Dehalogenation of dichloroethene in a contaminated soil: fatty acids and alcohols as electron donors and an apparent requirement for tetrachloroethene.

Environmental soil contamination at an industrial site in Marion, Ohio (USA) with tetrachloroethene (perchloroethene, PCE) resulted in residual cis-1, 2-dichloroethene (DCE) contamination that had not declined after more than 15 years. Microcosm slurries containing 2.6% soil from this site were supplemented with different electron donors, i.e., individual fatty acids or alcohols. None of the microcosms supported complete DCE dechlorination, unless PCE was added to the microcosm at initiation. The addition of fresh PCE resulted in the dehalogenation of PCE to DCE in the microcosms supplemented with fatty acids having an even number of carbon atoms (acetate, butyrate, and caproate), but not in those with an odd number of carbon atoms (formate, propionate, and valerate), where negligible or no activity was detected. No significant further DCE degradation was observed in any of the microcosms supplied with fatty acids as electron donors. Microcosms supplemented with freshly added PCE bioconverted PCE to DCE and completely dehalogenated both the ex-novo and soil-supplied DCE within 60 days, but only if alcohols having an even number of carbon atoms (ethanol or butanol) were also added as electron donors. Odd-numbered alcohols either did not produce dehalogenation (as with methanol) or only dehalogenated PCE to DCE (as with propanol).

Effect of vitamins on the aerobic degradation of 2-chlorophenol, 4-chlorophenol, and 4-chlorobiphenyl.

The effect of vitamins on the aerobic degradation and dechlorination of 2-chlorophenol and 4-chlorophenol by Pseudomonas pickettii, strain LD1, and 4-chlorobiphenyl by Pseudomonas sp. strain CPE1 was determined. These microorganisms are capable of using the target compounds as the sole carbon and energy source, but do not need vitamins to metabolize them. The addition to the culture medium of a vitamin solution containing biotin, folic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, niacin, pantothenic acid, cyanocobalamin, p-aminobenzoic acid, and thioctic acid (total final concentration: < or = 600 ppb) resulted in a 7%-16% increase in the amount of target compounds degraded over the incubation period required for the concentration of the compound in the cultures to drop to approximately zero. A corresponding increase in the amount of chloride ion produced was also detected during the same period, indicating active (and often stoichiometric) dechlorination of the target compounds.

Aerobic degradation and dechlorination of 2-chlorophenol, 3-chlorophenol and 4-chlorophenol by a Pseudomonas pickettii strain.

A Gram-negative aerobic bacterium capable of using 2-chlorophenol (2-CP), 3-chlorophenol (3-CP) and 4-chlorophenol (4-CP) as sole carbon sources was isolated and characterized. The bacterium, designated LD1, was identified to be a Pseudomonas pickettii strain. LD1 was able to totally degrade and dechlorinate 2-CP (initial concentration: 1.51 mmol l-1), 3-CP (initial concentration: 0.57 mmol l-1) and 4-CP (initial concentration: 0.75 mmol l-1) within 30, 30 and 40 h of incubation, respectively, under growing-cell batch conditions. LD1 was also found to be able to metabolize chlorocatechols in growing- and resting-cell conditions. This suggests that the bacterium degrades monochlorophenols through a chlorocatechol pathway. In addition, LD1 was found to be capable of readily metabolizing other organic compounds such as phenol, benzoic acid, hydroxybenzoic acids and hydroquinone. Because of the broad spectrum of monochlorophenols and organic compounds that LD1 can degrade, this bacterium appears to have the potential for being successfully used in the biotreatment of wastewaters and in soil decontamination.

Effect of yeast extract on growth kinetics during aerobic biodegradation of chlorobenzoic acids.

