Isolation and Characterization of Tetrahydrofuran-Degrading Bacteria ...

Journal of Water and Environment Technology, Vol. 11, No.1, 2013

Isolation and Characterization of TetrahydrofuranDegrading Bacteria for 1,4-Dioxane-Containing Wastewater Treatment by Co-Metabolic Degradation Kazunari SEI*, Masao OYAMA**, Takashi KAKINOKI**, Daisuke INOUE*, Michihiko IKE** *Department of Health Science, Kitasato University, 1-15-1 Kitasato, Sagamihara-Minami, Kanagawa 252-0373, Japan **Division of Sustainable Energy and Environmental Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan ABSTRACT 1,4-Dioxane is a cyclic ether mainly utilized in various chemical and pharmaceutical industries as solvent and reactant. Due to its toxic and persistent nature, 1,4-dioxane is a serious pollutant in the aquatic environment. Although 1,4-dioxane is quite recalcitrant to biodegradation, recent researches have shown 1,4-dioxane biodegradation as a sole carbon and energy source or by co-metabolism with tetrahydrofuran (THF). This study isolated and characterized THF-degrading bacteria to develop a biological process of 1,4-dioxane-containing wastewater treatment. Among five THF-degrading bacteria that were isolated, strain T1 from landfill soil and strains T3 and T5 from activated sludge showed stable co-metabolic degradation of 100 mg/L of 1,4-dioxane when coexisting with 100 mg/L of THF. Strains T1 and T5, identified as Rhodococcus ruber, were further characterized. Both strains could utilize a wide range of carbon sources, and grow at 15 – 35°C and pH 6 – 8. They demonstrated to have inducible THF degrading enzymes, and degraded up to 400 mg/L of THF as a growth substrate although they could not mineralize it. The optimum THF/1,4-dioxane ratios for the co-metabolic 1,4-dioxane degradation by strains T1 and T5 were determined to be 2 to 4. Our results would be useful for the development of biological 1,4-dioxane-containing wastewater treatment system. Keywords: co-metabolic degradation, 1,4-dioxane, tetrahydrofuran-degrading bacteria

INTRODUCTION 1,4-Dioxane is a cyclic ether mainly used as a solvent and a reactant in a variety of chemical and pharmaceutical industries. It is also formed as an undesired by-product in many industrial processes, especially in polyester synthesis. This xenobiotic is highly soluble and mobile in water, and a little volatilizable to the atmosphere and adsorptive to solid. In addition, since 1,4-dioxane does not have functional groups that are susceptible to hydrolysis (Wolfe and Jeffers, 2000) and does not absorb light in the environmental spectrum (> 290 nm), it is considered not to be hydrolyzed and photodegraded in the aquatic environment (Agency for Toxic Substances and Disease Registry (ATSDR), 2007). Thus, the fate of 1,4-dioxane depends basically on biodegradation in the aquatic environment. In fact, 1,4-dioxane has been widely detected in wastewater from industries (up to 4,020 g/L in Japan), surface water (up to 260 g/L in USA, 46 g/L in Japan and 10 g/L in the Netherlands), groundwater (up to 220 mg/L in USA) (ATSDR, 2007) and landfill leachate (up to 2,000 g/L in Japan) (Namegaya et al., 2002). This means that 1,4-dioxane will persist for a long time once released into the environment, hence it is considered to be quite recalcitrant to biodegradation. Thus, the treatment in its source is essential to prevent 1,4-dioxane contamination in the natural environment. Advanced oxidation processes (AOPs) such Address correspondence to Kazunari Sei, Department of Health Science, Kitasato University, Email: [email protected] Received May 10, 2012, Accepted September 5, 2012. - 11 -

