- Email: [email protected]
Metabolism of diphenylurea by a Marinobacter sp. isolated from a contaminated ephemeral stream bed in the Negev Desert
FEMS Microbiology Letters 213 (2002) 199^204
www.fems-microbiology.org
Metabolism of diphenylurea by a Marinobacter sp. isolated from a contaminated ephemeral stream bed in the Negev Desert Sebastian R. Srensen a
a;
, Ziv Arbeli
b;1
, Jens Aamand a , Zeev Ronen
b
Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS), Copenhagen, ster Voldgade 10, DK-1350 Copenhagen K, Denmark b Department of Environmental Hydrology and Microbiology, Ben Gurion University of the Negev, Jacob Blaustein Institute for Desert Research, Sede-Boker, Israel Received 8 February 2002; received in revised form 11 June 2002 ; accepted 12 June 2002 First published online 12 July 2002
Abstract A moderate halophilic Marinobacter sp. (designated strain DPUZ) able to metabolize 1,3-diphenylurea (DPU) was isolated from a contaminated ephemeral desert stream bed near an industrial complex in the northern part of the Negev Desert (Israel). Metabolism of DPU was accompanied by a transient accumulation of a metabolite identified as aniline using gas chromatography^mass spectrometry, thus indicating a metabolic pathway involving cleavage of the urea bridge between the phenyl structures. Aniline was further degraded without detection of other metabolites suggesting a complete degradation. Strain DPUZ grows at NaCl concentrations between 0.2 and 2.6 M with an optimum at 0.51 M. It grows at a temperature range between 20 and 40‡C with an optimum at 35‡C. This is the first study on bacterial metabolism of DPU. 7 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Diphenylurea ; Halophilic; Desert stream; Biodegradation ; Marinobacter
1. Introduction As a consequence of agricultural practice and accidental spillage during production of chemicals such as propellants, various phenylurea compounds are detected as environmental pollutants [1^3]. Studies on the environmental fate of phenylurea compounds have mainly been focused on phenylurea herbicides [4,5], and little is known about the fate of diphenylurea compounds in contaminated environments. For related compounds such as diphenylamine, biodegradation is the predominant mechanism for dissipation from environments such as sewage sludge [6] and marine sediments [1,2]. The aim of this study was to isolate and characterize diphenylurea-degrading organisms from a contaminated desert environment, choosing 1,3-diphenylurea (DPU) as a representative model compound.
* Corresponding author. Tel.: +45 (3814) 2317; Fax: +45 (3814) 2050. E-mail address : [email protected] (S.R. Srensen). 1 Present address: Laboratorio Ambiental, Instituto de Biotecnolog|¤a ^ Edi¢cio Manuel Ancizar, Universidad Nacional de Colombia, Bogota¤, Colombia.
By inoculating sediment from a ephemeral desert stream bed into a mineral medium provided DPU as sole source of carbon, nitrogen and energy, we enriched and isolated a Marinobacter sp. The stream was contaminated with industrial wastewater and located in the vicinity of an industrial area located in the northern part of the Negev Desert in Israel. Among the most abundant contaminants of the wastewater were various diphenylurea compounds including DPU. This is the ¢rst isolation of a DPU-degrading bacterium and attempt to elucidate the metabolic pathway for DPU.
2. Materials and methods 2.1. Media A carbon- and nitrogen-free medium (MM) was used for the enrichment and degradation studies. MM contained (l31 ): 0.5 g KH2 PO4 , 1.0 g Na2 HPO4 W2H2 O and 30 g NaCl. pH was adjusted to 7.5 before autoclaving (121‡C, 20 min). After cooling 2.5 ml of a ¢lter-sterilized MgSO4 W7H2 O solution (200 g l31 ) was added. DPU ( 6 100 mg l31 water solubility at 18‡C, purity s 98%,
0378-1097 / 02 / $22.00 7 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 8 2 0 - 0
FEMSLE 10572 5-8-02
200
S.R. S6rensen et al. / FEMS Microbiology Letters 213 (2002) 199^204
Sigma Chemicals, St. Louis, MO, USA) (Fig. 2A, inset) was added to the media from a stock solution (0.1 g ml31 ) in dimethylsulfoxide (DMSO). In all experiments with DPU in liquid media (20^100 mg l31 ) DPU crystals were initially visible in the media. A substrate rich medium (RM) was made by adding 1.0 g l31 casamino acids (Difco, Detroit, MI, USA) to the MM medium. A DPU-containing (20 mg l31 ) MM-based agar (MMA) was prepared by adding 15 g l31 Bacto Agar (Difco, Detroit, MI, USA), and DPU was added to the medium just before preparation of the plates. R2A (Remel, Lenexa, KS, USA) supplemented with 30 g l31 NaCl (R2A-salt) was used for enumeration of cells. 2.2. Enrichment and isolation Sediment was sampled from a small stream located in the vicinity of an industrial area in the northern part of the Negev Desert (Israel) [7,8]. Sediments from three different locations approximately 500 m apart were sampled from the wet part of the stream bed in July 1999. An amount of 5 g wet sediment was inoculated into 50 ml MM with 100 mg l31 DPU and incubated at 25‡C under either oxic or anoxic conditions. Oxic experiments were preformed in 250-ml Erlenmeyer £asks, and the anoxic experiments in 160-ml rubber-sealed serum bottles with a gas phase of 94% N2 and 6% H2 placed in a anaerobic chamber (Forma Scienti¢c, Marietta, OH, USA). Abiotic controls were inoculated with autoclaved sediment (121‡C, 20 min). After degradation of DPU was observed, subsamples (10% vol.) were transferred to fresh DPU-containing MM medium. After three transfers the enrichment culture was plated on MMA containing 20 mg l31 DPU. Following incubation for 2 weeks a DPU-degrading isolate (designated strain DPUZ) was isolated. Strain DPUZ was passed from DPU-containing MM to R2A-salt three times to ensure purity. 2.3. Characterization of strain DPUZ A stock of strain DPUZ was maintained at 380‡C in 40% glycerol. Before each experiment, strain DPUZ was thawed and grown in 250-ml Erlenmeyer £asks with 100 ml RM on a platform shaker (180 rpm) at 30‡C. Cells were harvested in the late exponential growth phase by centrifugation (10 min, 3500Ug), washed twice in MM and inoculated to a ¢nal density of 1U106 cells ml31 . Temperature and salinity ranges for growth of strain DPUZ in RM were determined. Optimal salinity was estimated by changing the NaCl concentration from 10.0 to 150.0 g l31 (0.17^2.57 M). Growth was measured at temperatures from 20.0 to 45.0‡C ( R 0.1‡C) in a Termaks incubator (model KBP 6395, Lytzen Lab, Denmark). Cell density was monitored by optical density measurements (600 nm) (Model 8452A Spectrophotometer, Hewlett-Packard, Palo Alto, CA, USA) during incubation for
