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Applied Microbiology and Biotechnology (v.47, #3)
Biotechnological production of prostaglandins by M. Lamačka; J. Šajbidor (pp. 199-206).
The metabolism of 20-carbon polyunsaturated fatty acids, particularly arachidonic acid, by prostaglandin H synthase results in a wide range of oxidized products with potent biological activities. Among these metabolites, a group of compounds called prostaglandins has drawn the attention of both scientists and medical practitioners. Prostaglandins can be manufactured from polyunsaturated fatty acids with the help of enzymes from mammals. The yield of the desired prostaglandin can be increased by means of various activators, enzyme recycling, immobilised enzymes or semibatch processes with either continuous or stepwise addition of the substrate.
Saponified palm kernel oil and its major free fatty acids as carbon substrates for the production of polyhydroxyalkanoates in Pseudomonas putida PGA1 by I. K. P. Tan; K. Sudesh Kumar; M. Theanmalar; S. N. Gan; B. Gordon III. (pp. 207-211).
The synthesis of polyhydroxyalkanoates (PHA) by Pseudomonas putida PGA1, using saponified palm kernel oil (SPKO), was investigated. The PHA produced from SPKO was compared with those produced by the major free fatty acids found in the palm kernel oil. Owing to the absence of lipase activity in P.␣putida, palm kernel oil did not support cell growth. However, SPKO could support cell growth and produced relatively high yield of both dry cells and PHA. The polyester produced was similar in properties to those derived from lauric (C12:0) and myristic (C14:0) acids, while oleic acid (C18:1) gave rise to PHA that was sticky and of broader molecular mass distribution. Nuclear magnetic resonance and gas chromatography showed that these PHA were copolymers consisting mainly of n-alkanoate monomers ranging from C6 to C14, with C8 as the predominant component. PHA derived from SPKO and oleic acid also contained a small amount of unsaturated monomers.
Fermentability of hemicelluloses extracted from municipal waste and commercial xylans by Clostridium sp. by M. V. S. Murty; T. S. Chandra (pp. 212-217).
The fermentability of commercial xylans and municipal waste hemicelluloses in the presence of Clostridium sp. (C.SAIV; ATCC 700188) has been evaluated. Teak, deal wood, banana stalk and bagasse of the municipal waste contained significant amounts (approx. 12 %–23 %) of hemicellulose. Under optimized growth conditions, the growth rate of C.SAIV was improved as indicated by an increase in the concentration of ethanol in the culture broth. Commercial xylans were utilized fairly efficiently and ethanol formed from larch wood xylan and bagasse hemicellulose was at least 64 mM. The amount of ethanol formed from the bagasse hemicellulose was at least three times higher than any other reported value. The current study also indicated that the source and composition of hemicellulose played an important role in determining the fermentability of the substrate for some microorganisms.
Improvement of tempe fermentations by application of mixed cultures consisting of Rhizopus sp. and bacterial strains by I. Wiesel; H. J. Rehm; B. Bisping (pp. 218-225).
Tempe fermentations using mixed cultures of Rhizopus oligosporus MS5, R. oryzae EN, Citrobacter freundii, and Brevibacterium epidermidis were investigated. Consumption of 150 g tempe, produced with a pure fungal mixed culture out of strains MS5 and EN, is sufficient to cover the daily requirements of niacin, vitamin K, ergosterol, and tocopherol as well as half of the daily requirement of pyridoxine, riboflavin, and biotin. Moreover, one-fourth of the recommended amount of folate is supplied. Supplementation of the fungal inoculum with C. freundii results in tempe enriched with vitamin B12. Menachinone was produced as a typical bacterial vitamin K derivative. Metabolic activity of C.␣freundii led to an additional decrease of the α-galactosides stachyose and raffinose compared to pure fungal fermentations. No bacterial formation of factor 2 could be observed.
Application of agro-industrial by-products for riboflavin production by Eremothecium ashbyii NRRL 1363 by A. E. Kalingan; M. R. V. Krishnan (pp. 226-230).
Riboflavin production is significantly determined by the type and initial concentration of the carbon and nitrogen sources and also by other flavinogenic stimulants. Using an optimum carbon and nitrogen concentration, an industrial fermentation medium has been designed with molasses as the carbon source and peanut seed cake as the nitrogen source. In addition the stimulatory effect of some of the low-cost agro-industrial by-products on riboflavin yield was investigated.
