Biodegradation of organophosphorus insecticides by bacteria isolated from turf green soil

JOURNALOF FERMENTATION AND BIOENOINEERING Vol. 82, No. 3, 299-305. 1996

Biodegradation of Orga,nophosphorus Insecticides by Bacteria Isolated from Turf Green Soil KAZUPUMI OHSHIRO,‘* TAKASUKE KAKUTA,2 TAKASHI SAKAI,3 HIDENORI HIROTA, TSUTOMU HOSHIN0,2 AND TAKE0 UCHIYAMA2 Department of Biosystem Science, Graduate School of Science and Technology,’ Department of Applied Biological Chembtry,2 Niigata University, Hukudagumi Co. Ltd., 3-10 Ichibanboridouri,3 and Department of Agrobiology, Niigata University, 2-8050 Ikarashi,4 Niigata 950-21, Japan Received 9 February 19%/Accepted

26 June 1996

Several organophosphate-degrading bacteria were isolated from test turf green soil using clear zones formed around their colonies on plates supplemented with organophosphate isoxathion. The degrading activity of the isolates for isoxathion was tested by incubation in liquid cultures and evaluated by gas chromatography. Strain B-5 exhibited the highest isoxathion degrading ability in the isolates and it was identified as an Arthrobacter sp. A high concentration of nutrients in the media afkted the isoxathion degrading activity of strain B-5. The bacterium could not utilize isoxathion as a sole source of carbon and phosphorus. The degradation products of isoxathion by B-5 washed cells were identified as its hydrolysis preducts, 3-hydroxy&phenylisoxazole and diethylthiophosphoric acid, suggesting that strain hydrolyzes the heterocycle ester bond in isoxathion. Arthrobacter sp. strain B-5 also hydrolyzed diion, parathion, EF+N,fenitrothion, isofenphos, chlorpyrifos, and ethoprophos at rates dependent on the substrate. Of the organophosphorus insecticides examined, isofenphos was affected most by the hydrolytic activity of the bacterium, which completely removed the compound (10 mg/l) from cultures within l-h incubation. [Key words: biodegradation,

organophosphorus

insecticides,

Soil microorganisms collectively decompose various xenobiotic compounds and return elements to the mineral state utilized by plants. They also play important roles in the dissipation of xenobiotic pesticides in the soil. Organophosphates have been extensively applied as alternatives to organochlorine compounds which possess longterm persistence and high toxicity. Organophosphorus compounds rapidly undergo degradation by soil microorganisms, so they do not persist in the environment. However, repeated applications of degradable organophosphates occasionally cause a significant reduction of their pesticidal effect. This phenomenon, which results from microbial adaptation to pesticide degradation and is called enhanced biodegradation, has often been observed in degradable pesticides such as organophosphates and carbamates. Most enhanced degradation in the field occurs after pesticide applications for two or more consecutive years (1). Changes in the amounts of pesticides in water drained through a test green set up in Niigata have previously been reported (2, 3). A variety of pesticides to protect the sward from insects and fungi were applied to the test green between 1990 and 1992. The organophosphorus insecticides and fungicides used were isoxathion, diazinon, fenitrothion, isofenphos, acephate, trichlorfon, and tolclofos-methyl. Isoxathion was frequently applied to the green. Almost all the organophosphates in groundwater which effused through the test green soil were found in trace amounts or not detected in previous reports (2, 3), indicating that organophosphate-degrading microorganisms might be enriched and that microbial degradation of organophosphates was enhanced in the test green soil. To understand and control the enhanced biodegradation of organophosphates, it is important to know the individ-

Arthrobacter]

ual degradation ability of microorganisms, the process of their acquisition of the ability, and their behavior in soil. Several insecticides, fungicides, and herbicides undergo enhanced biodegradation by soil microorganisms (1). Based on the results of the comparative degradation of six organophosphorus insecticides in soil, Racke and Coats (11) noted that the enhanced degradative phenomenon may exhibit some degree of specificity. They isolated an Arthrobacter sp. which could not metabolize any insecticide except isofenphos. We also isolated an Arthrobacter sp. with high degradation activity toward isofenphos. Here, we describe the isolation and characterization of an organophosphorus insecticide degrading bacteria from test green soil treated with pesticides, and the degradative specificity of the isolated bacterium for organophosphorus compounds. MATERIALS