The Monod or Andrews kinetic parameters describing the growth of Pseudomonas sp. CPE2 strain on 2,5-dich!orobenzoic acid and 2-chlorobenzoic acid, and Al-caligenes sp. CPE3 strain on 3,4-dichlorobenzoic acid, 4-chlorobenzoic acid, and 3-chlorobenzoic acid were determined from batch and continuous growth experiments conducted in the presence or absence of yeast extract (50 mg/L). Strain CPE2 displayed inhibitory growth kinetics in the absence of yeast extract and a noninhibitory kinetics in the presence of yeast extract. Similar results were obtained for CPE3. The presence of yeast extract also resulted in a significant increase in the affinity of the strains for the chlorobenzoic acids they degraded. (c) 1995 John Wiley & Sons, Inc.

Influence of organic and inorganic growth supplements on the aerobic biodegradation of chlorobenzoic acids.

The effect of yeast extract and its less complex substituents on the rate of aerobic dechlorination of 2-chlorobenzoic acid (2-ClBZOH) and 2,5-dichlorobenzoic acid (2,5-Cl2BZOH) by Pseudomonas sp. CPE2 strain, and of 3-chlorobenzoic acid (3-ClBZOH), 4-chlorobenzoic acid (4-ClBZOH) and 3,4-dichlorobenzoic acid (3,4-Cl2BZOH) by Alcaligenes sp. CPE3 strain were investigated. Yeast extract at 50 mg/l increased the average dechlorination rate of 200 mg/l of 4-ClBZOH, 2,5-Cl2BZOH, 3,4-Cl2BZOH, 3-ClBZOH and 2-ClBZOH by about 75%, 70%, 55%, 7%, and 1%, respectively. However, in the presence of yeast extract the specific dechlorination activity of CPE2 and CPE3 cells (per unit biomass) was always lower than without yeast extract, although it increased significantly during the exponential growth phase. When a mixed vitamin solution or a mixed trace element solution was used instead of yeast extract the rate of 4-ClBZOH dechlorination increased by 30%-35%, whereas the rate of 2,5-Cl2BZOH and 3,4-Cl2BZOH dechlorination increased by only 2%-10%. The presence of vitamins or trace elements also resulted in a specific dechlorination activity that was generally higher than that observed for the same cells grown solely on chlorobenzoic acid. The results of this work indicate that yeast extract, a complex mixture of readily oxidizable carbon sources, vitamins, and trace elements, enhances the growth and the dechlorination activity of CPE2 and CPE3 cells, thus resulting in an overall increase in the rate of chlorobenzoic acid utilization and dechlorination.

Cancer therapy with chemically modified enzymes. I. Antitumor properties of polyethylene glycol-asparaginase conjugates.

The covalent attachment of monomethoxypolyethylene glycol (PEG) to asparaginases from Escherichia coli and Vibrio succinogenes by new coupling methodology produced conjugates that are active, stable, without significant immune response, and with greatly extended plasma half-lives in mice. Therapeutic efficacies were greater for the PEG-asparaginases than for the unmodified asparaginases in mice infected with the L5178Y lymphosarcoma or the 6C3HED tumor. Large single doses of native or modified enzymes were more effective against tumors than the same amount of enzyme given in smaller doses over several days.

Eucaryote thermophily: role of lipids in the growth of Talaromyces thermophilus.

The effects of growth temperature on the fatty acid composition of the phospholipids of the fungus Talaromyces thermophilus were investigated. This thermophilic organism was unable to increase the degree of unsaturation of its fatty acids when shifted from high to low growth temperatures. Inhibition of fatty acid synthesis by the antibiotic cerulenin was reversed by the addition of a mixture of palmitic, stearic, and oleic acids and ergosterol. The data obtained were consistent with the hypothesis that the thermophilic character of T. thermophilus is due to metabolic limitations that restrict its ability to regulate membrane fluidity.

Enzyme-induced asparagine and glutamine depletion and immune system function.