Journal of Water and Environment Technology, Technology, Vol. 11, No.1, 2013

as a combination of ozone and hydrogen peroxide offer effective treatment for 1,4-dioxane although less economical and uses more energy than conventional biological process. Recently, 1,4-dioxane biodegradation as a sole carbon and energy source or by co-metabolism with tetrahydrofuran (THF) has been reported in both pure and mixed cultures. THF-degrading bacteria capable of degrading 1,4-dioxane by co-metabolism, which have been isolated in several studies (Kohlweyer et al., 2000; Vainberg et al., 2006; Sun et al., 2011), can be applied to the treatment of wastewater containing 1,4-dioxane by adding adequate concentration of THF as a primary substrate to the target wastewater. In this case, control of THF/1,4-dioxane concentration ratio is necessary to prevent from secondary pollution by THF. Another strategy is to induce THF degradation enzyme, which can also attack 1,4-dioxane, in the THF-degrading bacteria culturing tank, and to augment them into the treatment tank. In any of the cases, characterization, especially on THF/1,4-dioxane degradation properties, of THF-degrading bacteria which have 1,4-dioxane co-metabolic degradation ability is essential. However, there have been few studies on THF and co-metabolic 1,4-dioxane degradation properties of THF-degrading bacteria, including the THF/1,4-dioxane ratio that enhances the co-metabolic 1,4-dioxane degradation. This study aims to accumulate the useful information for developing biological 1,4-dioxane treatment technologies using THF-degrading bacteria as an option. Five THF-degrading bacteria, which have co-metabolic 1,4-dioxane degrading ability, were isolated from landfill soil and activated sludge samples and characterized on their THF and 1,4-dioxane degrading characteristics in detail. MATERIALS AND METHODS Enrichment and isolation of THF-degrading bacteria with 1,4-dioxane co-metabolic degradation ability To isolate THF-degrading bacteria, enrichment cultures that could degrade THF were developed using landfill soil (kindly provided by the National Institute for Environmental Studies, Japan) and activated sludge samples (kindly provided by a municipal wastewater treatment plant in Osaka, Japan) as bacterial seeds. A 50 mg/L (for landfill soil) or 100 mg/L (for activated sludge) of THF was added to 100 mL of basal salt medium (BSM) (Parales et al., 1994) along with 100 mg/L of 1,4-dioxane for the enrichment. The flasks were incubated at 28°C in a rotary shaker at 120 rpm. After confirming the significant degradation of THF and 1,4-dioxane, the cultures were successively inoculated into fresh media with the same component at 10% (v/v). After several times of repeated enrichment, the cultures were spread on BSM agar plate supplemented with THF as a vapor. The obtained colonies were checked in terms of their THF degrading ability in liquid BSM containing 100 mg/L of THF, and purified on 10 times diluted (1/10) CGY medium (casitone 0.5 g/L, glycerin 0.5 g/L, yeast extract 0.1 g/L). Physiological characterization and phylogenetic identification Cell morphology and motility, gram staining and catalase, oxidase and OF tests were performed to characterize the isolates according to the diagnostic tables of bacteria proposed by Cowan and Steel (1974). Carbon source utilization were examined with API 20E (Bio-Mérieux, Marcy I’Etoile, France) and Biolog GN2 plate (Biolog,

