Evaluation of the biodegradation potential of 1,4-dioxane in river, soil and activated sludge samples

To evaluate the biodegradation potential of 1,4-dioxane in natural environments, a total of 20 environmental samples including river water, activated sludge, soil from the drainage area of a chemical factory and garden soil were subjected to a 1,4-dioxane degradation test. The five soil samples from the drainage area of the chemical factory were capable of reducing 100 mg l^?1 of 1,4-dioxane to below the detection limit (0.8 mg l^?1) within 33 days. In one activated sludge sample, 100 mg l^?1 of 1,4-dioxane decreased by 69% within 14 days via cometabolic degradation in the presence of 100 mg l^?1 of tetrahydrofuran (THF). The ability of all samples to degrade 1,4-dioxane degradation with or without THF increased after repeated enrichment, except for one soil sample from the drainage area of the chemical factory that was no longer able to degrade 1,4-dioxane after the third cycle of enrichment. However, most of the samples (14/20) were not able to degrade 1,4-dioxane degradation. Thus, it can be concluded that the potential for 1,4-dioxane degradation is not ubiquitously distributed in natural environment.     

Biodegradation of Ether Pollutants by Pseudonocardia sp. Strain ENV478

A bacterium designated Pseudonocardia sp. strain ENV478 was isolated by enrichment culturing on tetrahydrofuran (THF) and was screened to determine its ability to degrade a range of ether pollutants. After growth on THF, strain ENV478 degraded THF (63 mg/h/g total suspended solids [TSS]), 1,4-dioxane (21 mg/h/g TSS), 1,3-dioxolane (19 mg/h/g TSS), bis-2-chloroethylether (BCEE) (12 mg/h/g TSS), and methyl tert-butyl ether (MTBE) (9.1 mg/h/g TSS). Although the highest rates of 1,4-dioxane degradation occurred after growth on THF, strain ENV478 also degraded 1,4-dioxane after growth on sucrose, lactate, yeast extract, 2-propanol, and propane, indicating that there was some level of constitutive degradative activity. The BCEE degradation rates were about threefold higher after growth on propane (32 mg/h/g TSS) than after growth on THF, and MTBE degradation resulted in accumulation of tert-butyl alcohol. Degradation of 1,4-dioxane resulted in accumulation of 2-hydroxyethoxyacetic acid (2HEAA). Despite its inability to grow on 1,4-dioxane, strain ENV478 degraded this compound for >80 days in aquifer microcosms. Our results suggest that the inability of strain ENV478 and possibly other THF-degrading bacteria to grow on 1,4-dioxane is related to their inability to efficiently metabolize the 1,4-dioxane degradation product 2HEAA but that strain ENV478 may nonetheless be useful as a biocatalyst for remediating 1,4-dioxane-contaminated aquifers.     

Biodegradation of ether pollutants by Pseudonocardia sp. strain ENV478

A bacterium designated Pseudonocardia sp. strain ENV478 was isolated by enrichment culturing on tetrahydrofuran (THF) and was screened to determine its ability to degrade a range of ether pollutants. After growth on THF, strain ENV478 degraded THF (63 mg/h/g total suspended solids [TSS]), 1,4-dioxane (21 mg/h/g TSS), 1,3-dioxolane (19 mg/h/g TSS), bis-2-chloroethylether (BCEE) (12 mg/h/g TSS), and methyl tert-butyl ether (MTBE) (9.1 mg/h/g TSS). Although the highest rates of 1,4-dioxane degradation occurred after growth on THF, strain ENV478 also degraded 1,4-dioxane after growth on sucrose, lactate, yeast extract, 2-propanol, and propane, indicating that there was some level of constitutive degradative activity. The BCEE degradation rates were about threefold higher after growth on propane (32 mg/h/g TSS) than after growth on THF, and MTBE degradation resulted in accumulation of tert-butyl alcohol. Degradation of 1,4-dioxane resulted in accumulation of 2-hydroxyethoxyacetic acid (2HEAA). Despite its inability to grow on 1,4-dioxane, strain ENV478 degraded this compound for > 80 days in aquifer microcosms. Our results suggest that the inability of strain ENV478 and possibly other THF-degrading bacteria to grow on 1,4-dioxane is related to their inability to efficiently metabolize the 1,4-dioxane degradation product 2HEAA but that strain ENV478 may nonetheless be useful as a biocatalyst for remediating 1,4-dioxane-contaminated aquifers.     