up to 10 days. Speci¢c growth rates were calculated from a minimum of four data points measured in the exponential growth phase. An identi¢cation of strain DPUZ based on fatty acid analysis, partial sequencing of the 16S rRNA gene (477 bp) and di¡erent physiological tests were performed by Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). The substrate utilization pattern was tested in MM. Alignment of the partial 16S rRNA sequence was performed with sequences from the GenBank Database (National Center for Biotechnology Information, USA) using the BLAST algorithm [9]. The partial 16S rRNA sequence of strain DPUZ has been deposited in the GenBank Database under the accession no. AF461181, and the strain has been deposited at the Institute Pasteur Collection (Paris, France) under the number CIP107463. 2.4. Metabolism of DPU Degradation was estimated in aliquots of sediment slurries or culture media by a reverse-phase high-performance liquid chromatograph (HPLC) equipped with a diode array detector (model DAD-400, Kontron Instruments, Milan, Italy) and a Supelco LC-18 column (Supelco, Bellfonte, PA, USA) with a mixture of 65% methanol and 35% ammonium acetate bu¡er (18 mM) as the mobile phase (£ow rate 1.5 ml min31 ). Prior to analysis the aliquots were diluted 1:1 with DMSO to ensure dissolution of DPU crystals, and centrifuged (10 min, 14 154Ug) before HPLC analysis of the supernatant. To identify a DPU metabolite detected by HPLC the culture medium was analyzed by gas chromatography^mass spectrometry (GC^MS) (Magnum, Finnigan Mat, San Jose, CA, USA). Compounds were separated using a DB-5 column (30 mU0.32 mm, inside diameter of 0.25 Wm) with an initial temperature of 50‡C for 10 min increased to 280‡C at a rate of 3‡C min31 . Identi¢cation was made by comparison with a GC^MS spectrum obtained with aniline (99.8% purity, Acros Organics, NJ, USA).
3. Results 3.1. Enrichment and isolation Sediment was inoculated into a DPU-containing mineral medium and incubated under oxic and anoxic conditions. Degradation of DPU was observed in oxic slurries after 40 days. No degradation was observed in the anoxic slurries or in the autoclaved controls within 4 months (data not shown). Oxic DPU-degrading slurries were subcultured several times by transferring aliquots to fresh DPU-containing MM before plating on MMA. Following 2 weeks of incubation a DPU-degrading isolate was obtained (designated strain DPUZ).
FEMSLE 10572 5-8-02
S.R. S6rensen et al. / FEMS Microbiology Letters 213 (2002) 199^204
201
3.2. Characterization and identi¢cation of strain DPUZ Alignment of a partial 16S rRNA gene sequence (477 bp) from strain DPUZ showed 100% similarity to the type strain of Marinobacter hydrocarbonoclasticus [10]. A whole-cell fatty acid pro¢le showed 26.5% 18:1g9c, 23.6% 16:0, 11.2% 16:1g9c, 10.1% 12:0 3OH and 5.5% 12:0 as the dominant fatty acids, furthermore indicating that strain DPUZ is related to M. hydrocarbonoclasticus [11]. Di¡erent physiological tests con¢rmed these results, however, the substrate utilization pattern di¡ers on glucose, adipate, citrate, gluconate, and hydroxybenzoate (Table 1). Besides the substrates presented in Table 1 strain DPUZ utilized n-octanoic acid and malate and was unable to utilize n-decanoic acid, phenylacetate, Lphenylalanine, glycine betaine, ethanolamine. Growth on agar was restricted to R2A-salt and MMA (both contained 30 g l31 NaCl). No growth was observed in R2A, Bacto nutrient agar (Difco, Detroit, MI, USA) or Luria^ Bertani agar made according to the manufacturer’s instructions. When grown on R2A-salt strain DPUZ formed white colonies within 24 h at 30‡C. Table 1 Phenotypic characteristics of M. hydrocarbonoclasticus (type strain) and Marinobacter sp. strain DPUZ Characterization
Strain DPUZ
M. hydrocarbonoclasticus ATCC 49840a
Gram reaction Shape of cells Width (Wm) Length (Wm) Motility Flagella Oxidase Catalase Urease NO2 from NO3 Hydrolysis of: Gelatin Esculin Starch Tween 80 Utilization as carbon source: D-Glucose D-Mannose D-Arabinose D-Fructose Adipate Mannitol Lactose Citrate Acetate D-Gluconate N-acetylglucosamine Succinate L-Glutamate L-Proline p-Hydroxybenzoate
3 rods 0.5^0.6 1.8^3.0 + 1, polar + + 3 +
3 rods 0.3^0.6 2.0^3.0 + 1, polar + + 3 +
3 3 + +
3 3 3 +
+ 3 3 3 3 3 3 3 + + 3 + + + 3
3 3 3 3 + 3 3 + + 3 3 + + + +
a
Data from [10].
Fig. 1. E¡ect of temperature (A) (NaCl 30 g l31 , pH 7.5) and salinity (B) (35‡C, pH 7.5) on the speci¢c growth rate of Marinobacter sp. strain DPUZ. Data are mean values (n = 3). The bars indicate the standard deviation.
Fast and exponential growth of strain DPUZ to an optical density of 0.2 (600 nm) was observed in RM, and growth data from the exponential phase were used to calculate speci¢c growth rates under various temperatures and NaCl concentrations (Fig. 1A,B). Growth was observed at temperatures from 20 to 40‡C with an optimum at 35‡C (NaCl 30 g l31 ) (Fig. 1A). The strain grows at NaCl concentrations ranging from 20 to 130 g l31 with an optimum at 30 g l31 (Fig. 1B). No growth was observed at 150 g l31 NaCl within 10 days, however viability was maintained, as shown by a rapid growth after transfer of subsamples to RM with 30 g l31 NaCl. In contrast, no viable cells were recovered after inoculation in RM without NaCl, either by plating on R2A-salt or transfer of subsamples to fresh RM with 30 g l31 NaCl. Phase-contrast microscopy strongly indicated that strain DPUZ lysed by this treatment. The speci¢c growth rate under optimal conditions in RM (35‡C, NaCl 30 g l31 ) was 0.33 R 0.03 h31 giving a doubling time of 2.13 R 0.20 h (n = 3). Growth of strain DPUZ was con¢rmed at pH 7.0^12.0, however precipitation of components in MM disrupted exact growth measurements and the pH range for strain DPUZ remains unknown. The results however indicate that strain DPUZ had optimum growth at pH 7.5 (data not shown).