AND METHODS

Analytical grade chlorpyrifos (99.9% Chemicals purity), diazinon (99.8% purity), EPN (99.8% purity), fenitrothion (99.4% purity), isofenphos (98.9% purity), isoxathion (99.7% purity), parathion (99.7% purity), and tolclofos-methyl (99.7% purity) were purchased from Wako Pure Chemical Industries Ltd. (Osaka). Analytical grade ethoprophos (97.0% purity) was purchased from Riedel-de Ha6n. 3-Hydroxy-5-phenylisoxazole (HPI) was a gift from Sankyo Co. Ltd. Diethylthiophosphoric acid (DETP) was synthesized by a modification of the method described in Methoden der Organischen Chemie (4). As DETP is not sufficiently volatile for gas chromatography, it was methylated with diazomethane dissolved in diethyl ether. Test turf green soil A test turf green (150 m2) was set up in sand dunes of suburban Niigata, Japan, in

* Corresponding author. 299

300

J. FERMENT.BIOENG.,

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1990 to investigate changes in the amounts of pesticides that drained through it into the soil. The vertical-layer soil structure consisted of a first layer (O-15 cm) of peat moss (4 Nm2), zeolites (5 kg/m2), and active carbon (6 I/ m2), a second layer (15-30cm) of sand, and a third layer (30-50 cm) of gravel. Pencross bentgrass was planted in the soil. The test turf green was treated with fourteen fungicides and nine insecticides, once or several times, between 1990 and 1992. Isolation and identification of bacteria Soil was collected 10 cm below the surface of the test turf green and stored at 4°C until bacteria were isolated. One gram of a well-mixed soil sample was suspended in 5 ml sterile deionized water, followed by dilution to an appropriate concentration. Diluted aliquots (0.1 ml) were plated on MSPY medium (KH2P04, 0.2 g; K2HP04, 0.5 g; MgS04. 7H20, O.2g; NaCl, 0.2 g; CaC12.2H20, 0.05 g; FeS04. 7H20, 0.025 g; NazMoO*, 0.005 g; MnS04, 0.0005 g; NaW02, 0.0005 g; peptone, 1 g; yeast extract, 2 g; distilled water, 1 (; pH 7.0) supplemented with isoxathion (50mg/l). Isoxathion was added as a methanol solution (10 mg/ml) after sterilization through a membrane (millipore) with a 0.2 pm pore size. The plates were incubated at 37°C and bacteria that formed clear zones around the colonies were selectively isolated. The isolates were further purified by transferring them to MSPY agar plates containing 50mg of isoxathion per liter several times. All strains were stocked at room temperature on an agar slant of MSPY medium containing 50 ppm isoxathion. The bacterium exhibiting the highest activity in isoxathion degradation was identified according to Bergey’s Manual of Systematic Bacteriology (5). The shape and morphology of bacterial cells were determined by light and electron microscopy. Diamino acids in cell wall peptidoglycan were determined in whole-cell hydrolysates according to Boone and Pine (6). Analytical procedure The ability of the isolates to degrade isoxathion was assessed in MSPY medium containing 10mg organophosphate per liter. Bacterial cells scraped 3 times from stock slants were suspended in 5 ml of sterile water. A portion (0.2ml) of each suspension was inoculated in 6 ml of medium and incubated on a reciprocal shaker at 30°C. Three and seven days thereafter, residual organophosphate in the culture was extracted 3 times with ethyl acetate and the amounts of organophosphates were determined by on a Shimadzu GC-14A gas chromatograph (GLC) equipped with a flame photometric detector. The column contained 1% OV-1 (1.6 m x 3.0 mm i.d.) and the temperature was set at 260°C. The temperature of the detector and injector was set at 280°C. The metabolites of isoxathion were identified in washed cell suspensions of strain B-5. The cells were obtained from 11 of MSPY medium by centrifugation (12,000 Xg, 15 min). The cell pellets were washed 3 times with 0.85% NaCl and resuspended in 50mM potassium phosphate buffer (pH 7.0) containing 0.2% Triton X-100 to solubilize isoxathion. Isoxathion (1 mg) was added to the washed cell suspension (5 ml) and the mixture was incubated on a reciprocal shaker at 30°C for 6, 12, or 18 h. After the washed cell suspension was acidified with 2 N HCI, isoxathion and metabolites were extracted 4 times with 3 ml ethyl acetate. The extract was dried with anhydrous sodium sulfate followed by filtration through glass-fiber paper (Whatman GF/B). The filtrate was evaporated to dryness under reduced pressure and redis-

