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.     

Anaerobic ethylene glycol degradation by microorganisms in poplar and willow rhizospheres

Although aerobic degradation of ethylene glycol is well documented, only anaerobic biodegradation via methanogenesis or fermentation has been clearly shown. Enhanced ethylene glycol degradation has been demonstrated by microorganisms in the rhizosphere of shallow-rooted plants such as alfalfa and grasses where conditions may be aerobic, but has not been demonstrated in the deeper rhizosphere of poplar or willow trees where conditions are more likely to be anaerobic. This study evaluated ethylene glycol degradation under nitrate-, and sulphate-reducing conditions by microorganisms from the rhizosphere of poplar and willow trees planted in the path of a groundwater plume containing up to 1.9 mol l⁻¹ (120 g l⁻¹) ethylene glycol and, the effect of fertilizer addition when nitrate or sulphate was provided as a terminal electron acceptor (TEA). Microorganisms in these rhizosphere soils degraded ethylene glycol using nitrate or sulphate as TEAs at close to the theoretical stoichiometric amounts required for mineralization. Although the added nitrate or sulphate was primarily used as TEA, TEAs naturally present in the soil or CO₂ produced from ethylene glycol degradation were also used, demonstrating multiple TEA usage. Anaerobic degradation produced acetaldehyde, less acetic acid, and more ethanol than under aerobic conditions. Although aerobic degradation rates were faster, close to 100% disappearance was eventually achieved anaerobically. Degradation rates under nitrate-reducing conditions were enhanced upon fertilizer addition to achieve rates similar to aerobic degradation with up to 19.3 mmol (1.20 g) of ethylene glycol degradation l⁻¹ day⁻¹ in poplar soils. This is the first study to demonstrate that microorganisms in the rhizosphere of deep rooted trees like willow and poplar can anaerobically degrade ethylene glycol. Since anaerobic biodegradation may significantly contribute to the phytoremediation of ethylene glycol in the deeper subsurface, the need for “pump and treat” or an aerobic treatment would be eliminated, hence reducing the cost of treatment.     

Degradation of 1,4-dioxane and cyclic ethers by an isolated fungus

By using 1,4-dioxane as the sole source of carbon, a 1,4-dioxane-degrading microorganism was isolated from soil. The fungus, termed strain A, was able to utilize 1,4-dioxane and many kinds of cyclic ethers as the sole source of carbon and was identified as Cordyceps sinensis from its 18S rRNA gene sequence. Ethylene glycol was identified as a degradation product of 1,4-dioxane by the use of deuterated 1,4-dioxane-d8 and gas chromatography-mass spectrometry analysis. A degradation pathway involving ethylene glycol, glycolic acid, and oxalic acid was proposed, followed by incorporation of the glycolic acid and/or oxalic acid via glyoxylic acid into the tricarboxylic acid cycle.     

Degradation of 1,4-Dioxane and Cyclic Ethers by an Isolated Fungus

By using 1,4-dioxane as the sole source of carbon, a 1,4-dioxane-degrading microorganism was isolated from soil. The fungus, termed strain A, was able to utilize 1,4-dioxane and many kinds of cyclic ethers as the sole source of carbon and was identified as Cordyceps sinensis from its 18S rRNA gene sequence. Ethylene glycol was identified as a degradation product of 1,4-dioxane by the use of deuterated 1,4-dioxane-d₈ and gas chromatography-mass spectrometry analysis. A degradation pathway involving ethylene glycol, glycolic acid, and oxalic acid was proposed, followed by incorporation of the glycolic acid and/or oxalic acid via glyoxylic acid into the tricarboxylic acid cycle.     

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.     

Toxic effects of some alcohol and ethylene glycol derivatives on Cladosporium resinae.

Eleven commercially available alcohol and ethylene glycol derivatives were tested for their toxicity toward a problem organism in jet fuel, Cladosporium resinae. In the presence of glucose, 20% (vol/vol) ethylene glycol monomethyl ether prevented spore germination and mycelial growth, and 10% (vol/vol) 2-ethoxybutanol, 10% 2-isopropoxyethanol, 10% 3-methoxybutanol, 5% 2-butyloxyethanol, 5% ethylene glycol dibutyl ether, and 5% diethylene glycol monobutyl ether were found to have similar effects. In a biphasic kerosene-water system, 3-methoxybutanol, 2-butyloxyethanol, and diethylene glycol monobutyl ether were again found to be more toxic than ethylene glycol monomethyl ether. Considerable potassium efflux, protein leakage, and inhibition of endogenous respiration were observed in the presence of the more toxic compounds. 2-Butyloxyethanol also caused loss of sterols from cells.     