FEMSLE 10572 5-8-02
202
S.R. S6rensen et al. / FEMS Microbiology Letters 213 (2002) 199^204
( R 0.38) mg day31 , 7.17 ( R 0.99) mg day31 and 6.61 ( R 0.42) mg day31 (n = 3) were calculated by linear regression (R2 = 0.93^0.98) for DPU concentrations of 100, 50, and 25 mg l31 , respectively. No exponential growth was observed in DPU-supplemented MM medium (Fig. 2B). At day 9 growth of strain DPUZ corresponding to 3.7 ( R 1.1)U106 , 4.2 ( R 1.6)U106 and 4.0 ( R 0.7)U106 cells ml31 (n = 3) were observed with initial DPU concentrations of 100, 50, and 25 mg l31 , respectively (Fig. 2B). In comparison 1.1 ( R 0.2)U106 cells ml31 were detected in controls containing only DMSO (Fig. 2B). Complete dissipation of DPU (100 mg l31 ) within 24 h was observed under optimal conditions when strain DPUZ was grown in RM supplemented with DPU (data not shown). HPLC analysis of culture media during DPU metabolism by strain DPUZ revealed transient accumulation of a metabolite in trace amount. This metabolite was subsequently identi¢ed as aniline by GC^MS analysis (Fig. 3).
4. Discussion
Fig. 2. Metabolism of 1,3-diphenylurea (DPU) by Marinobacter sp. strain DPUZ. A: Degradation of DPU (inset) in concentrations of 100 (R), 50 (b) and 25 mg l31 (F) by Marinobacter sp. DPUZ or in uninoculated controls (a). B: Growth of Marinobacter sp. DPUZ measured by plating during metabolism of 100, 50 or 25 mg l31 DPU (R, b, F) and in controls provided DMSO without DPU (a). Data are mean values (n = 3). The bars indicate the standard deviation.
3.3. Metabolism of DPU by strain DPUZ A constant DPU degradation rate by strain DPUZ was observed in MM (Fig. 2A), and degradation rates of 8.09
Biodegradation of DPU was observed in sediment sampled from an ephemeral desert stream contaminated with industrial wastewater. The stream is located above a fractured chalk aquifer with some fractures exposed directly to the stream bed [7], potentially leading to a fast and widespread transport of contaminants to the groundwater. Inoculating sediment into a mineral medium containing DPU as sole carbon and nitrogen source leads to the enrichment and isolation of a Marinobacter sp. Marinobacter spp. are ubiquitous in the marine environment [12] and several strains with broad catabolic capacities have been enriched from coastal sediments [10,13^15], the deep-sea £oor [12,16] and the ballast water of a tanker ship [17]. Recently a Marinobacter sp. (designated strain
Fig. 3. Mass spectrum of metabolite occurring during the metabolism of diphenylurea by Marinobacter sp. DPUZ. The spectrum is identical to that of aniline (inset).
FEMSLE 10572 5-8-02
S.R. S6rensen et al. / FEMS Microbiology Letters 213 (2002) 199^204
MB) was isolated from a hypersaline lake in the Sinai desert in Egypt [18], which together with our results indicates that Marinobacter spp. are more than strictly marine bacteria. Within the genus Marinobacter two species have so far been described, M. hydrocarbonoclasticus [10] and M. aquaeolei [14]. A third Marinobacter species has been suggested, ‘M. arcticus’ [17], however this name has never been validated. Sequencing of the 16S rRNA gene and fatty acid analysis indicate that strain DPUZ is a M. hydrocarbonoclasticus or alternatively represents a member of a closely related species. No experiments were performed with the type strain of M. hydrocarbonoclasticus in this study, and it remains to be elucidated whether DPU metabolism is a common feature of M. hydrocarbonoclasticus and closely related strains. The ephemeral desert stream from where strain DPUZ was isolated has £uctuating salinity, ranging from the dry stream bed with salt precipitated on the exposed sediment, to the short winter season where the stream is £owing and the salinity is reduced. Sustaining activity in this changing desert environment requires the ability to survive and proliferate within a range of environmental conditions. Several Marinobacter spp. tolerate a wide range of NaCl concentrations (e.g. [13,14,16]) in contrast to the more narrow salt range generally described for marine bacteria [19]. The broad salt range suggests that adaptation to changing saline conditions is a characteristic feature of the genus Marinobacter. The type strain of M. hydrocarbonoclasticus isolated from marine coastal sediment [10] grows at NaCl concentrations from 0.1 to 3.5 M with an optimum at 0.6 M. Strain DPUZ grows in a slightly narrower range of NaCl concentrations and with a lower optimum (0.2^ 2.6 M with an optimum at 0.51 M). Strain DPUZ can be categorized as a moderate halophilic bacterium [19] due to the broad range of NaCl concentrations and the absolute requirement for NaCl. Lysis of cells in the absence of Naþ ions has been described for M. hydrocarbonoclasticus [10] and another closely related strain [16]. A similar requirement might explain the lysis of strain DPUZ observed without NaCl in the media. The Marinobacter sp. strain MB isolated from the Sinai desert was also closely related to M. hydrocarbonoclasticus based on 16S rRNA similarities, however strain MB did not require NaCl for growth [18] in contrast to both strain DPUZ and the type strain of M. hydrocarbonoclasticus. The occurrence of aniline-based metabolites as endproducts or intermediates during bacterial metabolism of phenylurea or related compounds has previously been described [1,2,4^6]. The GC^MS analysis of culture liquid indicates that the metabolism of DPU by strain DPUZ involves cleavage of the urea bridge between the phenyl structures giving rise to aniline. Drzyzga and Blotevogel [2] constructed a coculture consisting of a Desulfovibrio sp. co-metabolically transforming diphenylamine to aniline, and a Desulfobacterium anilini utilizing this metabolite resulting in complete mineralization of diphenylamine. Mi-
203
crobial communities may readily adapt to degradation of aniline, and several aniline-degrading bacteria have been isolated from various environments (e.g. [20,21]). Strain DPUZ degraded aniline further and no DPU, aniline or unidenti¢ed peaks were measured at the end of the experiments. The production of approximately the same amount of cells without exponential growth after metabolism of 25^100 mg l31 DPU (Fig. 2) and the fact that DPU degradation was stimulated by casamino acids strongly indicate that strain DPUZ has unknown requirements limiting the growth in DPU-supplemented MM. In conclusion, we isolated a moderate halophilic Marinobacter sp. able to metabolize DPU from a desert stream bed in the Negev Desert. The metabolism of DPU involves cleavage of the urea bridge giving rise to intermediate occurrence of aniline, which subsequently was further degraded.