A new arabinofuranohydrolase from Bifidobacterium adolescentis able to remove arabinosyl residues from double-substituted xylose units in arabinoxylan by K. M. J. Van Laere; G. Beldman; A. G. J. Voragen (pp. 231-235).
An arabinofuranohydrolase (AXH-d3) was purified from a cell-free extract of Bifidobacterium adolescentis DSM 20083. The enzyme had a molecular mass of approximately 100 kDa as determined by gel filtration. It displayed maximum activity at pH 6 and 30 °C. Using an arabinoxylan-derived oligosaccharide containing double-substituted xylopyranosyl residues established that the enzyme specifically released terminal arabinofuranosyl residues linked to C-3 of double-substituted xylopyranosyl residues. In addition, this arabinofuranohydrolase released arabinosyl groups from wheat flour arabinoxylan polymer but showed no activity towards p-nitrophenyl α-l-arabinofuranoside or towards sugar-beet arabinan, soy arabinogalactan, arabino-oligosaccharides and arabinogalacto-oligosaccharides.
Application of the polymerase chain reaction (PCR) and reverse transcriptase / PCR for determining the fate of phenol-degrading Pseudomonas putida ATCC 11172 in a bioaugmented sequencing batch reactor by S. Selvaratnam; B. A. Schoedel; B. L. McFarland; C. F. Kulpa (pp. 236-240).
The impact of bioaugmentation on the efficacy of an existing microbial population to detoxify phenol in a laboratory-scale sequencing batch reactor was evaluated. Phenol degradation and the persistence and expression of the catabolic dmpN gene were studied for 44 days using a combination of conventional monitoring approaches and molecular techniques. Following addition of the phenol-degrading bacterium, Pseudomonas putida ATCC 11172, which converts phenol to catechol by the aerobic meta-cleavage pathway, phenol removal␣in the bioaugmented reactor increased and was maintained at 95 %–100 %. In the unaugmented control reactor, decreased phenol removal was observed. Correspondingly, dmpN DNA, characteristic of P. putida ATCC 11172, and its expression were detected in activated sludge biomass from the bioaugmented reactor for over 41 days. The results of this study show that (i) bioaugmentation provides stability in phenol degradation, and (ii) monitoring with molecular techniques such as the polymerase chain reaction (PCR) and reverse transcriptase/PCR can successfully assess the state of a bacterium used in bioaugmentation.
Development of HIV-1 protease expression methods using the T7 phage promoter system by T. Komai; Y. Ishikawa; R. Yagi; H. Suzuki-Sunagawa; T. Nishigaki; H. Handa (pp. 241-245).
New and simple human immunodeficiency virus type 1 (HIV-1) protease expression methods in Escherichia coli were developed using the T7 phage promoter system. In order to suppress leaky HIV-1 protease expression under the control of the T7 polymerase, two new methods were tested. One involved the introduction of supplementary T7 promoter regions into host cells [E. coli BL-21(DE3)] containing the HIV-1 protease gene under the control of the T7 promoter. It was expected that the supplementary T7 promoter regions would compete with the HIV-1 protease expression vector for the T7 polymerase binding. The other involved the infection of late-log-phase cultures of E.␣coli JM109 harboring the same HIV-1 protease expression vector with the M13 phage expressing T7 polymerase. Both methods were effective, and transformants with the mature HIV-1 protease expression vector showed ten times higher HIV-1 protease activity than activities obtained with the autoprocessing vector. The expression systems described here are convenient and are also easily applicable for the expression of other proteins toxic for E. coli.
Northern analysis of aflatoxin biosynthesis genes in Aspergillus parasiticus and Aspergillus sojae by M. A. Klich; B. Montalbano; K. Ehrlich (pp. 246-249).
RNAs from three Aspergillusparasiticus and three Aspergillus sojae isolates were probed with seven genes involved in aflatoxin biosynthesis. Previously published work and preliminary work in this study demonstrated that these aflatoxin biosynthesis genes were present in the DNA of the isolates. RNA from aflatoxin-producing and O-methylsterigmatocystin-producing A. parasiticus strains SRRC 143 and SRRC 2043 hybridized to all of the gene probes tested. However, RNA from a strain of A. parasiticus that had lost its ability to produce aflatoxin in culture (SRRC 77) and RNA from one of the A. sojae isolates did not hybridize to any of the gene probes. Two of the A. sojae isolates hybridized to the regulatory gene aflR and the structual gene uvm8, which is believed to code for a fatty acid synthase involved in an early step in aflatoxin biosynthesis, but not to any of the other five genes of the aflatoxin pathway tested. These results suggest that most of the genes involved in aflatoxin production are transcriptionally blocked in A. parasiticus SRRC 77 and all of the A. sojae isolates. The cause of this blockage is unknown.