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.     

Cryoprotectants for Crithidia fasciculata Stored at -20 C, with Notes on Trypanosoma gambiense and T. conorhini

SYNOPSIS. Cryoprotectants were tested in both complex and semidefined media for the trypanosomatid Crithidia fasciculata. Near log-phase or end-of-log-phase cultures were frozen for 24–48 hr at ∼ -20 C, then warmed in air to room temperature. Immediate motility was correlated with viability. The best protectant of the 83 tested was glycerol at ∼ 10% (w/v). Survival without cryoprotectant was rare. Outstanding cryoprotectants (perhaps also useful solvents for drugs poorly soluble in water) were: ethylene glycol; 2,2'-dioxyethanol (diethylene glycol); 1,2,4-butanetriol; 1,4-cyclohexanediol; dimethylsulfoxide; propylene glycol; and N -acetylethanolamine. Several sugars were active, e.g., D-arabinose, sucrose, and sorbitol. Trypanosomes tolerated cryoprotectants much less; tolerance was better in growth media than in suspension media. Trypanosoma gambiense was grown in blood-enriched media + 2-2.5% glycerol, suspended in 20% (w/v) glycerol. then frozen; this permitted 3-week survival. T. conorhini survived 4 weeks after growth in media containing glycerol 2.5%+ ethylene glycol 4%+ rutin 1.0 mg per 100 ml.     

Identification and characterization of 1,4-dioxane-degrading microbe separated from surface seawater by the seawater-charcoal perfusion apparatus

To determine the concentration of soluble 1,4-dioxane during biodegradation, a new method using of high-performance liquid chromatography equipped with a hydrophilic interaction chromatography column was developed. The developed method enabled easy and rapid determination of 1,4-dioxane, even in saline medium. Microbes capable of degrading 1,4-dioxane were selected from the seawater samples by the seawater-charcoal perfusion apparatus. Among 32 candidate 1,4-dioxane degraders,, strain RM-31 exhibited the strongest 1,4-dioxane degradation ability. 16S rDNA sequencing and the similarity analysis of strain RM-31 suggested that this organism was most closely related to Pseudonocardia carboxydivorans. This species is similar to Pseudonocardia dioxanivorans, which has previously been reported as a 1,4-dioxane degrader. Strain RM-31 could degrade 300 mg/L within 2 days. As culture incubation times increasing, the residual 1,4-dioxane concentration was decreasing and the total protein contents extracted from growth cells were increasing. The optimum initial pH of the broth medium and incubation temperature for 1,4-dioxane degradation were pH 6–8 and 25 °C. The biodegradation rate of 1,4-dioxane by strain RM-31 at 25 °C in broth medium with 3 % NaCl was almost 20 % faster than that without NaCl. It was probably a first bacteria from the seawater that can exert a strong degrading ability.     

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.     

Mineralization of 1,4-dioxane in the presence of a structural analog

A mixed culture with the ability to aerobically biodegrade 1,4-dioxane in the presence of tetrahydrofuran (THF) was enriched from a 1,4-dioxane contaminated aquifer. This consortium contained 3–4 morphologically different types of colonies and was grown in mineral salts media. Biodegradation of 1,4- dioxane began when THF concentrations in batch experiments became relatively low. No biodegradation of 1,4-dioxane was observed in the absence of THF and the measured cell yield was similar during degradation of 1,4-dioxane with THF or with THF alone. However, when the consortium was grown in the presence of ¹⁴C-1,4-dioxane plus THF, 2.1% of the radiolabeled 1,4-dioxane was present in the particulate fraction. The majority of the ¹⁴C (78.1%) was recovered as ¹⁴CO₂, while 5.8% remained in the liquid fraction. This activity is interesting since the non-growth substrate is mineralized, yet only minimally assimilated into biomass. Using THF as the growth substrate, 1,3-dioxane, methyl t-butyl ether, ethyl t-butyl ether and t-amyl methyl ether.     