Acknowledgements This study was supported by the EC Environment and Climate Research Programme (Contract: ENV4-CT970441), a bilateral Danish^Israeli Project (FRACFLUX) and a research fellowship awarded to S.R.S. from the EC (Human Potential Programme). We thank Patricia Simpson for helpful comments on the manuscript.
References [1] Drzyzga, O., Schmidt, A. and Blotevogel, K.-H. (1996) Cometabolic transformation and cleavage of nitrodiphenylamines by three newly isolated sulfate-reducing bacterial strains. Appl. Environ. Microbiol. 62, 1710^1716. [2] Drzyzga, O. and Blotevogel, K.-H. (1997) Microbial degradation of diphenylamine under anoxic conditions. Curr. Microbiol. 35, 343^ 347. [3] Stangroom, S.J., Collins, C.D. and Lester, J.N. (1999) Sources of organic micropollutants to lowland rivers. Environ. Technol. 19, 643^666. [4] Cullington, J.E. and Walker, A. (1999) Rapid biodegradation of diuron and other phenylurea herbicides by a soil bacterium. Soil Biol. Biochem. 31, 677^686. [5] Srensen, S.R., Ronen, Z. and Aamand, J. (2001) Isolation from agricultural soil and characterization of a Sphingomonas sp. able to mineralize the phenylurea herbicide isoproturon. Appl. Environ. Microbiol. 67, 5403^5409. [6] Gardner, A.M., Alvarez, G.H. and Ku, Y. (1982) Microbial degradation of 14 C-diphenylamine in a laboratory model sewage sludge system. Bull. Environ. Contam. Toxicol. 28, 91^96. [7] Nativ, R., Adar, E.M. and Becker, A. (1999) Designing a monitoring network for contaminated groundwater in fractured chalk. Ground Water 37, 38^47. [8] Ronen, Z. and Abeliovich, A. (2000) Anaerobic^aerobic process for microbial degradation of tetrabromobisphenol A. Appl. Environ. Microbiol. 66, 2373^2377. [9] Altschul, S.F., Madden, T.L., Scha«¡er, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSIBLAST : a new generation of protein database search programs. Nucleic Acids Res. 25, 3389^3402.
FEMSLE 10572 5-8-02
204
S.R. S6rensen et al. / FEMS Microbiology Letters 213 (2002) 199^204
[10] Gauthier, M.J., Lafay, B., Christen, R., Fernandez, L., Acquaviva, M., Bonin, P. and Betrand, J.-C. (1992) Marinobacter hydrocarbonoclasticus gen. nov., sp. nov., a new, extremely halotolerant, hydrocarbon-degrading marine bacterium. Int. J. Syst. Bacteriol. 42, 568^ 576. [11] Spro«er, C., Lang, E., Hobeck, P., Burghardt, J., Stackebrandt, E. and Tindall, B.J. (1998) Transfer of Pseudomonas nautica to Marinobacter hydrocarbonoclasticus. Int. J. Syst. Bacteriol. 42, 568^576. [12] Kaye, J.Z. and Baross, J.A. (2000) High incidence of halotolerant bacteria in Paci¢c hydrothermal-vent and pelagic environments. FEMS Microbiol. Ecol. 32, 249^260. [13] Rontani, J.-F., Gilewicz, M.J., Michotey, V.D., Zheng, T.L., Bonin, P.C. and Bertrand, J.-C. (1997) Aerobic and anaerobic metabolism of 6,10,14-trimethylpentadecan-2-one by a denitrifying bacterium isolated from marine sediments. Appl. Environ. Microbiol. 63, 636^643. [14] Huu, N.B., Denner, E.B.M., Ha, D.T.C., Wanner, G. and Stan-Lotter, H. (1999) Marinobacter aquaeolei sp. nov., a halophilic bacterium from a Vietnamese oil-producing well. Int. J. Syst. Bacteriol. 42, 568^ 576. [15] Hedlund, B.P., Geiselbrecht, A.D. and Staley, J.T. (2001) Marinobacter strain NCE312 has a Pseudomonas-like naphthalene dioxygenase. FEMS Microbiol. Lett. 201, 47^51.
[16] Kasuya, K., Mitomo, H., Nakahara, M., Akiba, A., Kudo, T. and Doi, Y. (2000) Identi¢cation of a marine benthic P(3HB)-degrading bacterium isolate and characterization of its P(3HB) depolymerase. Biomacromolecules 1, 194^201. [17] Button, D.K., Robertson, B.R., Lepp, P.W. and Schmidt, T.M. (1998) A small, dilute-cytoplasm, high-a⁄nity, novel bacterium isolated by extinction culture and having kinetic constants compatible with growth at ambient concentrations of dissolved nutrients in seawater. Appl. Environ. Microbiol. 64, 4467^4476. [18] Sigalevich, P., Baev, M.V., Teske, A. and Cohen, Y. (2000) Sulfate reduction and possible aerobic metabolism of the sulfate-reducing bacterium Desulfovibrio oxyclinae in a coculture with Marinobacter sp. strain MB in an aerated sulfate-depleted chemostat. Appl. Environ. Microbiol. 66, 5019^5023. [19] Larsen, H. (1986) Halophilic and halotolerant microorganisms ^ an overview and historical perspective. FEMS Microbiol. Rev. 39, 3^7. [20] Gheewala, S.H. and Annachhatre, A.P. (1997) Biodegradation of aniline. Wat. Sci. Tech. 36, 53^63. [21] Konopka, A. (1993) Isolation and characterization of a subsurface bacterium that degrades aniline and methylanilines. FEMS Microbiol. Lett. 111, 93^100.