Extracellular pH affects regulation of the pcbAB gene in Penicillium chrysogenum by Y.-W. Chu; D. Renno; G. Saunders (pp. 250-254).
The effect of extracellular pH and dissolved oxygen on regulation of the pcbAB gene in P.␣chrysogenum was examined, using Northern analysis and a reporter gene fusion. It was found that ambient pH markedly affected levels of pcbAB mRNA whereas maintenance of dissolved oxygen concentration above 10 % had no detectable effect. The presence of a DNA-binding protein, which binds upstream of the pcbAB translational start codon, was also related to ambient pH. In all fermentations, pcbAB mRNA was most abundant at around the late exponential/early stationary phase of a culture.
Large-scale production and characterization of Bacillus thuringiensis subsp. tenebrionis insecticidal protein from Escherichia coli by M. E. Gustafson; R. A. Clayton; P. B. Lavrik; G. V. Johnson; R. M. Leimgruber; S. R. Sims; D. E. Bartnicki (pp. 255-261).
Bacillus thuringiensis subsp. tenebrionis insecticidal protein was produced in recombinant Escherichia coli and purified to near homogeneity to provide quantities of protein for safety-assessment studies associated with the registration of transgenic potato plants. The 68-kDa protein is produced naturally by Bacillus thuringiensis subsp. tenebrionis by translation initiation at an internal initiation site in the native DNA sequence. The gene sequence specific for this truncated protein was expressed in E. coli strain JM 101 and fermented at the 1000-l scale. The protein accumulated as insoluble inclusion bodies, and was purified by extraction at pH␣10.8 with carbonate buffer, selective precipitation at pH 9.0, and differential centrifugation. No chromatography steps were required to produce over 50 g purified protein as a lyophilized powder with a purity greater than 95 % and demonstrating full insecticidal activity against Colorado potato beetle larvae. The protein was further characterized to assure identity and suitability for use in safety-assessment studies.
Cloning of the Bacillus pumilusβ-xylosidase gene (xynB ) and its expression in Saccharomyces cerevisiae by D. C. La Grange; I. S. Pretorius; W. H. van Zyl (pp. 262-266).
A genomic DNA library of the bacterium Bacillus pumilus PLS was constructed and the β-xylosidase gene (xynB) was amplified from a 3-kb genomic DNA fragment with the aid of the polymerase chain reaction technique. The amplified xynB gene was inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2 P ) and terminator (ADH2 T ) sequences on a multicopy episomal plasmid (pDLG11). The xynB gene was also fused in-frame to the secretion signal sequence of the yeast mating pheromone α-factor (MFα1 S ) before insertion between the ADH2 P and ADH2 T sequences on a similar multicopy episomal plasmid (pDLG12). The resulting construct ADH2 P -MFα1 S -xynB-ADH2 T was designated XLO1. Both plasmids pDLG11 and pDLG12 were introduced into Saccharomyces cerevisiae but only the expression of the XLO1 gene yielded biologically functional β-xylosidase. The total β-xylosidase activity remained cell-associated with a maximum activity of 0.09 nkat/ml obtained when the recombinant S. cerevisiae strain was grown for 143 h in synthetic medium. The temperature and pH optima of the recombinant Xlo1 enzyme were 45–50 °C and pH 6.6 respectively. The enzyme was thermostable at 45 °C; however, at 60 °C most of the Xlo1 was inactive after 5 min.
Induction of xylanase in Aspergillus tamarii by methyl β-d-xyloside by R. C. Simão; C. G. M. Souza; R. M. Peralta (pp. 267-271).
Aspergillus tamarii produced extracellular xylanase and intracellular β-xylosidase inductively in washed glucose-grown mycelia incubated with xylan and methyl β-d-xyloside, a synthetic glycoside. Methyl β-d-xyloside was a more effective inducer than xylan at the same concentration for both enzymes. Glucose and cycloheximide were found to inhibit xylanase production by methyl β-d-xyloside. Methyl β-d-xyloside was hydrolyzed to xylose by mycelial extract in vitro.