Anaerobic biodegradation of 1,4-dioxane by sludge enriched with iron-reducing microorganisms

The potential on anaerobic biodegradation of 1,4-dioxane was evaluated by use of enriched Fe(III)-reducing bacterium sludge from Hangzhou municipal wastewater treatment plant. The soluble Fe(III) supplied as Fe(III)-EDTA was more available for the Fe(III)-reducing bacterium in the sludge compared to insoluble Fe(III) oxide. The addition of humic acid (HA) further stimulated the anaerobic degradation of 1,4-dioxane accompanying with apparent reduction of Fe(III) which is believed that HA could stimulate the activity of Fe(III)-reducing bacterium by acting as an electron shuttle between Fe(III)-reducing bacterium and Fe(III), especially for insoluble Fe(III) oxides. After 40-day incubation, the concentration of 1,4-dioxane dropped up to 90% in treatment of Fe(III)-EDTA+HA. Further study proved that more than 50% of the carbon from 1,4-dioxane was converted to CO2 and no organic products other than biomass accumulated in the growth medium. The results demonstrated that, under the appropriate conditions, 1,4-dioxane could be biodegraded while serving as a sole carbon substrate for the growth of Fe(III)-reducing bacterium. It might be possible to design strategies for anaerobic remediation of 1,4-dioxane in contaminated subsurface environments.     

Effect of PEG modification on subtilisin Carlsberg activity, enantioselectivity, and structural dynamics in 1,4-dioxane

The employment of enzymes as catalysts within organic media has traditionally been hampered by the reduced enzymatic activities when compared to catalysis in aqueous solution. Although several complementary hypotheses have provided mechanistic insights into the causes of diminished activity, further development of biocatalysts would greatly benefit from effective chemical strategies (e.g., PEGylation) to ameliorate this event. Herein we explore the effects of altering the solvent composition from aqueous buffer to 1,4-dioxane on structural, dynamical, and catalytic properties of the model enzyme subtilisin Carlsberg (SBc). Furthermore, we also investigate the effects of dissolving the enzyme in 1,4-dioxane through chemical modification with poly(ethylene)-glycol (PEG, M(W) = 20 kDa) on these enzyme properties. In 1,4-dioxane a 10(4)-fold decrease in the enzyme's catalytic activity was observed for the hydrolysis reaction of vinyl butyrate with D(2)O and a 50% decrease in enzyme structural dynamics as evidenced by reduced amide H/D exchange kinetics occurred. Attaching increasing amounts of PEG to the enzyme reversed some of the activity loss. Evaluation of the structural dynamic behavior of the PEGylated enzyme within the organic solvent revealed an increase in structural dynamics at increased PEGylation. Correlation analysis between the catalytic and structural dynamic parameters revealed that the enzyme's catalytic activity and enantioselectivity depended on the changes in protein structural dynamics within 1,4-dioxane. These results demonstrate the importance of protein structural dynamics towards regulating the catalytic behavior of enzymes within organic media.     

FIELD NOTE: IRRIGATION OF TREE STANDS WITH GROUNDWATER CONTAINING 1,4-DIOXANE

Coniferous and deciduous tree stands totaling 14 ha were recently planted on a closed landfill, and when mature, the stands are expected to be part of a natural treatment system for recovered groundwater. The trees would be irrigated at the rate of 189 L/min year-round with water containing 1,4-dioxane (< 10 mg/L), a compound that would be taken up and phytovolatilized by the trees. The water is moderately saline and contains elevated levels of manganese. This paper describes a concurrent series of preliminary studies, performed prior to the full-scale planting, to assess the feasibility of the phytoremediation system. Greenhouse experiments were carried out to identify tree species that can take up 1,4-dioxane and are tolerant of the water. Estimates were made of the area of the tree stand necessary to transpire the irrigation water plus precipitation. The landfill matrix was characterized in terms of its percolation rate and water holding capacity and based on those results salinity-modeling studies were carried out to estimate the fate and leaching potential of the various inorganic species that would accumulate in the root-zone of the trees. A pilot study, currently in progress on the landfill, suggested that the landfill cap is a suitable matrix for the establishment of large trees, and that the stands could be irrigated without the production of excess drainage.     