- 12 -


Hayward, CA, USA). Gelatin hydrolysis, Voges-Proskauer reaction, nitrate reduction and indole production tests were performed using API 20NE (Bio-Mérieux). Utilization of cyclic and straight-chain ethers was examined with 7-day incubation of isolates in liquid BSM containing 200 mg/L of each compound. Phylogenetic analyses based on partial 16S rRNA gene sequences were performed as described previously (Inoue et al., 2008). The partial 16S rRNA gene sequences (1,354 bp) of strains T1 and T5 were registered in DDBJ/EMBL/GenBank as accession numbers AB715146 and AB715147, respectively. THF degradation studies with isolated bacteria Isolated bacteria were precultured in liquid BSM containing 200 mg/L of THF. As a basal test condition, the precultured bacteria were inoculated into 15 mL of BSM containing 100 mg/L of THF at a final cell density (determined by optical density at 600 nm (OD600)) of 0.03 and aerobically incubated by rotary shaking at 120 rpm at 28°C. Control tests without inoculating isolated bacteria were also conducted to evaluate the evaporation of THF under the incubating conditions. Effects of THF concentration, pH and temperature on THF degradation were evaluated by calculating the specific growth rate of each bacterium that occurred with THF degradation by appropriately changing the experimental condition. Cell yield (mg-cell protein/mg-THF degraded) was calculated by determining the cell protein concentration using BCATM Protein Assay Kit (Thermo Fisher Scientific, MA, USA). Co-metabolic 1,4-dioxane degradation studies with isolated bacteria Isolated bacteria were precultured as mentioned above. For 1,4-dioxane degradation tests, basal test condition shown above was applied with slight modification that THF and 1,4-dioxane were added to liquid BSM at 50 mg/L each, and precultured bacteria were inoculated at an OD600 of 0.15. Control tests were also performed. To evaluate the effect of THF and 1,4-dioxane concentrations on 1,4-dioxane degradation, these concentrations were varied appropriately to give different THF/1,4-dioxane ratio. Analytical procedures THF and 1,4-dioxane concentrations were determined by gas chromatography (GC-14B, Shimadzu, Kyoto, Japan) equipped with a flame-ionization detector as described previously (Sei et al., 2010). Total organic carbon (TOC) concentration was determined by a TOC analyzer (TOC-5000A, Shimadzu). RESULTS AND DISCUSSION Confirmation of co-metabolic 1,4-dioxane degradation by isolated bacteria Five THF-degrading bacteria were successfully isolated from landfill soil (strains T1, T2 and T4) and activated sludge samples (strains T3 and T5). Strains T1, T3 and T5 showed stable co-metabolic degradation of 100 mg/L of 1,4-dioxane when coexisting with 100 mg/L of THF, whereas strains T2 and T4 could not completely degrade 100 mg/L of 1,4-dioxane under the same condition, but rather weakened their 1,4-dioxane degradation ability during repeated batch degradation tests (Fig. 1). In Fig. 1, two typical co-metabolic 1,4-dioxane degradation profiles are shown. The profile by strain T2 represents the incomplete and unstable co-metabolic 1,4-dioxane degradation, and the profile by strain T4 was almost the same (data not shown). Similarly, the profile by

- 13 -


strain T5 represents the complete and stable co-metabolic 1,4-dioxane degradation, and the profiles by strains T1 and T3 were almost the same (data not shown). Physiological characterization and phylogenetic identification of isolated bacteria Further characterization was performed against strains T1 and T5 because they showed a high co-metabolic 1,4-dioxane degradation ability. Both strains were rod-shaped, non-motile, gram- and catalase-positive and oxidase-negative bacteria with aerial mycelium formation ability. They showed the highest 16S rRNA gene sequence identity (100% of 1,354 bp) with Rhodococcus ruber DSM43338T (X80625) and R. ruber M2 (AY247275), and thus identified as R. ruber. R. ruber and its relatives have been reported to degrade ether compounds, like THF, 1,4-dioxane and methyl tert-butyl ether (Daye et al., 2003; Goodfellow et al., 2004; Mahendra and Alvarez-Cohen, 2006). Both strains could utilize a wide range of carbon source, including sugars, organic acids, and amino acids (Table 1). D-Glucose, D-fructose, D-sucrose and D-sorbitol are especially good substrates for their growth. Although strains T1 and T5 were also capable of utilizing cyclic ethers (e.g., THF, tetrahydropyran, γ-butyrolactone, γ-valerolactone, and 1,3-dioxolan-2-one), they could not utilize straight-chain ethers such as ethoxyacetic acid.