Depletion of nonessential amino acids and its effect on the immune system can be studied by the administration of bacterial enzymes. Escherichia coli asparaginase hydrolyzes both asparagine and glutamine: administration of this enzyme to mice is rapidly immunosuppressive. Vibrio succinogenes asparaginase hydrolyzes only asparagine and has no apparent effect on immune system function. When the enzymes are rendered nonantigenic and nonimmunogenic by covalent attachment of polyethylene glycol, the effects on immune system function remain the same as described above with the native (nonmodified) enzymes. We believe the data reviewed justify the conclusion that glutamine deficiency is specifically immunosuppressive whereas asparagine deficiency is not. We further believe that enzymatic depletion of nonessential amino acids can be a useful tool for nutritional investigations.

Inhibition of blastogenesis by native and polyethylene glycol-modified asparaginases from Escherichia coli and Vibrio succinogenes.

The in vitro blastogenic response of rat splenocytes to concanavalin A stimulation is inhibited by inclusion of asparaginase in the culture medium. The glutaminase-free asparaginase from Vibrio succinogenes is as potent an inhibitor as the Escherichia coli enzyme which has 2% glutaminase activity. The polyethylene glycol-modified forms of both enzymes are also inhibitory. We suggest that previously proposed explanations for the ability of asparaginases to inhibit blastogenesis are not likely to be correct and propose that asparaginase interacts with a mitogenic factor.

Immunological effects of native and polyethylene glycol-modified asparaginases from Vibrio succinogenes and Escherichia coli in normal and tumour-bearing mice.

The immunosuppressive effects of polyethylene glycol-modified asparaginases from Vibrio succinogenes (PEG-asparaginase VS) and Escherichia coli (PEG-asparaginase EC) have been investigated in mice. Measurements of the mitogen-induced blastogenic responses of splenocytes, harvested 5 days after in vivo administration of the PEG-enzymes, show that PEG-asparaginase VS is not immunosuppressive, whereas PEG-asparaginase EC does cause immunosuppression. Both enzymes cause the spleen to be smaller than the control mice. In mice carrying the L5178Y tumour and its associated LDH-elevating virus, which causes the circulation life of asparaginase VS to be comparable to that of PEG-asparaginase VS, tumour regression and its attendant immunological changes are identical in animals treated with either the native or the modified enzyme. The data presented in this paper, along with independent immunological evidence presented by other workers strongly suggest that PEG-asparaginase VS may be the enzyme of choice for clinical use.

Asparaginase production by human clinical isolates of Vibrio succinogenes.

Three human isolates of Vibrio succinogenes produced asparaginase. Apparent Km's were 87,220, and 320 microM. The rate of glutamine hydrolysis was between 2.8 and 3.5% of the rate of asparagine hydrolysis. Asparaginase production was not induced by ammonium ions, and enzyme yields were lower than those obtained with the rumen strain.

A rapid purification procedure for L-asparaginase from Vibrio succinogenes.

A simple procedure has been developed for the purification of L-asparaginase from Vibrio succinogenes. Only two steps of ion-exchange chromatography are required. A higher yield and higher specific activity are obtained than previously reported.

Purification and characterization of L-asparaginase with anti-lymphoma activity from Vibrio succinogenes.

Homogeneols L-asparaginase with anti-lymphoma activity was prepared from Vibrio succinogenes, an anaerobic bacterium from the bovine rumen. An overall yield of pure L-asparaginase of 40 to 45% and a specific activity of 200 +/- 2 IU per mg of protein was obtained. The pure enzyme can be stored at -20 degrees for at least 3 months with no loss of activity. The isoelectric point of the L-asparaginase is 8.74. No carbohydrate, phosphorus, tryptophan, disulfide, or sulfhydryl groups were detected. The enzyme has a molecular weight of 146,000 and a subunit weight of approximately 37,000. The Km of the enzyme for L-asparagine is 4.78 X 10(-5) M and the pH optimum of the L-asparaginase reaction is 7.3. D-Asparagine was hydrolyzed at 6.5% of the rate found with the L isomer. L-Glutamine and a variety of other amides were not hydrolyzed at significant rates; the activity of the enzyme for L-glutamine was 130- to 600-fold less than that of other therapeutically effective L-asparaginases of bacterial origin. The L-asparaginase from V. succinogenes is immunologically distinct from the L-asparaginase (EC-2) of Escherichia coli.

