Degradation by Bacteria

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Letters in Applied Microbiology1999,28, 6165

The degradation of sodium O,O-diethyl dithiophosphate bybacteria from metalworking fluids

R.E. Sherburn and P.J. LargeDepartment of Biological Sciences, University of Hull, UK

1885/98: received and accepted 8 October 1998

R.E. SHE RBURN AND P .J. LARG E. 1999.The breakdown of sodium O,O-diethyl

dithiophosphate (O,O-diethyl phosphorodithioate) by four bacterial strains

(tentatively identified as strains ofAeromonas,Pseudomonas,Flavobacteriumand Bacillus)

isolated from contaminated metalworking fluids was shown to involve the

successive formation of ethanol, aldehyde and orthophosphate. An acid

phosphodiesterase was identified in cell-free extracts that was five- to sevenfold enhanced in

specific activity in bacteria grown on O,O-diethyl dithiophosphate as sole phosphorus

source, compared with bacteria grown on orthophosphate. This is thought to

initiate the breakdown process.

I N T R O D U C T I O N

The insecticide malathion (S-(1,2-dicarbethoxyethyl)-O,O-

dimethyl dithiophosphate) (Matsumura and Boush 1966) and

other substituted dithiophosphate (phosphorodithioate)

derivatives have been shown to act as carbon (Munnecke

and Hsieh 1976), phosphorus (Cook etal. 1978) and sulphur

(Cook etal. 1980) sources for bacteria isolated from soil or

sewage sludge, and some information is available on theirbiodegradation (Kerteszetal. 1994).

Zinc dialkyldithiophosphates have been used for many

years as antioxidants (Bridgewater etal. 1980) and anti-wear

additives (Rounds 1975) in lubricating oils, where they pro-

duce protective films on sliding surfaces (Yin etal. 1993).

The sodium salts have been used in the separation of metals

from sulphide minerals (Valli etal. 1994). The chemical

breakdown of dialkyl dithiophosphates has been studied

extensively (Burnetal. 1990).

The present work reports the degradation of the soluble

diethyl dithiophosphate salt sodiumO,O-diethyl dithiophos-

phate (sodium O,O-diethyl phosphorodithioate) (NaDDP)by extracts of four bacterial species, Gram-negative strains

FM2, SP1 and AV1 and Gram-positive strain CL1, ten-

tatively identified as strains of Aeromonas, Pseudomonas,

Flavobacterium and Bacillus, respectively, isolated from con-

taminated cutting oils. Evidence is presented that the break-

down involves, in each case, the successive formation of

Correspondence to: Dr Peter J. Large, Department of Biological Sciences,

University of Hull, Hull HU6 7RX, UK (e-mail:

[email protected]).

1999 The Society for Applied Microbiology

ethanol, acetaldehyde and orthophosphate from NaDDP, and

an acid phosphodiesterase.

M A T E R I A L S A N D M E T H O D S

Materials

Sodium diethyl dithiophosphate and 3-methyl-2-benzo-thiazolinone hydrazone hydrochloride (MBTH) were from

Aldrich. All other chemicals and enzymes were from Sigma.

Soluble oil (a model metalworking fluid containing 100 sol-

vent neutral, distilled tall oil, naphthenic acid, Texafor M6,

C16 a-olefin, tall oil amide, potassium hydroxide and water)

and microbially-contaminated metalworking fluids were con-

tributed by Castrol International (Pangbourne, UK).

Isolation of bacteria from contaminated

metalworking fluids

Bacteria were isolated from a 100 ml sample of a 105 dilution

of contaminated metalworking fluids by repeated sub-

culturing of colonies initially cultured on either malt extract-,

nutrient- or LB-agar (5 g yeast extract, 10 g NaCl, 10 g tryp-

tone and 15 g bacteriological agar in 1 litre distilled water).

All agar was sterilized by autoclaving at 121 C for 15 min

before use. Isolated organisms were tentatively identified

using the standard biochemical tests in the API 20NE test

kits (bioMerieux) and further examined by pyrrolysis mass-

spectroscopy (Hindmarch etal. 1990). The 20 tests in the

API 20NE kit are nitrate reduction, indole production, acid


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62 R . E. SH ER BU R N AN D P. J. LAR GE

production, activities of arginine dihydrolase, urease, b-glu-

cosidase, protease, b-galactosidase and assimilation of

glucose, arabinose, mannose, mannitol,N-acetylglucosamine,

maltose, gluconate, caproate, adipate, malate, citrate and

phenylacetate.

Growth media

For the growth in liquid batch culture of bacteria which were

not acclimatized to NaDDP, a medium was used containing

(l1 distilled water): 04 g MgSO4.7H2O, 70g (NH4)SO4,

25 m g FeSO4.7H2O, 41 mg MnSO4.4H2O, 44 mg

ZnSO4.7H2O, 079 mg CuSO4.5H2O, 734mg CaCl2.2H2O,

80mmol l1 potassium-sodium phosphate buffer, pH 70,

and 50 mmol l1 glycerol. The potassium-sodium phosphate

buffer was replaced by 50 mmol l1 sodium diethyl dithio-

phosphate from a 55% aqueous solution and 80 mmol l1

Tris-(hydroxymethylmethylamine) buffer, pH 70, for the

liquid batch culture of bacteria acclimatized to sodium diethyldithiophosphate. The distilled water was replaced by a 5%

solution of soluble oil for liquid batch culture within a soluble

oil emulsion (see Materials). All media were sterilized by

autoclaving at 121 C for 15 min. Glycerol was autoclaved

separately as a concentrate and added to the rest of the media

after sterilization.