1,4-Dioxane degradation potential of members of the genera <Emphasis Type="Italic">Pseudonocardia</Emphasis> and <Emphasis Type="Italic">Rhodococcus</Emphasis>

In recent years, several strains capable of degrading 1,4-dioxane have been isolated from the genera Pseudonocardia and Rhodococcus. This study was conducted to evaluate the 1,4-dioxane degradation potential of phylogenetically diverse strains in these genera. The abilities to degrade 1,4-dioxane as a sole carbon and energy source and co-metabolically with tetrahydrofuran (THF) were evaluated for 13 Pseudonocardia and 12 Rhodococcus species. Pseudonocardia dioxanivorans JCM 13855^T, which is a 1,4-dioxane degrading bacterium also known as P. dioxanivorans CB1190, and Rhodococcus aetherivorans JCM 14343^T could degrade 1,4-dioxane as the sole carbon and energy source. In addition to these two strains, ten Pseudonocardia strains could degrade THF, but no Rhodococcus strains could degrade THF. Of the ten Pseudonocardia strains, Pseudonocardia acacia JCM 16707^T and Pseudonocardia asaccharolytica JCM 10410^T degraded 1,4-dioxane co-metabolically with THF. These results indicated that 1,4-dioxane degradation potential, including degradation for growth and by co-metabolism with THF, is possessed by selected strains of Pseudonocardia and Rhodococcus, although THF degradation potential appeared to be widely distributed in Pseudonocardia. Analysis of soluble di-iron monooxygenase (SDIMO) α-subunit genes in THF and/or 1,4-dioxane degrading strains revealed that not only THF and 1,4-dioxane monooxygenases but also propane monooxygenase-like SDIMOs can be involved in 1,4-dioxane degradation.     

Kinetics of methyl t-butyl ether cometabolism at low concentrations by pure cultures of butane-degrading bacteria

Butane-oxidizing Arthrobacter (ATCC 27778) bacteria were shown to degrade low concentrations of methyl t-butyl ether (MTBE; range, 100 to 800 microg/liter) with an apparent half-saturation concentration (K(s)) of 2.14 mg/liter and a maximum substrate utilization rate (k(c)) of 0.43 mg/mg of total suspended solids per day. Arthrobacter bacteria demonstrated MTBE degradation activity when grown on butane but not when grown on glucose, butanol, or tryptose phosphate broth. The presence of butane, tert-butyl alcohol, or acetylene had a negative impact on the MTBE degradation rate. Neither Methylosinus trichosporium OB3b nor Streptomyces griseus was able to cometabolize MTBE.     

Metabolism of Radiolabeled β-Guaiacyl Ether-Linked Lignin Dimeric Compounds by Phanerochaete chrysosporium

Phanerochaete chrysosporium metabolized the radiolabeled lignin model compounds [γ-¹⁴C]guaiacylglycerol-β-guaiacyl ether and [4-methoxy-¹⁴C]veratrylglycerol-β-guaiacyl ether (VI) to ¹⁴CO₂ in stationary and in shaking cultures. ¹⁴CO₂ evolution was greater in stationary culture. ¹⁴CO₂ evolution from [γ-¹⁴C]guaiacyl-glycerol-β-guaiacyl ether and [4-methoxy-¹⁴C]veratrylglycerol-β-guaiacyl ether in stationary cultures was two- to threefold greater when 100% O₂ rather than air (21% O₂) was the gas phase above the cultures. ¹⁴CO₂ evolution from the metabolism of the substrates occurred only as the culture entered the stationary phase of growth. The presence of substrate levels of nitrogen in the medium suppressed ¹⁴CO₂ evolution from both substrates in stationary cultures. [¹⁴C]veratryl alcohol and 4-ethoxy-3-methoxybenzyl alcohol were formed as products of the metabolism of VI and 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether, respectively.     

Degradation of 1,4-dioxane by an actinomycete in pure culture.