solved in a small amount of ethyl acetate. Isoxathion metabolites were detected by thin-layer chromatography (TLC) using silica gel GF plates (Analtech, Inc.) which were developed with hexane-ethyl acetate-formic acid (70 : 30 : 2). Spots were detected by UV light or by spraying with 0.5% palladium chloride in 1 N HCl, which visualizes metabolites with a P-S bond. Gas chromatography-mass spectrometry (GC-MS) of the metabolites was carried out using a Hitachi M-60 equipped with a 3% OV-17 column (2.0 m x 3.0 mm i.d.) at an ionization energy of 70 eV. The column temperature was set at 22O”C, and that of the injector and the interface at 240°C. The ability of strain B-5 to degrade diazinon, parathion, EPN, fenitrothion, isofenphos chlorpyrifos, ethoprophos (insecticides), and tolclofos-methyl (fungicide) while growing was determined using l/IO-diluted NB supplemented with 10 mg of insecticides per liter. After the bacterium was grown on MSPY agar medium at 37°C for 24 h, 3 loopfuls of cells were suspended in 5 ml sterile water. The suspension (0.2ml) was inoculated in 6ml of the medium and incubated on a reciprocal shaker at 30°C. Residual organophosphorus compounds were extracted with ethyl acetate and measured by GLC. The recovery of organophosphorus compounds was determined using autoclaved B-5 cells. Cultures incubated in l/lo-diluted NB for 8 or 20 h were autoclaved at 121°C for 15 min. The autoclaved cultures were incubated with 10mg organophosphorus compounds per liter for 8 or 20 h. The amounts of pesticides were measured by GLC as described above. RESULTS Evaluation of the ability of isolates to degrade isoxathion Based on the properties of bacteria that

formed a clear zone around the colonies on MSPY medium supplemented with isoxathion (Fig. l), 8 were readily obtained from the test turf green soil. The activities of the isolates in degrading isoxathion are shown in Table 1. The amounts of isoxathion remaining in the cultures of strains B-l, 2, 3, 6, 7, and 8 after 7-d incubation were 75.2, 84.8, 85.5, 85.7, 77.9, and 60.7%, respectively. Strains B-4 and 5 metabolized more isoxathion after 7 d

FIG. 1. Clear zones formed by microbial colonies grown on an MSPY agar plate supplemented with isoxathion (50 mg/f). The plate was incubated at 37°C for 5 d. Arrows indicate clear zones around the colonies.

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VOL. 82, 1996 TABLE 1. Days 3d Id