160

THF and 1,4-dioxane (mg/L)

THF and 1,4-dioxane (mg/L)

180 160 140 120 100 80 60 40 20

140 120 100 80 60 40 20 0

0 0

20

40

60 80 Time (days)

100

120

0

20

40 Time (days)

60

80

Fig. 1 - Typical 1,4-dioxane co-metabolic degradation profiles of THF-degrading bacterial strains T2 (A) and T5 (B). Closed square, 1,4-dioxane; open square, THF; closed circle, 1,4-dioxane (control); open circle, THF (control); closed arrow, culture transfer into fresh medium; open arrow, addition of THF. Table 1 - Assimilable carbon sources of strains T1 and T5 in the Biolog assay. C a te g o ry Sugars

Organic acids

Amino acids Alcohols

St r a i n T 1 adonitol, L-arabinose, i-erythritol, D-fructose, α-D-glucose, m-inositol, α-D-lactose, lactulose, maltose, D-melibiose, L-rhamnose, D-sorbitol, sucrose, D-trehalose, turanose, xylitol acetic acid, citric acid, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, γ-hydroxybutyric acid, itaconic acid, D,L-lactic acid, malonic acid, quinic acid glucuronamide, D-alanine, L-alanine, L-asparagine, L-glutamic acid, glycyl-L-aspartic acid, L-proline, L-serine, uridine, 2,3-butanediol, glycerol

- 14 -

St r a in T 5 L-arabinose,

i-erythritol, D-fructose, gentiobiose, α-D-glucose, m-inositol, α-D-lactose, maltose, D-mannitol, D-raffinose, L-rhamnose, D-sorbitol, sucrose, D-trehalose, turanose, xylitol acetic acid, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, α-hydroxybutyric acid, β-hydroxybutyric acid, γ-hydroxybutyric acid, α-keto valeric acid, D,L-lactic acid, propionic acid, quinic acid, D-saccharic acid D-alanine, L-alanine, L-aspartic acid, L-glutamic acid, glycyl-L-aspartic acid, L-ornithine, L-proline, D,L-carnitine, 2-aminoethanol 2 ,3 - b u t a n e d i o l , g l y c e r o l D-galactose,


THF degradation properties of strains T1 and T5 Typical time courses of THF degradation and TOC removal by strains T1 and T5 are shown in Figs. 2A and 2D, respectively. In control experiments without inoculating strains T1 and T5, TOC concentration declined only slightly and around 80% of initial TOC remained at the end of the experiments, indicating that the evaporation did not largely affect the evaluation of biodegradation abilities of test strains. Strains T1 and T5 completely degraded 50 mg/L of THF within 7 days. However, approximately 50% and 30% of initial TOC remained for a long period for strains T1 and T5, respectively. With the GC analysis applied in this study, no intermediate peaks were detected throughout the experimental period. In the proposed THF degradation pathway, non-volatile compounds such as succinic acid are produced during THF biodegradation (Thiemer et al., 2003), and we confirmed that strains T1 and T5 could not utilize succinic acid. Thus, the remaining TOC would be due to the accumulation of such intermediates. Because a lag phase was observed before the initiation of THF degradation for both strains when precultured on glucose instead of THF (data not shown), the strains were suggested to have inducible THF degrading enzymes. Figure 3 shows the time courses of THF degradation and concomitant cell growth of strains T1 and T5 at various THF concentrations. Both strains were capable of degrading up to 400 mg/L of THF as a growth substrate, although strain T5 could not completely degrade 400 mg/L of THF. THF concentration of higher than 100 mg/L and 300 mg/L clearly inhibited the THF degradation ability and growth of strains T5 and T1, respectively, probably due to the toxicity of THF and/or intermediates. Strains T1 and T5 were capable of growing on THF at pH 6 to 8 and at temperature of 15°C to 35°C. The optimum pH and temperature for their growth were 7.0 and 35°C, respectively. The cell yields of both strains on THF were 0.22 mg-protein/mg-THF. 70

(A)

40 30 20 10 0

50 40 30 20 10 0

5

70

10 15 20 Time (days)

40 30 20 10 0


50 40 30 20 10 0

0

5


25

60 40 20

25

0

5

100

(E)

60 Concentration (mg/L)

50

5

70

(D)

60

(C)

80

0 0

25

Concentration (mg/L)

0


100

(B)

60


50



60


25

(F)

80 60 40 20 0

0

5


25

0

5


25

Fig. 2 - THF and 1,4-dioxane degradation profiles of strains T1 (A, B and C) and T5 (D, E and F). Results of THF (A and D), 1,4-dioxane (B and E), and THF and 1,4-dioxane (C and F) supplemented systems are shown. Closed circle, THF; closed triangle, TOC; open triangle, TOC (control: without bacteria); closed square, 1,4-dioxane.