An actinomycete capable of sustained aerobic growth on 1,4-dioxane was isolated from a dioxane-contaminated sludge samples. The actinomycete, CB1190, grows on 1,4-dioxane as the sole carbon and energy source with a generation time of approximately 30 h. CB1190 degrades 1,4-dioxane at a rate of 0.33 mg of dioxane min-1 mg of protein-1 and mineralizes 59.5% of the dioxane to CO2. CB1190 also grows with other cyclic and linear ethers as the sole carbon and energy sources, including 1,3-dioxane, 2-methyl-1,3-dioxolane, tetrahydrofuran, tetrahydropyran, diethyl ether, and butyl methyl ether. CB1190 is capable of aerobic autotrophic growth on H2 and CO2.     

Lithocholic Acid Side-chain Cleavage to Produce 17-Keto or 22-Aldehyde Steroids by Pseudomonas putida strain ST-491 Grown in the Presence of an Organic Solvent,Diphenyl Ether

We devised a method to screen for microorganisms capable of growing on bile acids in the presence of organic solvents and producing organic solvent-soluble derivatives. Pseudomonas putida biovar A strain ST-491 isolated in this study produced decarboxylated derivatives from the bile acids. Strain ST-491 grown on 0.5% lithocholic acid catabolized approximately 30% of the substrate as a carbon source, and transiently accumulated in the medium androsta-1,4-diene-3,17-dione in an amount of corresponding to 5% of the substrate added. When 20% (v/v) diphenyl ether was added to the medium, 60% of the substrate was converted to 17-keto steroids (androst-4-ene-3,17-dione-like steroid, androsta-1,4-diene-3,17-dione) or a 22-aldehyde steroid (pregna-1,4-dien-3-on-20-al). Amounts of the products were responsible for 45, 10, and 5% of the substrate, respectively. In the presence of the surfactant Triton X-100 instead of diphenyl ether, 40% of the substrate was converted exclusively to androsta-1,4-diene-3,17-dione.     

Biodegradation of 1,4-dioxane and transformation of related cyclic compounds by a newly isolated Mycobacterium sp. PH-06

A new bacterial strain PH-06 was isolated using enrichment culture technique from river sediment contaminated with 1,4-dioxane, and identified as belonging to the genus Mycobacterium based on 16S rRNA sequencing (Accession No. EU239889). The isolated strain effectively utilized 1,4-dioxane as a sole carbon and energy source and was able to degrade 900 mg/l 1,4-dioxane in minimal salts medium within 15 days. The key degradation products identified were 1,4-dioxane-2-ol and ethylene glycol, produced by monooxygenation. Degradation of 1,4-dioxane and concomitant formation of metabolites were demonstrated by GC/MS analysis using deuterium labeled 1,4-dioxane (1,4-dioxane-d8). In addition to 1,4-dioxane, this bacterium could also transform structural analogues such as 1,3-dioxane, cyclohexane and tetrahydrofuran when pre-grown with 1,4-dioxane as the sole growth substrate. Our results suggest that PH-06 can maintain sustained growth on 1,4-dioxane without any other carbon sources.     

Isolation of a Bacterial Culture That Degrades Methyl t-Butyl Ether

We have isolated a mixed bacterial culture (BC-1) which is capable of degrading the gasoline oxygenate methyl t-butyl ether (MTBE). BC-1 was developed from seed microorganisms present in a chemical plant biotreater sludge. This enrichment culture has been maintained in continuous culture treating high concentrations of MTBE (120 to 200 mg/liter) as the sole carbon source in a simple feed containing NH₄⁺, PO₄^3-, Mg²⁺, and Ca²⁺ nutrients. The unit had a stable MTBE removal rate when maintained with a long cell retention time (ca. 80 to 90 days); however, when operated at a ≤50-day cell waste rate, loss of MTBE-degrading activity was observed. The following three noteworthy experimental data show that MTBE is biodegraded extensively by BC-1: (i) the continuous (oxygen-sparged) culture was able to sustain a population of autotrophic ammonia-oxidizing bacteria which could nitrify influent NH₄⁺ concentrations at high rates and obtain CO₂ (sole carbon source for growth) from the metabolism of the alkyl ether, (ii) BC-1 metabolized radiolabeled either (¹⁴CH₃O-MTBE) to ¹⁴CO₂ (40%) and ¹⁴C-labeled cells (40%), and (iii) cell suspensions of the culture were capable of degrading (substrate depletion experiments) MTBE to t-butyl alcohol, a primary metabolite of MTBE. BC-1 is a mixed culture containing several bacterial species and is the first culture of its kind which can completely degrade an alkyl ether.     