Improved growth media for Vibrio succinogenes.

Five new culture media for Vibrio succinogenes are described. Of these, a medium composed of 0.4% yeast extract, 100 mM ammonium formate, 120 mM sodium fumarate, and 0.05% sodium thioglycolate, pH 7.3, supports the best growth.

L-Asparaginase production by the rumen anaerobe Vibrio succinogenes.

The rumen anaerobe Vibrio succinogenes possesses a constitutive L-asparaginase. The amount of enzyme produced is affected by the compound supplied to the organism to generate the fumaric acid it requires as a terminal electron acceptor. When nitrate is provided as the terminal electron acceptor, the amount of enzyme produced is affected by the compound provided to satisfy the nutritional requirement of the organism for succinic acid. Specific activities of up to 8.4 IU/mg of protein in cell-free extracts have been obtained. This specific activity is higher than has been previously reported for any organism. The enzyme has an apparent K(m) of 1.7 x 10(-5) M and low activity towards L-glutamine when assayed at pH 8.5.

Glucose fermentation products in Ruminococcus albus grown in continuous culture with Vibrio succinogenes: changes caused by interspecies transfer of H 2 .

The influence of a H(2)-utilizing organism, Vibrio succinogenes, on the fermentation of limiting amounts of glucose by a carbohydrate-fermenting, H(2)-producing organism, Ruminococcus albus, was studied in continuous cultures. Growth of V. succinogenes depended on the production of H(2) from glucose by R. albus. V. succinogenes used the H(2) produced by R. albus to obtain energy for growth by reducing fumarate in the medium. Fumarate was not metabolized by R. albus alone. The only products detected in continuous cultures of R. albus alone were acetate, ethanol, and H(2). CO(2) was not measured. The only products detected in the mixed cultures were acetate and succinate. No free H(2) was produced. No formate or any other volatile fatty acid, no succinate or other dicarboxylic acids, lactate, alcohols other than ethanol, pyruvate, or other keto-acids, acetoin, or diacetyl were detected in cultures of R. albus alone or in mixed cultures. The moles of product per 100 mol of glucose fermented were approximately 69 for ethanol, 74 for acetate, 237 for H(2) for R. albus alone and 147 for acetate and 384 for succinate for the mixed culture. Each mole of succinate is equivalent to the production of 1 mol of H(2) by R. albus. Thus, in the mixed cultures, ethanol production by R. albus is eliminated with a corresponding increase in acetate and H(2) formation. The mixed-culture pattern is consistent with the hypothesis that nicotinamide adenine dinucleotide (reduced form), formed during glycolysis by R. albus, is reoxidized during ethanol formation when R. albus is grown alone and is reoxidized by conversion to nicotinamide adenine dinucleotide and H(2) when R. albus is grown with V. succinogenes. The ecological significance of this interspecies transfer of H(2) gas and the theoretical basis for its causing changes in fermentation patterns of R. albus are discussed.

An anaerobic chemostat that permits the collection and measurement of fermentation gases.

A chemostat was designed to allow anaerobic growth in the culture vessel in the absence of a continuous stream of O(2)-free gas. Produced gases were collected within the culture and collection vessels, and pressure build-up was prevented by allowing gases to expand into a collapsed football bladder. The culture overflow was collected in a flask, held at 0 C, that was emptied by applying a positive CO(2) pressure to the system. Ruminococcus albus, a H(2) and CO(2)-producing anaerobe, was used to test the operation of the apparatus. H(2) production was measured by sampling the various gas spaces of known volume and measuring H(2) concentration by gas chromatography. Measurement of accumulated fermentation gases and the effects of the accumulation on fermentations can be studied with the apparatus.