FEMSLE 10572 5-8-02
www.fems-microbiology.org
Metabolism of diphenylurea by a Marinobacter sp. isolated from a contaminated ephemeral stream bed in the Negev Desert Sebastian R. Srensen a
a;
, Ziv Arbeli
b;1
, Jens Aamand a , Zeev Ronen
b
Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS), Copenhagen, ster Voldgade 10, DK-1350 Copenhagen K, Denmark b Department of Environmental Hydrology and Microbiology, Ben Gurion University of the Negev, Jacob Blaustein Institute for Desert Research, Sede-Boker, Israel Received 8 February 2002; received in revised form 11 June 2002 ; accepted 12 June 2002 First published online 12 July 2002
Abstract A moderate halophilic Marinobacter sp. (designated strain DPUZ) able to metabolize 1,3-diphenylurea (DPU) was isolated from a contaminated ephemeral desert stream bed near an industrial complex in the northern part of the Negev Desert (Israel). Metabolism of DPU was accompanied by a transient accumulation of a metabolite identified as aniline using gas chromatography^mass spectrometry, thus indicating a metabolic pathway involving cleavage of the urea bridge between the phenyl structures. Aniline was further degraded without detection of other metabolites suggesting a complete degradation. Strain DPUZ grows at NaCl concentrations between 0.2 and 2.6 M with an optimum at 0.51 M. It grows at a temperature range between 20 and 40‡C with an optimum at 35‡C. This is the first study on bacterial metabolism of DPU. 7 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Diphenylurea ; Halophilic; Desert stream; Biodegradation ; Marinobacter
1. Introduction As a consequence of agricultural practice and accidental spillage during production of chemicals such as propellants, various phenylurea compounds are detected as environmental pollutants [1^3]. Studies on the environmental fate of phenylurea compounds have mainly been focused on phenylurea herbicides [4,5], and little is known about the fate of diphenylurea compounds in contaminated environments. For related compounds such as diphenylamine, biodegradation is the predominant mechanism for dissipation from environments such as sewage sludge [6] and marine sediments [1,2]. The aim of this study was to isolate and characterize diphenylurea-degrading organisms from a contaminated desert environment, choosing 1,3-diphenylurea (DPU) as a representative model compound.
* Corresponding author. Tel.: +45 (3814) 2317; Fax: +45 (3814) 2050. E-mail address : [email protected] (S.R. Srensen). 1 Present address: Laboratorio Ambiental, Instituto de Biotecnolog|¤a ^ Edi¢cio Manuel Ancizar, Universidad Nacional de Colombia, Bogota¤, Colombia.
By inoculating sediment from a ephemeral desert stream bed into a mineral medium provided DPU as sole source of carbon, nitrogen and energy, we enriched and isolated a Marinobacter sp. The stream was contaminated with industrial wastewater and located in the vicinity of an industrial area located in the northern part of the Negev Desert in Israel. Among the most abundant contaminants of the wastewater were various diphenylurea compounds including DPU. This is the ¢rst isolation of a DPU-degrading bacterium and attempt to elucidate the metabolic pathway for DPU.
2. Materials and methods 2.1. Media A carbon- and nitrogen-free medium (MM) was used for the enrichment and degradation studies. MM contained (l31 ): 0.5 g KH2 PO4 , 1.0 g Na2 HPO4 W2H2 O and 30 g NaCl. pH was adjusted to 7.5 before autoclaving (121‡C, 20 min). After cooling 2.5 ml of a ¢lter-sterilized MgSO4 W7H2 O solution (200 g l31 ) was added. DPU ( 6 100 mg l31 water solubility at 18‡C, purity s 98%,
0378-1097 / 02 / $22.00 7 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 8 2 0 - 0
FEMSLE 10572 5-8-02
200
S.R. S6rensen et al. / FEMS Microbiology Letters 213 (2002) 199^204
Sigma Chemicals, St. Louis, MO, USA) (Fig. 2A, inset) was added to the media from a stock solution (0.1 g ml31 ) in dimethylsulfoxide (DMSO). In all experiments with DPU in liquid media (20^100 mg l31 ) DPU crystals were initially visible in the media. A substrate rich medium (RM) was made by adding 1.0 g l31 casamino acids (Difco, Detroit, MI, USA) to the MM medium. A DPU-containing (20 mg l31 ) MM-based agar (MMA) was prepared by adding 15 g l31 Bacto Agar (Difco, Detroit, MI, USA), and DPU was added to the medium just before preparation of the plates. R2A (Remel, Lenexa, KS, USA) supplemented with 30 g l31 NaCl (R2A-salt) was used for enumeration of cells. 2.2. Enrichment and isolation Sediment was sampled from a small stream located in the vicinity of an industrial area in the northern part of the Negev Desert (Israel) [7,8]. Sediments from three different locations approximately 500 m apart were sampled from the wet part of the stream bed in July 1999. An amount of 5 g wet sediment was inoculated into 50 ml MM with 100 mg l31 DPU and incubated at 25‡C under either oxic or anoxic conditions. Oxic experiments were preformed in 250-ml Erlenmeyer £asks, and the anoxic experiments in 160-ml rubber-sealed serum bottles with a gas phase of 94% N2 and 6% H2 placed in a anaerobic chamber (Forma Scienti¢c, Marietta, OH, USA). Abiotic controls were inoculated with autoclaved sediment (121‡C, 20 min). After degradation of DPU was observed, subsamples (10% vol.) were transferred to fresh DPU-containing MM medium. After three transfers the enrichment culture was plated on MMA containing 20 mg l31 DPU. Following incubation for 2 weeks a DPU-degrading isolate (designated strain DPUZ) was isolated. Strain DPUZ was passed from DPU-containing MM to R2A-salt three times to ensure purity. 2.3. Characterization of strain DPUZ A stock of strain DPUZ was maintained at 380‡C in 40% glycerol. Before each experiment, strain DPUZ was thawed and grown in 250-ml Erlenmeyer £asks with 100 ml RM on a platform shaker (180 rpm) at 30‡C. Cells were harvested in the late exponential growth phase by centrifugation (10 min, 3500Ug), washed twice in MM and inoculated to a ¢nal density of 1U106 cells ml31 . Temperature and salinity ranges for growth of strain DPUZ in RM were determined. Optimal salinity was estimated by changing the NaCl concentration from 10.0 to 150.0 g l31 (0.17^2.57 M). Growth was measured at temperatures from 20.0 to 45.0‡C ( R 0.1‡C) in a Termaks incubator (model KBP 6395, Lytzen Lab, Denmark). Cell density was monitored by optical density measurements (600 nm) (Model 8452A Spectrophotometer, Hewlett-Packard, Palo Alto, CA, USA) during incubation for
