-chlorophenol (2CP), 3-chlorobenzoate (3CB), phenol, and benzoate was examined. Three effects were observed, including extended acclimation periods (0.1 to 2.0 ppm), reduced dechlorination or biodegradation rates (0.1 to 2.0 ppm), and failure to dechlorinate or biodegrade the target compound (0.5 to 5.0 ppm). 3CB biodegradation was most sensitive to Cd(II) and Cr(VI). Biodegradation of benzoate and phenol was most sensitive to Cu(II) and Hg(II), respectively. Adding Cr(VI) at 0.01 ppm increased biodegradation rates of phenol (177%) and benzoate (169%), while Cd(II) and Cu(II) at 0.01 ppm enhanced biodegradation rates of benzoate (185%) and 2CP (168%), respectively. Interestingly, with Hg(II) at 1.0 to 2.0 ppm, 2CP and 3CB were biodegraded 133 to 154% faster than controls after an extended acclimation period, suggesting adaptation to Hg(II). Metal ions were added at inhibitory, but sublethal, concentrations to investigate effects on metabolic intermediates and end products. Phenol accumulated to concentrations higher than those in controls only in the 2CP consortium with added Cu(II) at 1.2 ppm but was subsequently degraded. There was no effect on benzoate, and little effect on acetate intermediates was observed. In most cases, methane yields were reduced by 23 to 97%. Thus, dehalogenation, aromatic degradation, and methanogenesis in these anaerobic consortia showed differential sensitivities to the heavy metal ions added. These data indicate that the presence of heavy metals can affect the outcome of anaerobic bioremediation of aromatic pollutants. In addition, a potential exists to use combinations of anaerobic bacterial species to bioremediate sites contaminated with both heavy metals and aromatic pollutants.     

The tris-catalyzed isomerization of potassium D-glucose 6-O-sulfate.

Potassium D-glucose 6-O-[35S]sulfate was partially converted by Tris (greater than or equal 25 mM, pH 7.5) to 35SO4(2-), fructose, and a 35S-labelled compound which was tentatively identified as fructose 6-O-sulfate. The isomerization reaction was also catalyzed by ethanolamine and 2-dimethylaminoethanol but not by glycine, triethanolamine, or pentaerythritol. The significance of these findings in relation to kinetic studies of the enzyme glycosulfatase is discussed.     

Carboxylation of o-cresol by an anaerobic consortium under methanogenic conditions.

The metabolism of o-cresol under methanogenic conditions by an anaerobic consortium known to carboxylate phenol to benzoate was investigated. After incubation with the consortium at 29 degrees C for 59 days, o-cresol was transformed to 3-methylbenzoic acid, which was not further metabolized by the consortium. Proteose peptone in the culture medium was essential for the transformation of o-cresol. In addition, a transient compound detected in the culture was identified as 4-hydroxy-3-methylbenzoic acid. o-Cresol-6d was transformed by the consortium to deuterated hydroxy-methylbenzoic acid and deuterated methylbenzoic acid. These results demonstrate that o-cresol is carboxylated in the para position relative to the phenolic hydroxyl group and dehydroxylated by the anaerobic consortium.