13C NMR cross polarization and magic angle spinning (CPMAS) and gas chromatography/mass spectrometry analysis of the products from a soda pulp mill effluent decolourised by two Streptomyces strains by M. Hernández; J. Rodríguez; M. I. Pérez; A. S. Ball; M. E. Arias (pp. 272-278).
Two Streptomyces strains, UAH 30 and UAH 51, have been shown to decolourise a paper-mill effluent obtained after semichemical alkaline pulping of wheat straw. Fractionation of the effluent decolourised by strains UAH 30 and UAH 51 showed that 60% and 80% respectively of the alkali-lignin fraction have been removed from the effluent after 7 days of growth. 13C NMR cross polarization and magic angle spinning (CPMAS) spectra of the alkali-lignin remaining in the effluent after decolourisation revealed a decrease in the relative amount of aromatic lignin units compared to that obtained from the untreated effluent along with a reduction in the ratio of syringyl:guaiacyl units. Gas chromatography/mass spectrometry analysis of the low-molecular-mass compounds extracted from the decolourised effluent revealed the presence of new aromatic lignin-related compounds that were not present in the untreated control effluent. This was linked to a general depolymerization of larger lignin molecules occurring during decolourisation by the two Streptomyces strains. Identification of low-molecular-mass aromatic compounds extracted from the decolourised effluent revealed only the presence of p-hydroxyphenyl units in effluents decolourised by the strain UAH 30 while p-hydroxyphenyl, guaiacyl and syringyl units were detected in effluents decolourised by Streptomyces strain UAH 51. The study indicates that, while decolourisation is a common feature of the two Streptomyces strains, the mechanisms involved in the degradation of the lignin fractions may be different and strain-specific.
Aptitude of cheese bacteria for volatile S-methyl thioester synthesis I. Effect of substrates and pH on their formation by Brevibacterium linens GC171 by G. Lamberet; B. Auberger; J. L. Bergère (pp. 279-283).
The aptitude of resting cells of Brevibacterium linens G171 to synthesize S-methyl thioesters was studied in presence of methanethiol and nine short-chain fatty acids individually or as a mixture. Esterification of acetic, propionic and methyl branched-chain acids occurred with methanethiol alone and was enhanced by fatty acid addition. Addition of n-chain, 3-hydroxybutyric and 2-hydroxyvaleric acids allowed synthesis of n-chain thioesters up to thiocaproate. The kinetics of production and the effect of concentrations of both substrates and of cells were tested. The optimum pH for synthesis varied according to the kind of thioesters produced. Results suggested that thioesters were derived mainly from acyl-CoA from different metabolic breakdowns, such as the degradation of fatty acids or some amino acids, and that several acyltransferases could be involved.
Inhibition of mesophilic solid-substrate anaerobic digestion by ammonia nitrogen by H. M. Poggi-Varaldo; R. Rodríguez-Vázquez; G. Fernández-Villagómez; F. Esparza-García (pp. 284-291).
This work focused on determining the effects of ammonia-nitrogen supplementation on the mesophilic solid-substrate anaerobic digestion of municipal wastes and waste activated sludge (biosolids). Bench-scale, semi-continuous, mesophilic reactors were operated with a 21-day mass-retention time and dosed with NH4Cl, such that the corresponding chemical O2 demand (COD)/N ratios in their feeds were 90, 80, 65 and 50 (reactors R1 or control, R2, R3 and R4 respectively). Reactor performance was evaluated in terms of the efficiency of volatile solid removal (efficiency for short), biogas productivity, methane content in the biogas, pH and volatile organic acid contents, among other monitoring and analytical parameters. The feedstock was a mixture of urban wastes with biosolids. It was found that the process performance deteriorated at increasing dosages of ammonia N, the process practically ceasing at COD/N = 50 (R4). Inhibition was characterized by efficiency and biogas productivity decreases and a more sudden drop of methane content in biogas and pH. A significant rise of propionic, butyric and valeric acid was found in reactors receiving the highest doses of ammonia N (R3 and R4). This suggested that inhibition of the syntrophic bacteria present in the anaerobic consortia also occurred. Luong and Pearson inhibition models were fitted to the data. Both models represented very well the acute effects of N supplementation on solid-substrate anaerobic digestion. However, the Luong model could also represent the process ceasing at a critical ammonia N concentration of 2800 mg/kg mixed solids.