Mineralization of Linear Alkylbenzene Sulfonate by a Four-Member Aerobic Bacterial Consortium

A bacterial consortium capable of linear alkylbenzene sulfonate (LAS) mineralization under aerobic conditions was isolated from a chemostat inoculated with activated sludge. The consortium, designated KJB, consisted of four members, all of which were gram-negative, rod-shaped bacteria that grew in pairs and short chains. Three isolates had biochemical properties characteristic of Pseudomonas spp.; the fourth showed characteristics of the Aeromonas spp. Cell suspensions were grown together in minimal medium with [¹⁴C]LAS as the only carbon source. After 13 days of incubation, more than 25% of the [¹⁴C]LAS was mineralized to ¹⁴CO₂ by the consortium. Pure bacterial cultures and combinations lacking any one member of the KJB bacterial consortium did not mineralize LAS. Three isolates carried out primary biodegradation of the surfactant, and one did not. This study shows that the four bacteria complemented each other and synergistically mineralized LAS, indicating catabolic cooperation among the four consortium members.     

Acetate as a source of reducing equivalents in the reductive dechlorination of 2,5-dichlorobenzoate

Desulfomonile tiedjei is the key dechlorinating organism in a three-tiered bacterial consortium that grows on the methanogenic degradation of 3-chlorobenzoate. 2,5-Dichlorobenzoate, however, is only converted to 2-chlorobenzoate and is not a methanogenic substrate for the consortium. The dechlorinator uses hydrogen produced from benzoate by the benzoate degrading member of consortium as its source of reducing equivalents for the dechlorination reaction. Incubation of 3-chlorobenzoate grown consortium cells with 2,5-dichlorobenzoate resulted in the consumption of acetate concurrent with the formation of 2-chlorobenzoate indicating that acetate can serve as an alternative source of reducing equivalents for reductive dechlorination. This interpretation was confirmed by the finding that the formation of ¹⁴CO₂ from 2-¹⁴C-labeled acetate was stoichiometric. The addition of hydrogen to 2,5-dichlorobenzoate metabolizing cells resulted in (i) an 2.7-fold increase in the rate of dechlorination, and (ii) a drop in the amount of label recovered as CO₂+CH₄ from methyl ¹⁴C-labeled acetate, indicating that hydrogen was the preferred source of reducing equivalents for reductive dechlorination. Benzoate, an indirect source of H₂ in the consortium, also inhibited the oxidation of acetate, while glucose, methanol, and butyrate did not affect labeled gas production and therefore were not suitable electron donors. Concomittant to dechlorination of 2,5-dichlorobenzoate 3- and 4-methoxybenzoate were converted to 3- and 4-hydroxybenzoate respectively. These conversions stimulated the rate of dechlorination 2-fold. Demethylation of 4-methoxybenzoate stimulated, but demethylation of 3-methoxybenzoate inhibited the oxidation of benzoate during the dechlorination of 2,5-dichlorobenzoate, suggesting that these isomers are metabolized through different pathways. Experiments with benzoate, 3-chlorobenzoate and 2,5-dichlorobenzoate metabolizing cells amended with ¹⁴CO₂ showed that actively dechlorinating cells catalyzed an exchange reaction between CO₂ and acetate.     

Mode of interaction of 1,4-dioxane agonists at the M2 and M3 muscarinic receptor orthosteric sites

The methyl group in cis stereochemical relationship with the basic chain of all pentatomic cyclic analogues of ACh is crucial for the agonist activity at mAChR. Among these only cevimeline (1) is employed in the treatment of xerostomia associated with Sjögren’s syndrome. Here we demonstrated that, unlike 1,3-dioxolane derivatives, in the 1,4-dioxane series the methyl group is not essential for the activation of mAChR subtypes. Docking studies, using the crystal structures of human M₂ and rat M₃ receptors, demonstrated that the 5-methylene group of the 1,4-dioxane nucleus of compound 10 occupies the same lipophilic pocket as the methyl group of the 1,3-dioxolane 4.     