- 15 -

Journal of Water and Environment Technology, Technology, Vol. 11, No.1, 2013 (B) 0.35 0.3

100

0.25

80

OD600

THF THFresidual removalratio ratio (%) (%)

(A) 120

60 40

0.1

20

0.05 0

0 0

5

(C) 120

10 15 Time (days)

20

0

25

(D)

5

10

15

20

25

20

25

Time (days)

0.4 0.35

100

0.3

80 OD600

THF THFresidual removalratio ratio (%)

0.2

0.15

60

0.25 0.2 0.15

40

0.1

20

0.05

0

0

0

5

10

15

20

25

Time (days)

0

5

10 15 Time (days)

Fig. 3 - THF degradation (A and C) and concomitant growth (B and D) profiles of THF-degrading bacterial strains T1 (A and B) and T5 (C and D). Closed circle, 25 mg/L; open circle, 50 mg/L; closed square, 100 mg/L; open square, 300 mg/L; closed triangle, 400 mg/L. Such low cell yields would be due to the requirement of a large quantity of energy for the scission of ether bonds in THF (White et al., 1996). In addition, the accumulation of some toxic intermediates may lower the cell yields of both strains. Co-metabolic 1,4-dioxane degradation properties of strains T1 and T5 Typical degradation profiles of 1,4-dioxane with and without THF by strains T1 and T5 are shown in Fig. 2 (B, C, E and F). In all control experiments without inoculation of strains T1 and T5, TOC declined only slightly within the experimental period. When 1,4-dioxane was added as the sole carbon source, both strains could not degrade it in the early stage of the experiments (Fig. 2B and 2E). However, strain T1 showed slight degradation of 1,4-dioxane after 10 days and concomitant decrease of TOC concentration. One of the possible reasons for this is that 1,4-dioxane weakly induced THF degrading enzymes in the strain. Also, the occurrence of a mutant of strain T1 which obtained 1,4-dioxane metabolizing ability could not be ruled out as reported by Parales et al. (1994). When both THF and 1,4-dioxane were added to the test systems, THF was degraded first within 7 days, which was followed by the degradation of 1,4-dioxane within 24 days (Fig. 2C and 2F). This is a typical profile of co-metabolic degradation with competitive inhibition. Zenker et al. (2000) reported the competitive inhibition of co-metabolic 1,4-dioxane degradation by THF. Even after both THF and 1,4-dioxane were completely degraded, 50 – 75% of TOC remained in the test systems. In the putative 1,4-dioxane degradation pathway (Vainberg et al., 2006), 1,4-dioxane is transformed to 2-hydroxyethoxyacetic acid (2-HEAA), which is a straight-chain ether, via 1,4-dioxane-ol and 2-hydroxyethoxy-2-acetoaldehyde. As mentioned above, strains T1 and T5 cannot utilize straight-chain ethers. Thus, the remaining TOC may be attributable to the accumulation of non-volatile compounds (e.g., succinic acid) and 2-HEAA, which are the intermediates of the THF and 1,4-dioxane degradation, respectively. - 16 -