Metabolic pathways of quinoline, indole and their methylated analogs by Desulfobacterium indolicum (DSM 3383) by S. S. Johansen; D. Licht; E. Arvin; H. Mosbæk; A. B. Hansen (pp. 292-300).
The transformation of quinoline, isoquinoline and 3-, 4-, 6- and 8-methylquinoline by Desulfobacterium indolicum was compared with that of the N-containing analogues indole and 1-, 2-, 3- and 7-methylindole. The metabolites were identified using high-performance liquid chromatography with UV detection, thin-layer chromatography, combined gas chromatography/mass spectrometry and proton NMR spectroscopy. All degraded compounds were initially hydroxylated at position 2 by D. indolicum. A new degradation product of quinoline was observed in the second transformation step, where 3,4-dihydro-2-quinolinone accumulated. This ring-reduced compound was further transformed into unidentified products. The transformation pathway of indole was characterized by well-known steps through oxindole, isatin, and anthranilic acid. No further transformation of the hydroxylated methyl analogues: 3- and 7-methyloxindole and 3- and 4-methyl-2-quinolinone, was observed within 162 days of incubation. These degradation products accumulated in stoichiometric amounts, while 6- and 8-methyl-2-quinolinone were further degraded to 6- and 8-methyl-3,4-dihydro-2-quinolinone in stoichiometric amounts. Isoquinoline, 2-methylquinoline and 1- and 2-methylindole were not degraded by D. indolicum. These observations indicate that a methyl group at or close to position 2 results in blockage of the microbial attack, and that transformation of hydroxyquinolines methylated at the heterocyclic ring also was blocked or sterically inhibited. An incomplete transformation of some methylated compounds was observed, e.g. for 3- and 6-methylquinoline and 3- and 7-methylindole, with residual concentrations of 0.5–4 mg/l in relation to initial concentrations of 10–15 mg/l.
Application of the Plackett-Burman experimental design to evaluate nutritional requirements for the production of Colletotrichum coccodes spores by X. Yu; S. G. Hallett; J. Sheppard; A. K. Watson (pp. 301-305).
Colletotrichum coccodes is being examined as a biological weed control agent for velvetleaf (Abutilon theophrasti). A modified Richard's solution containing V-8 juice has been used to produce C. coccodes spores for growth-chamber and field experiments. Although C. coccodes sporulates well in this medium, V-8 is not available as a bulk commodity and is too expensive for commercial production. Eight substrates were evaluated as replacements for V-8 juice in modified Richard's solution. Soy protein and casamino acids were equal to V-8 juice for sporulation of C. coccodes. The Plackett-Burman experimental design was used to test the relative importance of various components of a complex medium based on soy protein on mycelium biomass production and sporulation of C. coccodes. A new medium composed of sucrose (20 g/l), soy protein (5 g/l), KNO3 (5 g/l), KH2PO4 (5 g/l), MgSO4 (2 g/l), CaCl2 (0.5 g/l), and CuSO4 (0.05 g/l) was selected as the base medium for further study in the development of a low-cost and effective medium for C. coccodes spore production.
Catechol dioxygenase expression in a Pseudomonas fluorescens strain exposed to different aromatic compounds by G. Cenci; G. Caldini (pp. 306-308).
Batch cultures of Pseudomonas fluorescens (strain P2a) maintained under carbon-limiting conditions in the presence of chrysene and other aromatics, survived starvation with no detectable changes in cell number for at least 4 months. P2a also demonstrated high dioxygenase levels after growth on benzoate and catechol. To characterize this strain further, early stationary-phase cells were resuspended in fresh mineral medium containing different aromatics, to evaluate enzyme expression in the presence of high-molecular-mass (isocyclic and heterocyclic) compounds. Results demonstrated effects on catechol 1,2-dioxygenase modulation by the model compounds used, confirming a low substrate specificity for this enzyme. The increases of specific activity observed in the presence of heterocyclic compounds were higher than those observed with isocyclic compounds.
Growth and amino acid requirements of hyaluronic-acid-producing Streptococcus zooepidemicus by D. C. Armstrong; M. J. Cooney; M. R. Johns (pp. 309-312).