Isolation and characterization of bacterial strains that have high ability to degrade 1,4-dioxane as a sole carbon and energy source

Four novel metabolic 1,4-dioxane degrading bacteria possessing high ability to degrade 1,4-dioxane (designated strains D1, D6, D11 and D17) were isolated from soil in the drainage area of a chemical factory. Strains D6, D11 and D17 were allocated to Gram-positive actinomycetes, similar to previously reported metabolic 1,4-dioxane degrading bacteria, whereas strain D1 was allocated to Gram-negative Afipia sp. The isolated strains could utilize a variety of carbon sources, including cyclic ethers, especially those with carbons at position 2 that were modified with methyl- or carbonyl-groups. The cell yields on 1,4-dioxane were relatively low (0.179–0.223 mg-protein (mg-1,4-dioxane)^?1), which was likely due to requiring energy for C–O bond fission. The isolated strains showed 2.6–13 times higher specific 1,4-dioxane degradation rates (0.052–0.263 mg-1,4-dioxane (mg-protein)^?1 h^?1) and 2.3–7.8 fold lower half saturation constants (20.6–69.8 mg L^?1) than the most effective 1,4-dioxane degrading bacterium reported to date, Pseudonocardia dioxanivorans CB1190, suggesting high activity and affinity toward 1,4-dioxane degradation. Strains D1 and D6 possessed inducible 1,4-dioxane degrading enzymes, whereas strains D11 and D17 possessed constitutive ones. 1,4-Dioxane degradation (100 mg L^?1) by Afipia sp. D1 was not affected by the co-existence of up to 3,000 mg L^?1 of ethylene glycol. The effects of initial pH, incubation temperature and NaCl concentration on 1,4-dioxane degradation by the four strains revealed that they could degrade 1,4-dioxane under a relatively wide range of conditions, suggesting that they have a certain adaptability and applicability for industrial wastewater treatment.     

Degradation of biphenyl by methanogenic microbial consortium

Biphenyl was readily degraded and mineralized to CO₂ and CH₄ by a PCB-dechlorinating anaerobic microbial consortium. Degradation occurred when biphenyl was supplied as a sole source of carbon or as a co-metabolic substrate together with glucose and methanol. p-Cresol was detected and confirmed by mass spectroscopy as a transient intermediate. Production of ¹⁴ C-CO₂ and ¹⁴C-CH₄ from ¹⁴C-biphenyl was observed in the approximate ratio of 1:2. The results indicated the existence of novel pathways for biphenyl degradation in a natural anaerobic microbial community.     

Comparison of unitrophic and mixotrophic substrate metabolism by acetate-adapted strain of Methanosarcina barkeri

We examined the unitrophic metabolism of acetate and methanol individually and the mixotrophic utilization of these compounds by using detailed ¹⁴C-labeled tracer studies in a strain of Methanosarcina barkeri adapted to grow on acetate as the sole carbon and energy source. The substrate consumption rate and methane production rate were significantly lower on acetate alone than during the unitrophic or mixotrophic metabolism of methanol. Cell yields (in grams per mole of substrate) were identical during exponential growth on acetate and exponential growth on methanol. During unitrophic metabolism of acetate, the methyl moiety accounted for the majority of the CH₄ produced, but 14% of the CO₂ generated originated from the methyl moiety. This correlated with the concurrent reduction of equivalent amounts of the C-1 of acetate to CH₄. ¹⁴CH₄ was also produced from added ¹⁴CO₂, although to a lesser extent than from reduction of the C-1 of acetate. During mixotrophic metabolism, methanol and acetate were catabolized simultaneously. The rates of ¹⁴CH₄ and ¹⁴CO₂ generation from [2-¹⁴C]acetate were logarithmic and higher in mixotrophic than in unitrophic cultures at substrate concentrations of 50 mM. A comparison of the oxidoreductase activities in cell extracts of the acetate-adapted strain grown on acetate and of strain MS grown on methanol or on H₂ plus CO₂ indicated that the pyruvate, α-ketoglutarate, and isocitrate dehydrogenase activities remained constant, whereas the CO dehydrogenase activity was significantly higher (5,000 nmol/min per mg of protein) in the acetate-adapted strain. These results suggested that a significant intramolecular redox pathway is possible for the generation of CH₄ from acetate, that energy metabolism from acetate by M. barkeri is not catabolite repressed by methanol, and that the acetate-adapted strain is a metabolic mutant with derepressed CO dehydrogenase activity.     