Isoxathion-degrading

OF ORGANOPHOSPHATES

BY SOIL BACTERIA

301

activity of eight isolates

Residual isoxathion (%) B-l

B-2

B-3

B-4

B-S

B-6

B-l

B-8

84.3 75.2

91.6 84.8

92.2 85.5

71.8 26.8

3.0 0.6

92.9 85.7

90.4 71.9

78.4 60.1

than the other six strains, with B-5 degrading nearly all the isoxathion (96%) within 3 d. Consequently, strain B-5, which exhibited the highest isoxathion-degrading activity, was selected for further study. Identification of bacterium Strain B-5 was gram positive in the exponential growth phase but predominantly gram negative in the stationary phase. The bacterium was obligately aerobic, immotile, formed no endospores, and was catalase positive. No acid was produced from glucose and gelatin was hydrolyzed. Only biotin was required as an organic growth factor. Strain B-S showed a remarkable rod-coccus cycle during growth in nutrient broth. Cells in the exponential growth phase were long rods that frequently branched and formed a V shape. In the stationary phase, the cells were largely coccoid. The cell wall peptidoglycan of strain B-5 contained lysine as the diamino acid. Based on these morphological and biochemical characteristics, the isolate was identified as a member of the genus Arthrobucter according to Bergey’s manual (5). Effects of nutrients on the growth and isoxathion degrading activity of strain B-5 The growth and isoxathion degrading activity of strain B-5 were examined in five media: MSPY, MSP (mineral salts as in MSPY medium; peptone, 0.5 g; distilled water, 1 I), NB (meat extract, 10 g; peptone, log; NaCl, 5 g; distilled water, 1 I), l/10 diluted NB and MSB medium (mineral salts supplemented with 0.5 g of NHaOs; biotin, 0.1 g; distilled water, 1 0. MSB medium contained 50 mg isoxathion per liter, while 10mg isoxathion per liter was added to the other media. The pHs of all media was adjusted to 7.0. Growth was monitored as the optical density at 660nm and residual isoxathion were determined by GLC. There were marked differences in the growth of strain B-5 in four of the media (Fig. 2a). While the strain proliferated quite well in NB and MSPY, it grew less well in l/10-NB and MSP; in MSP medium in particular, its growth fell to l/3 of that in NB and MSPY media. On the other hand, the residual isoxathion levels were 40 and 45% in NB and MSPY media, and 4.5 and

01

0

1

I

12

18

Time (h) FIG. 3. Isoxathion strain B-5 cells.

degradation

by washed Arthrobacter

sp.

12% in l/10-NB and MSP media, respectively, after a 9h incubation with strain B-5 cells (Fig. 2b). These results indicated that the degradation of isoxathion by strain B5 was inhibited by enriching the medium. Strain B-5 neither proliferated nor degraded isoxathion in MSB or in MSB in which KH2P04 and K2HP04 were replaced with KCl, indicating that the strain could not use isoxathion as a sole source of carbon and phosphorus. To estimate the recovery of isoxathion, B-5 cells grown for 6, 12, or 24 h in MSPY and NB media were autoclaved at 121°C for 15 min. The autoclaved cultures were incubated with 10 mg isoxathion per liter for 6, 12, or 24 h. The concentrations of isoxathion were determined as described in Materials and Methods. The recovery of isoxathion from the cultures was about 100%. No isoxathion was lost by adsorption onto the bacterial cells or by volatilization from the cultures during the incubation. We then determined whether the isoxathion-degrading activity of strain B-5 was induced by addition of isoxathion. B-5 cells were subcultured 3 times on an agar medium containing mineral salts and glucose (10 g/f) with and without isoxathion (50mg/I), inoculated in l/IO-NB supplemented with isoxathion (10 mg/Z), and incubated at 30°C for 3, 6, 9, 12, or 15 h. The degradation activities were essentially similar in the cultures with and without isoxathion, indicating that the degrading activity is constitutive in B-5 cells.

ra

0

I

6

b

10

5

I5

Time(h) FIG. 2. Effects of nutrients on the growth (a) and isoxathion-degrading medium; A, l/10 diluted NB; 0, MSPY medium; q , NB.

Time (h) activity (b) of Arthrobacter

sp. strain B-5.