up to 10 days. Speci¢c growth rates were calculated from a minimum of four data points measured in the exponential growth phase. An identi¢cation of strain DPUZ based on fatty acid analysis, partial sequencing of the 16S rRNA gene (477 bp) and di¡erent physiological tests were performed by Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). The substrate utilization pattern was tested in MM. Alignment of the partial 16S rRNA sequence was performed with sequences from the GenBank Database (National Center for Biotechnology Information, USA) using the BLAST algorithm [9]. The partial 16S rRNA sequence of strain DPUZ has been deposited in the GenBank Database under the accession no. AF461181, and the strain has been deposited at the Institute Pasteur Collection (Paris, France) under the number CIP107463. 2.4. Metabolism of DPU Degradation was estimated in aliquots of sediment slurries or culture media by a reverse-phase high-performance liquid chromatograph (HPLC) equipped with a diode array detector (model DAD-400, Kontron Instruments, Milan, Italy) and a Supelco LC-18 column (Supelco, Bellfonte, PA, USA) with a mixture of 65% methanol and 35% ammonium acetate bu¡er (18 mM) as the mobile phase (£ow rate 1.5 ml min31 ). Prior to analysis the aliquots were diluted 1:1 with DMSO to ensure dissolution of DPU crystals, and centrifuged (10 min, 14 154Ug) before HPLC analysis of the supernatant. To identify a DPU metabolite detected by HPLC the culture medium was analyzed by gas chromatography^mass spectrometry (GC^MS) (Magnum, Finnigan Mat, San Jose, CA, USA). Compounds were separated using a DB-5 column (30 mU0.32 mm, inside diameter of 0.25 Wm) with an initial temperature of 50‡C for 10 min increased to 280‡C at a rate of 3‡C min31 . Identi¢cation was made by comparison with a GC^MS spectrum obtained with aniline (99.8% purity, Acros Organics, NJ, USA).
3. Results 3.1. Enrichment and isolation Sediment was inoculated into a DPU-containing mineral medium and incubated under oxic and anoxic conditions. Degradation of DPU was observed in oxic slurries after 40 days. No degradation was observed in the anoxic slurries or in the autoclaved controls within 4 months (data not shown). Oxic DPU-degrading slurries were subcultured several times by transferring aliquots to fresh DPU-containing MM before plating on MMA. Following 2 weeks of incubation a DPU-degrading isolate was obtained (designated strain DPUZ).
FEMSLE 10572 5-8-02
S.R. S6rensen et al. / FEMS Microbiology Letters 213 (2002) 199^204
201
3.2. Characterization and identi¢cation of strain DPUZ Alignment of a partial 16S rRNA gene sequence (477 bp) from strain DPUZ showed 100% similarity to the type strain of Marinobacter hydrocarbonoclasticus [10]. A whole-cell fatty acid pro¢le showed 26.5% 18:1g9c, 23.6% 16:0, 11.2% 16:1g9c, 10.1% 12:0 3OH and 5.5% 12:0 as the dominant fatty acids, furthermore indicating that strain DPUZ is related to M. hydrocarbonoclasticus [11]. Di¡erent physiological tests con¢rmed these results, however, the substrate utilization pattern di¡ers on glucose, adipate, citrate, gluconate, and hydroxybenzoate (Table 1). Besides the substrates presented in Table 1 strain DPUZ utilized n-octanoic acid and malate and was unable to utilize n-decanoic acid, phenylacetate, Lphenylalanine, glycine betaine, ethanolamine. Growth on agar was restricted to R2A-salt and MMA (both contained 30 g l31 NaCl). No growth was observed in R2A, Bacto nutrient agar (Difco, Detroit, MI, USA) or Luria^ Bertani agar made according to the manufacturer’s instructions. When grown on R2A-salt strain DPUZ formed white colonies within 24 h at 30‡C. Table 1 Phenotypic characteristics of M. hydrocarbonoclasticus (type strain) and Marinobacter sp. strain DPUZ Characterization
Strain DPUZ
M. hydrocarbonoclasticus ATCC 49840a
Gram reaction Shape of cells Width (Wm) Length (Wm) Motility Flagella Oxidase Catalase Urease NO2 from NO3 Hydrolysis of: Gelatin Esculin Starch Tween 80 Utilization as carbon source: D-Glucose D-Mannose D-Arabinose D-Fructose Adipate Mannitol Lactose Citrate Acetate D-Gluconate N-acetylglucosamine Succinate L-Glutamate L-Proline p-Hydroxybenzoate
3 rods 0.5^0.6 1.8^3.0 + 1, polar + + 3 +
3 rods 0.3^0.6 2.0^3.0 + 1, polar + + 3 +
3 3 + +
3 3 3 +
+ 3 3 3 3 3 3 3 + + 3 + + + 3
3 3 3 3 + 3 3 + + 3 3 + + + +
a
Data from [10].
Fig. 1. E¡ect of temperature (A) (NaCl 30 g l31 , pH 7.5) and salinity (B) (35‡C, pH 7.5) on the speci¢c growth rate of Marinobacter sp. strain DPUZ. Data are mean values (n = 3). The bars indicate the standard deviation.
Fast and exponential growth of strain DPUZ to an optical density of 0.2 (600 nm) was observed in RM, and growth data from the exponential phase were used to calculate speci¢c growth rates under various temperatures and NaCl concentrations (Fig. 1A,B). Growth was observed at temperatures from 20 to 40‡C with an optimum at 35‡C (NaCl 30 g l31 ) (Fig. 1A). The strain grows at NaCl concentrations ranging from 20 to 130 g l31 with an optimum at 30 g l31 (Fig. 1B). No growth was observed at 150 g l31 NaCl within 10 days, however viability was maintained, as shown by a rapid growth after transfer of subsamples to RM with 30 g l31 NaCl. In contrast, no viable cells were recovered after inoculation in RM without NaCl, either by plating on R2A-salt or transfer of subsamples to fresh RM with 30 g l31 NaCl. Phase-contrast microscopy strongly indicated that strain DPUZ lysed by this treatment. The speci¢c growth rate under optimal conditions in RM (35‡C, NaCl 30 g l31 ) was 0.33 R 0.03 h31 giving a doubling time of 2.13 R 0.20 h (n = 3). Growth of strain DPUZ was con¢rmed at pH 7.0^12.0, however precipitation of components in MM disrupted exact growth measurements and the pH range for strain DPUZ remains unknown. The results however indicate that strain DPUZ had optimum growth at pH 7.5 (data not shown).