Effect of THF concentration and THF/1,4-dioxane ratio on the co-metabolic 1,4-dioxane degradation by strains T1 and T5 Co-metabolic 1,4-dioxane degradation by strains T1 and T5 was further examined under the conditions that THF and 1,4-dioxane concentrations were varied against the fixed concentration of 1,4-dioxane and THF (100 mg/L), respectively (Fig. 4). When the 1,4-dioxane concentration was fixed to 100 mg/L, 1,4-dioxane was rapidly degraded at THF concentrations of 50 – 200 mg/L (Fig. 4A) and 25 – 100 mg/L (Fig. 4B) in strains T1 and T5, respectively. When THF concentrations increased within the above range, 1,4-dioxane degradation ratio increased. For strain T1, complete 1,4-dioxane degradation was observed with 200 mg/L of THF. For strain T5, the highest 1,4-dioxane degradation was observed with 100 mg/L of THF although the degradation ratio remained in 69.6%. The higher concentration of THF (higher than 200 mg/L and 100 mg/L for strains T1 and T5, respectively) diminished the ability of both strains to degrade 1,4-dioxane due to the competitive inhibition and toxicity of THF itself as mentioned above. When the THF concentration was fixed to 100 mg/L, rapid 1,4-dioxane degradation was observed at 1,4-dioxane concentrations of 25 – 50 mg/L in both strains (Fig. 4C and 4D), and strain T1 showed 94.8% degradation up to 100 mg/L of 1,4-dioxane. It is obvious that higher than 100 mg/L and 50 mg/L of 1,4-dioxane have inhibitive effect on 1,4-dioxane co-metabolic degradation by strains T1 and T5, respectively. Based on those results, the optimum THF/1,4-dioxane ratio was determined to be 2 to 4 for both strains T1 and T5. The acceptable THF and 1,4-dioxane (B) (B) 1,4-Dioxane residual ratio (%)

1,4-Dioxane residual ratio (%)

(A) 120 (A) 100 80 60 40 20

100 80 60 40 20 0

0 0

5

(C) (C) 120

10

15

20

0

25

(D) (D)

Time (days)



120

100 80 60 40 20

5

10

15

20

25

20

25

Time (days)

120 100 80 60 40 20 0

0 0

5

10

15

20

25

0

5

10

15

Time (days)

Time (days)

Fig. 4 - Effect of THF concentration on 1,4-dioxane degradation by strains T1 (A and B) and T5 (C and D). THF concentration was varied between 25 – 400 mg/L against 100 mg/L of 1,4-dioxane (A and C), and 1,4-dioxane concentration was varied between 25 – 400 mg/L against 100 mg/L of THF (B and D). Closed circle, 25 mg/L; open circle, 50 mg/L; closed square, 100 mg/L; open square, 200 mg/L; closed diamond, 400 mg/L. The data for 100 mg/L of both THF and 1,4-dioxane in A and B, and in C and D are the same data sets.

- 17 -


concentrations to maximize the co-metabolic 1,4-dioxane degradation with minimum competitive inhibition were 200 mg/L and 100 mg/L, respectively, for strain T1, and 100 mg/L and 50 mg/L, respectively, for strain T5. CONCLUSIONS Among five THF-degrading bacteria isolated from landfill soil and activated sludge samples, two strains (T1 and T5), which showed superior THF degradation ability and identified as R. ruber, were examined especially on their THF and co-metabolic 1,4-dioxane degradation properties. Strains T1 and T5 could completely degrade up to 400 mg/L and 300 mg/L of THF, respectively, although they could not completely mineralize it. Complete co-metabolic 1,4-dioxane degradation was confirmed when the added THF concentration was 100 – 200 mg/L with 1,4-dioxane concentration within 25 – 100 mg/L for strain T1. In the same way, strain T5 showed complete co-metabolic 1,4-dioxane degradation when the added THF concentration was 100 mg/L with 1,4-dioxane concentration within 25 – 50 mg/L. The optimum THF/1,4-dioxane ratio was determined to be 2 to 4 for both strains, T1 and T5, to maximize the co-metabolic 1,4-dioxane degradation with minimum competitive inhibition. It can be concluded that strains T1 and T5 would be applicable to treat 1,4-dioxane-containing wastewater, and that the results obtained in this study would be greatly helpful for the development and control of 1,4-dioxane-containing wastewater treatment system using THF-degrading bacteria. However, the accumulation of intermediates including 2-HEAA during 1,4-dioxane degradation is a critical issue to be solved in future studies. ACKNOWLEDGEMENT This study was supported in part by the Environment Research and Technology Development Fund (B-1201) of the Ministry of the Environment, Japan, and by a grant from Kitasato University School of Allied Health Sciences (Grant-in-Aid for Research Project, No. 2011-1005). REFERENCES Agency for Toxic Substances and Disease Registry (ATSDR) (2007) Toxicological profile for 1,4-Dioxane (Draft for Public Comment). U. S. Department of Health and Human Services, Public Health Service, Atlanta, USA. http://www.atsdr.cdc.gov/toxprofiles/tp187.pdf (accessed on May 2, 2012). Cowan S. T. and Steel K. J. (1974) Manual for identification of medical bacteria, 2nd edn. Cambridge University Press, Cambridge, UK. Daye K. J., Groff J. C., Kirpenkar A. C. and Mazumder R. (2003) High efficiency degradation of tetrahydrofuran (THF) using a membrane bioreactor: identification of THF-degrading cultures of Pseudonocardia sp. strain M1 and Rhodococcus ruber isolate M2. J. Ind. Microbiol. Biotechnol., 30(12), 705-714. Goodfellow M., Jones A. L., Maldonado L. A. and Salanitro J. (2004) Rhodococcus aetherivorans sp. nov., a new species that contains methyl t-butyl ether-degrading actinomycetes. Syst. Appl. Microbiol., 27(1), 61-65. Inoue D., Hara S., Kashihara M., Murai Y., Danzl E., Sei K., Tsunoi S., Fujita M. and Ike M. (2008) Degradation of bis(4-hydroxyphenyl)methane (bisphenol F) by