Symbols: 0, MSP

302

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J. FERMENT. BIOENG.,

Degradation products of isoxathion Washed B-5 cells decomposed 72% of the isoxathion within 18 h (Fig. 3). TLC revealed two isoxathion metabolites and their accumulation with incubation in the reaction mixture. The two compounds with Rf values of 0.48 and 0.17 were identified as HP1 and DETP, respectively, based on a comparison with standards. The identification of DETP was supported by the fact that the color of the metabolite with an Rf value of 0.17 became orange like authentic DETP when the TLC plate was sprayed with palladium chloride. The results were confirmed by GC-MS. The spots with Rf values of 0.48 and 0.17 were scraped from the plate and extracted with ethyl acetate. Gas chromatography of HP1 revealed two peaks (retention times: 0.45 and 1.18 min). Since HP1 has the reversible isomeric interconvertibility (y-lactam and r-lactim) known as tautomerism (Fig. 4c), 5-phenyl4-isoxazolin-3-one of y-lactam was present in the HP1 standard. The retention times and the mass spectra of the two peaks coincided with those of the two peaks of the authentic sample. The mass spectra of the two peaks

with retention times of 0.45 and 1.18 min are shown in Figs. 4a and b, respectively. We failed to determine from the tautomeric feature of HP1 which of the peaks, a or b, is the y-lactam or the r-lactim. The compound with an RI value of 0.17 was methylated by diazomethane and analyzed by GC-MS. The retention time and mass spectrum coincided with those of authentic DETP. The molecular ion peak of the methyiated metabolite was observed at 184m/z, which resulted in the introduction of a methyl group into DETP (Fig. 5). These results suggested that an enzyme(s) responsible for the degradation of isoxathion hydrolyzed the heterocycle ester bond in isoxathion. Comparative degradation of organophosphorus comWe determined the relationship between the pounds

degradation activities of organophosphorus compounds and the growth of strain B-5 (Fig. 6). The structures of the compounds are shown in Fig. 7. The growth of B-5 was essentially unaffected by the tested compounds, though chlorinated substrates (chlorpyrifos and tolclofos-methyl) slightly delayed the growth. The degradation

43

105

161

l.l& II’

I

I .I

,,,,,I’,‘,,,,,,I,,,,,,,,,i,,,,,~,,,I,’,,,’,’

I

4

0

20

40

60

80

100

120

140

160

180

200

0

20

40

60

80

100

120

140

160

180

200

m/z C

3-hydroxy-5.phenylisoxazole (y-lactim)

5.phenyl4isoxazolin-3.one

(y-lactam)

FIG. 4 Mass spectra and structures of HP1 and the tautomer. Compounds with retention times of 0.45 (a) and 1.18 (b) min on gas chromatography were examined by mass spectrometry. (c) Structures of 3-hydroxy-5-phenyiisoxazole and Sphenyl-4-isoxazolin-3-one.

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C&O,

OF ORGANOPHOSPHATES

BY SOIL BACTERIA

303

S

11

, P- OCH3

184

I

C2H50

156

47

0

20

60

40

80

120

100

160

140

180

200

m/z

FIG. 5.

Mass spectrum of methylated DETP.

activities of strain B-5 differed according to the organophosphorus compounds examined. Isoxathion, EPN, diazinon, isofenphos, and chlorpyrifos were readily decomposed by the strain. Isofenphos was completely removed from the medium within l-h incubation. On the other hand, parathion, fenitrothion, and ethoprophos were hardly decomposed and the ratios of residual parathion, fenitrothion and ethoprophos were 62.0, 77.0, and 90.6%, respectively, after 20h. Tolclo-

0

4

8

12

Time (h)

16

20

0

4

8

phos-methyl was not dissipated during the incubation period. These results suggested that Arthrobacter sp. strain B-5 exhibited substrate specificity for the degradation of organophosphorus compounds and the highest degradation activity for isofenphos. DISCUSSION

Bacteria that degrade organophosphorus

12

Time (h)

16

20

0

4

8

12

16

insecticides

20

Time (h)

FIG. 6. Comparative degradation of organophosphorus compounds by Arthrobacter sp. strain B-5. Strain B-5 cells were incubated on a reciprocal shaker at 30°C in l/10-diluted NB supplemented with organophosphorus insecticide (10 mg/f). Symbols: 0, growth; l , residual organophosphorus compound.