FEMSLE 10572 5-8-02
202
S.R. S6rensen et al. / FEMS Microbiology Letters 213 (2002) 199^204
( R 0.38) mg day31 , 7.17 ( R 0.99) mg day31 and 6.61 ( R 0.42) mg day31 (n = 3) were calculated by linear regression (R2 = 0.93^0.98) for DPU concentrations of 100, 50, and 25 mg l31 , respectively. No exponential growth was observed in DPU-supplemented MM medium (Fig. 2B). At day 9 growth of strain DPUZ corresponding to 3.7 ( R 1.1)U106 , 4.2 ( R 1.6)U106 and 4.0 ( R 0.7)U106 cells ml31 (n = 3) were observed with initial DPU concentrations of 100, 50, and 25 mg l31 , respectively (Fig. 2B). In comparison 1.1 ( R 0.2)U106 cells ml31 were detected in controls containing only DMSO (Fig. 2B). Complete dissipation of DPU (100 mg l31 ) within 24 h was observed under optimal conditions when strain DPUZ was grown in RM supplemented with DPU (data not shown). HPLC analysis of culture media during DPU metabolism by strain DPUZ revealed transient accumulation of a metabolite in trace amount. This metabolite was subsequently identi¢ed as aniline by GC^MS analysis (Fig. 3).
4. Discussion
Fig. 2. Metabolism of 1,3-diphenylurea (DPU) by Marinobacter sp. strain DPUZ. A: Degradation of DPU (inset) in concentrations of 100 (R), 50 (b) and 25 mg l31 (F) by Marinobacter sp. DPUZ or in uninoculated controls (a). B: Growth of Marinobacter sp. DPUZ measured by plating during metabolism of 100, 50 or 25 mg l31 DPU (R, b, F) and in controls provided DMSO without DPU (a). Data are mean values (n = 3). The bars indicate the standard deviation.
3.3. Metabolism of DPU by strain DPUZ A constant DPU degradation rate by strain DPUZ was observed in MM (Fig. 2A), and degradation rates of 8.09
Biodegradation of DPU was observed in sediment sampled from an ephemeral desert stream contaminated with industrial wastewater. The stream is located above a fractured chalk aquifer with some fractures exposed directly to the stream bed [7], potentially leading to a fast and widespread transport of contaminants to the groundwater. Inoculating sediment into a mineral medium containing DPU as sole carbon and nitrogen source leads to the enrichment and isolation of a Marinobacter sp. Marinobacter spp. are ubiquitous in the marine environment [12] and several strains with broad catabolic capacities have been enriched from coastal sediments [10,13^15], the deep-sea £oor [12,16] and the ballast water of a tanker ship [17]. Recently a Marinobacter sp. (designated strain
Fig. 3. Mass spectrum of metabolite occurring during the metabolism of diphenylurea by Marinobacter sp. DPUZ. The spectrum is identical to that of aniline (inset).
FEMSLE 10572 5-8-02
S.R. S6rensen et al. / FEMS Microbiology Letters 213 (2002) 199^204
MB) was isolated from a hypersaline lake in the Sinai desert in Egypt [18], which together with our results indicates that Marinobacter spp. are more than strictly marine bacteria. Within the genus Marinobacter two species have so far been described, M. hydrocarbonoclasticus [10] and M. aquaeolei [14]. A third Marinobacter species has been suggested, ‘M. arcticus’ [17], however this name has never been validated. Sequencing of the 16S rRNA gene and fatty acid analysis indicate that strain DPUZ is a M. hydrocarbonoclasticus or alternatively represents a member of a closely related species. No experiments were performed with the type strain of M. hydrocarbonoclasticus in this study, and it remains to be elucidated whether DPU metabolism is a common feature of M. hydrocarbonoclasticus and closely related strains. The ephemeral desert stream from where strain DPUZ was isolated has £uctuating salinity, ranging from the dry stream bed with salt precipitated on the exposed sediment, to the short winter season where the stream is £owing and the salinity is reduced. Sustaining activity in this changing desert environment requires the ability to survive and proliferate within a range of environmental conditions. Several Marinobacter spp. tolerate a wide range of NaCl concentrations (e.g. [13,14,16]) in contrast to the more narrow salt range generally described for marine bacteria [19]. The broad salt range suggests that adaptation to changing saline conditions is a characteristic feature of the genus Marinobacter. The type strain of M. hydrocarbonoclasticus isolated from marine coastal sediment [10] grows at NaCl concentrations from 0.1 to 3.5 M with an optimum at 0.6 M. Strain DPUZ grows in a slightly narrower range of NaCl concentrations and with a lower optimum (0.2^ 2.6 M with an optimum at 0.51 M). Strain DPUZ can be categorized as a moderate halophilic bacterium [19] due to the broad range of NaCl concentrations and the absolute requirement for NaCl. Lysis of cells in the absence of Naþ ions has been described for M. hydrocarbonoclasticus [10] and another closely related strain [16]. A similar requirement might explain the lysis of strain DPUZ observed without NaCl in the media. The Marinobacter sp. strain MB isolated from the Sinai desert was also closely related to M. hydrocarbonoclasticus based on 16S rRNA similarities, however strain MB did not require NaCl for growth [18] in contrast to both strain DPUZ and the type strain of M. hydrocarbonoclasticus. The occurrence of aniline-based metabolites as endproducts or intermediates during bacterial metabolism of phenylurea or related compounds has previously been described [1,2,4^6]. The GC^MS analysis of culture liquid indicates that the metabolism of DPU by strain DPUZ involves cleavage of the urea bridge between the phenyl structures giving rise to aniline. Drzyzga and Blotevogel [2] constructed a coculture consisting of a Desulfovibrio sp. co-metabolically transforming diphenylamine to aniline, and a Desulfobacterium anilini utilizing this metabolite resulting in complete mineralization of diphenylamine. Mi-
203
crobial communities may readily adapt to degradation of aniline, and several aniline-degrading bacteria have been isolated from various environments (e.g. [20,21]). Strain DPUZ degraded aniline further and no DPU, aniline or unidenti¢ed peaks were measured at the end of the experiments. The production of approximately the same amount of cells without exponential growth after metabolism of 25^100 mg l31 DPU (Fig. 2) and the fact that DPU degradation was stimulated by casamino acids strongly indicate that strain DPUZ has unknown requirements limiting the growth in DPU-supplemented MM. In conclusion, we isolated a moderate halophilic Marinobacter sp. able to metabolize DPU from a desert stream bed in the Negev Desert. The metabolism of DPU involves cleavage of the urea bridge giving rise to intermediate occurrence of aniline, which subsequently was further degraded.