- 18 -


Sphingobium yanoikuyae strain FM-2 isolated from river water. Appl. Environ. Microbiol., 74(2), 352-358. Kohlweyer U., Thiemer B., Schräder T. and Andreesen J. R. (2000) Tetrahydrofuran degradation by a newly isolated culture of Pseudonocardia sp. strain K1. FEMS Microbiol. Lett., 186(2), 301-306. Mahendra S. and Alvarez-Cohen L. (2006) Kinetics of 1,4-dioxane biodegradation by monooxygenase-expressing bacteria. Environ. Sci. Technol., 40(17), 5435-5442. Namegaya Y., Suzuki S., Yasuhara A., Mohri S., Yamada M. and Inoue Y. (2002) Concentrations of inorganic components, 1,4-dioxane, phenols, and phthalates in leachates from waste landfills and their treated waters. J. Environ. Chem., 12(4), 817-827. Parales R. E., Adamus J. E., White N. and May H. D. (1994) Degradation of 1,4-dioxane by an actinomycete in pure culture. Appl. Environ. Microbiol., 60(12), 4527-4530. Sei K., Kakinoki T., Inoue D., Soda S., Fujita M. and Ike M. (2010) Evaluation of the biodegradation potential of 1,4-dioxane in river, soil and activated sludge samples. Biodegradation, 21(4), 585-591. Sun B., Ko K. and Ramsay J. A. (2011) Biodegradation of 1,4-dioxane by a Flavobacterium. Biodegradation, 22(3), 651-659. Thiemer B., Andreesen J. R. and Schräder T. (2003) Cloning and characterization of a gene cluster involved in tetrahydrofuran degradation in Pseudonocardia sp. strain K1. Arch. Microbiol., 179(4), 266-277. Vainberg S., McClay K., Masuda H., Root D., Condee C., Zylstra G. J. and Steffan R. J. (2006) Biodegradation of ether pollutants by Pseudonocardia sp. strain ENV478. Appl. Environ. Microbiol., 72(8), 5218-5224. White G. F., Russell N. J. and Tidswell E. C. (1996) Bacterial scission of ether bonds. Microbiol. Rev., 60(1), 216-232. Wolfe N. L. and Jeffers P. M. (2000) Hydrolysis. In: Handbook of Property Estimation Methods for Chemicals: Environmental and Health Sciences, R. S. Bothering and D. Mackay (ed.). Lewis Publishers, Chelsea, USA, pp. 311-334. Zenker M. J., Borden R. C. and Barlaz M. A. (2000) Mineralization of 1,4-dioxane in the presence of a structural analog. Biodegradation, 11(4), 239-246.

- 19 -