304

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to isolate microbes from soils and enrichment cultures. Eight bacterial colonies were selected based on their ability to form large clear zones. However, only one isolate, strain B-5, degraded large amounts of isoxathion (Table 1). The results indicated that zone size did not always correspond to the degree of degradative activity. Differences in culture conditions (agar or liquid media) may explain this. We identified strain B-5 as a member of the Arthrobacter species. Many Arthrobacter have been isolated from various soils as microorganisms which degrade pesticide (8-15). The genus Arthrobacter contains many species which are ubiquitous in soil and which can utilize many aromatic hydrocarbons. Stevenson studied their ability to utilize about 40 aromatic hydrocarbons, and revealed that most of the isolates from soil could utilize a wide range of aromatic substrates as their sole carbon source (16). These features of the Arthrobacter genus will facilitate the acquisition of the ability to metabolize pesticides, thereby enabling the genus to play an important role in the degradation of pesticides in the natural en-

isoxathion

diazinon

fenitrothion

isofenphos

EPN

vironment .

parathion

chlorpyrifos

tolclofos-methyl

ethoprophos FIG. 7.

ii Cd-W - P W,H7),

Structures of the organophosphorus

compounds studied.

were isolated from turf green soil by screening colonies forming clear zones around them on plates containing isoxathion. We initially considered that the formation of clear zones would be due to the degradation of isoxathion having low water solubility to compounds having high solubility by an extracellular or intracellular enzyme(s) liberated from the microbial cells. However, no degrading activity for isoxathion and isofenphos was detected in the supernatant of liquid cultures of strain B-5. These findings suggested that the bacterium did not produce enzymes that degrade organophosphates in culture. On the other hand, Clark and Wright (7) isolated Arthrobacter and Achromobacter utilizing isopropyl Nphenylcarbamate from soil by this means and suggest that the process of clear zone formation was the gradual dissolution and diffusion of the herbicide in very small amounts of the organisms. The mechanism of clear zone formation by strain B-5 appeared to be the same as that proposed by Clark and Wright. Hans et al. (8) isolated Arthrobacter oxydans P52, which co-metabolizes phenmedipham by this procedure. Although few microorganisms that degrade pesticides have been isolated using clear zone formation, it is a simple and convenient means of detecting the ability to degrade pesticides and

The effects of nutrients on pesticide degradation by strain B-5 have been investigated. Adhya et al. (17) showed that glucose inhibited the hydrolysis of parathion by Pseudomonas sp., which also metabolizes 4-nitrophenol in medium. However, the metabolic pathway of 4-nitrophenol differed in media with and without glucose. Mycobacterium sp. and Flavobacterium sp. degraded the herbicide molinate from soil, and this process was suppressed by excess nutrients in the medium (18). Bacillus sp. degraded methyl parathion, parathion and fenitrothion, but fenitrothion was degraded only in the presence of yeast extract (19). The degradative rates of these compounds accelerated with increasing yeast-extract concentrations. The isoxathiondegrading activity of strain B-5 was lower in a medium enriched by nutrients than in a nutrient-poor medium, and it did not utilize isoxathion as a sole carbon or phosphorus source. In addition, strain B-5 did not utilize the degradation product of isoxathion, HPI, or isofenphos. Metabolites formed from isoxathion by strain B-5 were identified by TLC and GC-MS as the hydrolysis products HP1 and DETP. Nakagawa et al. (20) have isolated several metabolic products of isoxathion from soil and identified HP1 as the major metabolite. We showed here for the first time that HP1 is a degradation product of isoxathion produced by an isolated bacterium. Takahi et al. (21) have shown that HP1 may be a fungicide, since it inhibited the mycelial growth of several plant pathogens on potato-sucrose-agar medium. However, its fungicidal activity markedly decreases when repeatedly applied to soil. This may be due to the enhanced biodegradation of HP1 by its repeated application. Hydrolytic products of organophosphates undergo further microbial metabolism in the soil (22), and the phenomenon of enhanced biodegradation by the soil ecosystem may be important in detoxifying toxic products. Arthrobacter sp. strain B-5 degraded organophosphorus compounds such as chlorpyrifos, diazinon, EPN, fenitrothion, isofenphos, parathion, and ethoprophos. Although their metabolites remain to be determined, the type of degradation is most likely to be hydrolysis at the aryl or heterocyclic ester bond from the identification of the isoxathion metabolites. This view is supported by the formation of a yellow compound, possibly p-