Acknowledgements This study was supported by the EC Environment and Climate Research Programme (Contract: ENV4-CT970441), a bilateral Danish^Israeli Project (FRACFLUX) and a research fellowship awarded to S.R.S. from the EC (Human Potential Programme). We thank Patricia Simpson for helpful comments on the manuscript.
References [1] Drzyzga, O., Schmidt, A. and Blotevogel, K.-H. (1996) Cometabolic transformation and cleavage of nitrodiphenylamines by three newly isolated sulfate-reducing bacterial strains. Appl. Environ. Microbiol. 62, 1710^1716. [2] Drzyzga, O. and Blotevogel, K.-H. (1997) Microbial degradation of diphenylamine under anoxic conditions. Curr. Microbiol. 35, 343^ 347. [3] Stangroom, S.J., Collins, C.D. and Lester, J.N. (1999) Sources of organic micropollutants to lowland rivers. Environ. Technol. 19, 643^666. [4] Cullington, J.E. and Walker, A. (1999) Rapid biodegradation of diuron and other phenylurea herbicides by a soil bacterium. Soil Biol. Biochem. 31, 677^686. [5] Srensen, S.R., Ronen, Z. and Aamand, J. (2001) Isolation from agricultural soil and characterization of a Sphingomonas sp. able to mineralize the phenylurea herbicide isoproturon. Appl. Environ. Microbiol. 67, 5403^5409. [6] Gardner, A.M., Alvarez, G.H. and Ku, Y. (1982) Microbial degradation of 14 C-diphenylamine in a laboratory model sewage sludge system. Bull. Environ. Contam. Toxicol. 28, 91^96. [7] Nativ, R., Adar, E.M. and Becker, A. (1999) Designing a monitoring network for contaminated groundwater in fractured chalk. Ground Water 37, 38^47. [8] Ronen, Z. and Abeliovich, A. (2000) Anaerobic^aerobic process for microbial degradation of tetrabromobisphenol A. Appl. Environ. Microbiol. 66, 2373^2377. [9] Altschul, S.F., Madden, T.L., Scha«¡er, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSIBLAST : a new generation of protein database search programs. Nucleic Acids Res. 25, 3389^3402.
FEMSLE 10572 5-8-02
204
S.R. S6rensen et al. / FEMS Microbiology Letters 213 (2002) 199^204
[10] Gauthier, M.J., Lafay, B., Christen, R., Fernandez, L., Acquaviva, M., Bonin, P. and Betrand, J.-C. (1992) Marinobacter hydrocarbonoclasticus gen. nov., sp. nov., a new, extremely halotolerant, hydrocarbon-degrading marine bacterium. Int. J. Syst. Bacteriol. 42, 568^ 576. [11] Spro«er, C., Lang, E., Hobeck, P., Burghardt, J., Stackebrandt, E. and Tindall, B.J. (1998) Transfer of Pseudomonas nautica to Marinobacter hydrocarbonoclasticus. Int. J. Syst. Bacteriol. 42, 568^576. [12] Kaye, J.Z. and Baross, J.A. (2000) High incidence of halotolerant bacteria in Paci¢c hydrothermal-vent and pelagic environments. FEMS Microbiol. Ecol. 32, 249^260. [13] Rontani, J.-F., Gilewicz, M.J., Michotey, V.D., Zheng, T.L., Bonin, P.C. and Bertrand, J.-C. (1997) Aerobic and anaerobic metabolism of 6,10,14-trimethylpentadecan-2-one by a denitrifying bacterium isolated from marine sediments. Appl. Environ. Microbiol. 63, 636^643. [14] Huu, N.B., Denner, E.B.M., Ha, D.T.C., Wanner, G. and Stan-Lotter, H. (1999) Marinobacter aquaeolei sp. nov., a halophilic bacterium from a Vietnamese oil-producing well. Int. J. Syst. Bacteriol. 42, 568^ 576. [15] Hedlund, B.P., Geiselbrecht, A.D. and Staley, J.T. (2001) Marinobacter strain NCE312 has a Pseudomonas-like naphthalene dioxygenase. FEMS Microbiol. Lett. 201, 47^51.
[16] Kasuya, K., Mitomo, H., Nakahara, M., Akiba, A., Kudo, T. and Doi, Y. (2000) Identi¢cation of a marine benthic P(3HB)-degrading bacterium isolate and characterization of its P(3HB) depolymerase. Biomacromolecules 1, 194^201. [17] Button, D.K., Robertson, B.R., Lepp, P.W. and Schmidt, T.M. (1998) A small, dilute-cytoplasm, high-a⁄nity, novel bacterium isolated by extinction culture and having kinetic constants compatible with growth at ambient concentrations of dissolved nutrients in seawater. Appl. Environ. Microbiol. 64, 4467^4476. [18] Sigalevich, P., Baev, M.V., Teske, A. and Cohen, Y. (2000) Sulfate reduction and possible aerobic metabolism of the sulfate-reducing bacterium Desulfovibrio oxyclinae in a coculture with Marinobacter sp. strain MB in an aerated sulfate-depleted chemostat. Appl. Environ. Microbiol. 66, 5019^5023. [19] Larsen, H. (1986) Halophilic and halotolerant microorganisms ^ an overview and historical perspective. FEMS Microbiol. Rev. 39, 3^7. [20] Gheewala, S.H. and Annachhatre, A.P. (1997) Biodegradation of aniline. Wat. Sci. Tech. 36, 53^63. [21] Konopka, A. (1993) Isolation and characterization of a subsurface bacterium that degrades aniline and methylanilines. FEMS Microbiol. Lett. 111, 93^100.
FEMSLE 10572 5-8-02