In vitro biosynthesis of 1,4-β-galactan attached to rhamnogalacturonan I

 The biosynthesis of galactan was investigated using microsomal membranes isolated from suspension-cultured cells of potato (Solanum tuberosum L. var. AZY). Incubation of the microsomal membranes in the presence of UDP-[¹⁴C]galactose resulted in a radioactive product insoluble in 70% methanol. The product released only [¹⁴C]galactose upon acid hydrolysis. Treatment of the product with Aspergillus niger endo-1,4-β-galactanase released 65–70% of the radioactivity to a 70%-methanol-soluble fraction. To a minor extent, [¹⁴C]galactose was also incorporated into proteins, however these galactoproteins were not a substrate for Aspergillus niger endo-1,4-β-galactanase. Thus, the majority of the ¹⁴C-labelled product was 1,4-β-galactan. Compounds released by the endo-1,4-β-galactanase treatment were mainly [¹⁴C]galactose and [¹⁴C]galactobiose, indicating that the synthesized 1,4-β-galactan was longer than a trimer. In vitro synthesis of 1,4-β-galactan was most active with 6-d-old cells, which are in the middle of the linear growth phase. The optimal synthesis occurred at pH 6.0 in the presence of 7.5 mM Mn²⁺. Aspergillus aculeatus rhamnogalacturonase A digested at least 50% of the labelled product to smaller fragments of approx. 14 kDa, suggesting that the synthesized [¹⁴C]galactan was attached to the endogenous rhamnogalacturonan I. When rhamnogalacturonase A digests of the labelled product were subsequently treated with endo-1,4-β-galactanase, radioactivity was not only found as [¹⁴C]galactose or [¹⁴C]galactobiose but also as larger fragments. The larger fragments were likely the [¹⁴C]galactose or [¹⁴C]galactobiose still attached to the rhamnogalacturonan backbone since treatment with β-galactosidase together with endo-1,4-β-galactanase digested all radioactivity to the fraction eluting as [¹⁴C]galactose. The data indicate that the majority of the [¹⁴C]galactan was attached directly to the rhamnose residues in rhamnogalacturonan I. Thus, isolated microsomal membranes contain enzyme activities to both initiate and elongate 1,4-β-galactan sidechains in the endogenous pectic rhamnogalacturonan I. Received: 24 June 1999 / Accepted: 30 August 1999     

Influence of organic acids on the growth ofThiobacillus neapolitanus

An ether-soluble fraction of yeast extract was found to bring about the reduction of cytochromes of thec type in crude cell-free extracts of autotrophically grown cells ofT. neapolitanus. Re-extraction of the ether-soluble fraction with petroleum ether resulted in marked decrease in its ability to reduce the cytochromes ofT. neapolitanus. Among several organic acids tested only succinate was effective in reducing the cytochromes. Acetate at 20mm concentration was a potent inhibitor of growth, although thiosulfate oxidation by intact cells was only slightly affected. No poly-β-hydroxybutyric acid was found in cells grown on thiosulfate in the presence or absence of non-inhibitory concentrations of acetate. Glycollic acid was not inhibitory to growth at concentrations up to 60mm. Glycollic oxidase could not be demonstrated in crude extracts, however glyoxylic reductase was present in high concentrations. In cells fixing¹⁴CO₂ in the presence or α-hydroxypyridyl-methanosulfonic acid, (HPMSA), a specific inhibitor of glycollic acid oxidase, 18% of the total radioactivity extracted by 95% alcohol was present in glutamate, whereas in the absence of HPMSA, glutamate accounted only for about 9% of the total radioactivity. No accumulation of glycollic acid, glycine or serine was observed in the presence of HPMSA. It appears that glycollic acid formation does not participate to a significant extent in CO₂ fixation by this organism. α-Hydroxypyridylmethanosulfonic acid appears to act differently on this obligate autotroph than on photosynthetic organisms.