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nitrophenol, from the degradation of EPN and parathion. Bacterial isolates and enzymes that hydrolyze organophosphates and structural analogues have been described (23-25). The effects of alkyl and aryl substituents for the hydrolytic activity have been investigated (17, 24-26). Adhya et al. suggested that since the Pseudomonas sp. hydrolyzed parathion and diazinon but not methyl parathion, the alkyl constituent attached to phosphorus is more important than the aromatic portion in the degrading ability (17). Munnecke showed that the enzyme specificity for organophosphorus insecticides was more influenced by alkyl substitutions on the phosphorus atom than by aromatic ring substitution or changes (24). On the other hand, Donarski et al. demonstrated that the K,,, and V,, of phosphotriesterase from Pseudomonas diminuta for 16 paraoxon analogues differed only in the structure of the phenolic leaving group (26). Our results showed that strain B-5 hydrolyzed organophosphorus compounds in a substrate-dependent manner. However, the affects of the alkyl or aryl groups of the organophosphorus compounds on the degrading activity remain to be investigated in detail. Racke and Coats (11) isolated an Arthrobacter sp. that degrades isofenphos from cornfield soil in Iowa with an isofenphos history. The strain rapidly metabolized isofenphos, but not chlorpyrifos, fonofos, ethoprop, terbufos, or phorate. Arthrobacter sp. strain B-5 readily decomposed isoxathion, diazinon, EPN, isofenphos, and chlorpyrifos, with particularly high activity for isofenphos among these organophosphorus compounds. The two Arthrobacter spp. are similar in that they possess a high level of degrading activity for only isofenphos, and this specificity implies similar structures and kinetics of the degrading enzymes produced by these strains. As to the high specificity, the phosphoramide bond in isofenphos may cause a high degree of recognition by the enzymes, as described by Racke and Coats. However, further research is required to confirm this.

6. 7.

8.

9.

10. 11.

12. 13.

14.

15.

16.

17.

18. ACKNOWLEDGMENTS The authors express their appreciation to Dr. M. Kojima for assistance in identifying the bacterium. Thanks are also due to Mr. I. Nakanishi of Sankyo Co. Ltd. for providing the authentic sample of HP1 and Dr. M. Sasaki of Sumitomo Kagaku Co. Ltd. for suggestions regarding the synthesis of DETP. REFERENCES 1. Racke, K. D. and Coats, J. R.: Enhanced biodegradation of insecticides in midwestern corn soils, p. 68-82. In Racke, K. D. and Coats, J. R. (ed.), Enhanced biodegradation of pesticides in the environment. ACS Symposium Series 334. American Chemical Society, Washington, D.C. (1990). 2. Sakai, T., Ishixuka, S., Shimaxo, Y., Ohshiro, IL., Uchiyama, T., and Hirota, H.: Changes of agricultural chemicals in water drained out through the test-green sward II. J. Japan. Soci. Turfgrass Sci., 22, 197-207 (1994). (in Japanese) 3. Sakal, T., Ishizuka, S., Shimazu, Y., Uchiyama, T., and Hirota, H.: Run-off performance of drained water and agricultural chemicals in test-turf green. J. Japan. Soci. Turfgrass Sci., 20, 173-182 (1992). (in Japanese) 4. Sasse, K.: Organische phosphorverbindungen, p. 603. In Milller, E. (ed.), Methoden der Organischen Chemie, Band XII/Z. Georg Thieme Verlag, Stuttgart (1964). 5. Keddie, R. M., Collins, M.D., and Jones, D.: Genus Arthrobacter Conn and Dimmick, p. 1288-1301. In Sneath,

19.

20.

21.

22.

23. 24. 25. 26.

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