A chemically defined medium has been developed for anaerobic cultivation of hyaluronic-acid(HA)-producing Streptococcus zooepidemicus, which contains 11 amino acids essential to growth, and glutamine as a principal nitrogen source. The final HA concentration, the specific rate of HA production and HA-to-glucose yields were similar for growth in the chemically defined medium relative to growth in complex medium. Consequently cells cultivated on chemically defined medium can be expected to have similar activity regarding HA synthesis as compared to cells grown on complex media. However, the specific growth rate and volumetric HA production rate were found to be less favourable in the chemically defined media.
Selection of biochemical mutants of Aspergillus niger with enhanced catalase production by J. Fiedurek; A. Gromada (pp. 313-316).
The production of extracellular catalase in a submerged culture by a number of biochemical mutants has been evaluated. Eight of these mutants showed increased extracellular catalase, the level of which ranged widely in individual cases from 44% to over 94% in comparison with the parental strain. Studies of the relationship between a criterion of selection and the frequency of mutation showed that the highest frequency of positive mutations (15.8% and 24.2%) was obtained with respect to mutants resistant to ethidium bromide (1 mmol/l) and sodium gluconate (45%) respectively. The time course of growth and enzyme production by the most active mutant, AM-20, showed extra- and intracellular catalase activities increasing about 2- and 2.6-fold respectively, compared with the parental strain.
Anaerobic dechlorination of carbon tetrachloride by free-living and attached bacteria under various electron-donor conditions by R.-a. Doong; T.-f. Chen; Y.-w. Wu (pp. 317-323).
The dechlorination of carbon tetrachloride (CCl4) by free-living and attached bacteria under anaerobic conditions was studied to examine the relationship between porous media and electron donor. Two batch-type experiments, the free-living and attached bacterial systems, were conducted with and without addition of 0.5-mm glass beads. Glucose and acetate were selected as the primary electron donors because they are easily biodegradable. Direct epifluorescence technology, the DAPI (4′ 6-diamidino-2-phenylindole) method, was used for counting the microbial activities. Adding glass beads could accelerate the dechlorination rate of CCl4. Removals of 44 %–57 % were observed in free-living bacterial system. Whereas a two- to fivefold increase in the CCl4 dechlorination rate was observed in the attached system. Experimental results and thermodynamic calculations indicated that glucose is a better supplementary substrate than acetate for stimulating the dechlorinating capability of microorganisms because of its relatively high available free energy. A higher concentration of substrate provided more reducing power for attached bacteria to initiate the dechlorination reaction. The pseudo-first-order rate constants of CCl4 dechlorination ranged from 0.007 day−1 to 0.017 day−1 and from 0.011 day−1 to 0.0625 day−1 for free-living and attached bacterial systems respectively. Microscopic observation revealed a three- to eightfold difference of microbial number between the free-living and attached bacterial systems. On the basis of the results in this study, we can conclude that the presence of porous media and an electron donor can change the dechlorination capabilities of the microorganisms. This work will be valuable in the design of in situ bioremediation as it discusses the specific area of the medium and supplementation with an electron donor to stimulate the indigenous microflora.
Effect of chlorinated aliphatic hydrocarbons on the acetoclastic methanogenic activity of granular sludge by J. L. Sanz; N. Rodríguez; R. Amils (pp. 324-328).
The toxicity of chlorinated aliphatic hydrocarbons on acetoclastic methanogens in anaerobic granular sludge was determined using a standardized anaerobic bioassay method. Most of the chloroaliphatics tested were strong inhibitors of methanogenesis. Tri- and tetrachloride derivatives of methane and ethane were the most highly toxic compounds tested, with concentrations of less than 18 mg/l resulting in 50% inhibition (IC50) of the methanogenic activity. Dichlorinated compounds were less toxic, with IC50 values ranging from 40 mg/l to 100 mg/l. On the other hand, perchlorinated derivatives of ethane and ethene were scarcely inhibitory at concentrations near their maximum water solubility. The toxicity caused by chlorinated aliphatic hydrocarbons was reversible. The comparison of structurally related compounds indicated that unsaturated chloroaliphatics were less toxic than their saturated counterparts. A reverse correlation between the electric dipole moment of these compounds and their methanogenic toxicity is discussed.