Effect of oligomers of ethylene glycol on thermotropic phase transition of dipalmitoylphosphatidylcholine multilamellar vesicles.

The effect of oligomers of ethylene glycol (EG) on thermotropic phase transitions of dipalmitoylglycerophosphatidylcholine multilamellar vesicles (DPPC-MLV) were investigated. Diethylene glycol (di-EG) had a biphasic effect on transition temperature, reducing pre-transition temperature (Tp) at low concentrations but increasing main transition temperature (Tm) and extinguishing pre-transition at high concentration. Results of the X-ray diffraction method and the excimer method indicated that di-EG induced interdigitated gel phase (L beta 1 phase) in the DPPC membranes at high concentration. Phase diagram of temperature-di-EG concentration for DPPC-MLV was determined by use of X-ray diffraction and differential scanning calorimetry, which was similar to that of temperature-EG concentration. The minimum concentration of di-EG where L beta 1 phase was induced was 42%(w/v), which was larger than that of EG (30%(w/v)). On the other hand, in the presence of triethylene glycol (tri-EG), Tm and Tp increased with an increased in tri-EG concentration, as well as poly(ethylene glycol). These differences, between the effects of di-EG and those of tri-EG, might be due to the differences of their sizes.     

Enzymic acylation of 6-aminopenicillanic acid

The enzymic synthesis of benzylpenicillin from 6-aminopenicillanic acid in the presence of poly (ethylene glycol) has been studied. With equimolar initial concentrations (20 mM) of 6-aminopenicillanic acid and phenylacetic acid a 60% conversion to benzylpenicillin can be achieved at 10°C and pH 5.2 in the presence of 45% (w/v) poly(ethylene glycol). Under these conditions the lactam ring of the benzylpenicillin and 6-aminopenicillanic acid and the enzyme, penicillin acylase (penicillin amidase, penicillin amidohydrolase, EC 3.5.1.11), were more stable than in the absence of the polyol.     

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.     

Hybrid aspen is not preferred by the large poplar borer (Saperda carcharias)

An industrially valuable tree, aspen, suffers from several fungal diseases and insect pests such as the large poplar borer (Saperda carcharias). The role of the beetle as a pest of aspen and hybrid aspen was investigated in five aspen and three hybrid aspen stands in Finland. Approximately 50% of the trees in the study had signs of the large poplar borer. Of these trees, 5% had insect exit holes. Approximately 70% of the trees with larval galleries had only one or two larval galleries. The trees with larval galleries were on average 2 m shorter than those without larval galleries. No significant difference could be detected in the diameter growth between these two groups. The proportion of decay was greater in hybrid aspen (27%) than in aspen (14%). These results show that the large poplar borer is an important pest of both aspen and hybrid aspen in Finland. No significant difference was observed between the aspen and hybrid aspen stands in the number of trees with larval galleries. Hybrid aspen therefore does not appear to be more sensitive to damage caused by the large poplar borer. Thus, a change from hybrid aspen to regular aspen in aspen cultivation is unnecessary, presuming that healthy saplings and an optimal rotation time are used.     

Glycolic acid production using ethylene glycol-oxidizing microorganisms.

Screening for microorganisms oxidizing ethylene glycol to glycolic acid was carried out. Among stock cultures, several yeasts and acetic acid bacteria showed high glycolic acid producing activity. Pichia naganishii AKU 4267 formed the highest concentration of glycolic acid, 35.3 g/l, from 10% (v/v) ethylene glycol (molar conversion yield, 26.0%). Among soil isolates, Rhodotorula sp. 3Pr-126, isolated using propylene glycol as a sole carbon source, formed the highest concentration of glycolic acid, 25.1 g/l, from 10% (v/v) ethylene glycol (molar conversion yield, 18.5%). Rhodotorula sp. 3Pr-126 showed higher activity toward 20% (v/v) ethylene glycol than P. naganishii AKU 4267. Optimization of the conditions for glycolic acid production was investigated using P. naganishii AKU 4267 and Rhodotorula sp. 3Pr-126. Under the optimized conditions, P. naganishii AKU 4267 and Rhodotorula sp. 3Pr-126 formed 105 and 110 g/l of glycolic acid (corrected molar conversion yields, 88.0 and 92.2%) during 120 h of reaction, respectively.