Cell harvesting and preparation of extracts

Cells were harvested by centrifugation at 25 000g, washed in

10 mmol l1 potassium phosphate buffer and resuspended in

100mmol l1

Tris-HCl buffer, pH 70, containing 5 mmol l1

dithiothreitol. Cell extracts were prepared by passing

these suspensions through a French pressure cell twice and

centrifuging at 30 000gto remove cell debris.

Biotransformation of sodium diethyl

dithiophosphate

Ethanol, aldehyde and phosphate. For the enzymic pro-

duction of ethanol, aldehyde and phosphate, shaken 250 ml

flasks were used. Initial reaction volumes of 47 ml contained

85mmol l1 Tris-HCl buffer, pH 70, 56 mmol l1 sodium

diethyl dithiophosphate and1 mg cell extract protein in sterile

distilled water. Extracts were used from cells grown using the

following phosphorus sources: dipotassium orthophosphate,

sodium diethyl dithiophosphate and sodium diethyl dithio-

phosphate in soluble oil. Controls either used boiled extracts

or omitted either extract or sodium diethyl dithiophosphate.

Samples of 1 ml were removed at timed intervals, treated

with 03 ml 1 mol l1 perchloric acid and centrifuged at high

speed in a bench-top centrifuge to remove precipitated

protein. All experiments were carried out in triplicate and

results are expressed asmmol product mg protein1.

1999 The Society for Applied Microbiology,Letters in Applied Microbiology28, 6165

Hydrogen sulphide. For the enzymic production of hydrogen

sulphide, Dreschler bottles were used with 01 mol l1 zinc

acetate as a trapping agent. An initial reaction volume of

20 ml contained 10ml 01mol l1 Tris-HCl buffer, pH 70,

2 mg protein and 56 mmol l1 sodium diethyl dithiophos-

phate in sterile distilled water. Anaerobic experiments werecarried out under nitrogen. The zinc acetate was assayed for

trapped hydrogen sulphide at intervals over a 2 h period. All

experiments were carried out in triplicate.

Analysis

Ethanol was measured colorimetrically with alcohol oxidase

(Verduyn etal. 1984). Aldehydes were measured by the

MBTH method (Sawicki etal. 1961). Phosphate was mea-

sured by the method of Lowry and Lopez (1946). Hydrogen

sulphide was measured by the method of Truper and Schlegel

(1964). Acid phosphodiesterase activity was measured byincubating extract in 22mmol l1 sodium citrate buffer,

pH60, with 01 mmol l1 2?-deoxythymidine-3 ?-(4-nitro-

phenyl) phosphate for 5 min at 25 C, and stopping the reac-

tion by adding 29 ml 01 mol l1 NaOH. The absorbance was

measured at 405 nm and corrected for the absorbance of a

blank without enzyme. Alkaline phosphodiesterase activity

was measured by incubating extract in 02 mol l1 Tris-HCl,

pH 89, containing 33 mg bis-4-nitrophenyl phosphate ml1.

The rate of increase in absorbance at 405 nm was followed

spectrophotometrically at 25 C. One unit of acid phos-

phodiesterase activity is the amount of enzyme required to

catalyse the formation of one micromole of 4-nitrophenol

per minute. Protein concentrations were measured by the

Bradford (1976) method with a bovineg-globulin standard.

R E S U L T S

Isolation and growth of bacteria

Approximately 30 organisms were isolated from con-

taminated metalworking fluids. Most were Gram-negative

rod-shaped bacteria. Four organisms were selected for further

study, AV1, CL1, FM2 and SP1. AV1 reduced nitrate, had

urease activity, assimilated glucose, arabinose, mannose, N-

acetyl-glucosamine, maltose and malate. All the other bio-

chemical tests were negative or inconclusive. CL1 reduced

nitrate, had b-glucosidase, b-galactosidase activities, and

assimilated glucose, arabinose, mannose, mannitol, maltose,

gluconate, malate and citrate. Strain FM2 produced indole

and acid, had b-glucosidase activity and assimilated N-ace-

tylglucosamine, maltose and citrate. SP1 assimilated glucose,

arabinose, mannose, N-acetylglucosamine, maltose and

malate. On the basis of these tests and further characterization

by pyrrolysis mass spectroscopy, these organisms were ten-


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D I TH I OPH OSPH ATE BR EAKD OWN 63

60

10

00

Time (min)

Productformed(m

oll1)

50

8

6

4

2

10 20 30 40

(c)

60

7

00

Time (min)

Productformed(moll1)

50

6

5

4

2

10 20 30 40

(b)

3

1

60

2

00

Time (min)Productformed(moll1)

5010 20 30 40

(a)

1

Fig. 1 The biotransformation of sodium diethyldithiophosphateto yield ethanol (), aldehyde () and phosphate (R) by cell

extracts prepared from organism AV1 (tentatively identified as a

species ofFlavobacterium) grown on (a) dipotassium

orthophosphate, (b) sodium diethyl dithiophosphate and (c)

sodium diethyl dithiophosphate in soluble oil. The release of ethanol

(), aldehyde () and phosphate (r) by cell extracts in the

absence of the substrate is also shown. All results are expressed as

mmol product mg protein1


60

7

00

Time (min)


50

6

5

4

2

10 20 30 40

(c)

60

7

00

Time (min)


50

6

5

4

2

10 20 30 40

(b)

3

1

60

2

00

Time (min)Productformed(moll1)

5010 20 30 40

(a)

1

3

1

Fig. 2 The biotransformation of sodium diethyldithiophosphateto yield ethanol (), aldehyde (), and phosphate (R) by cell

extracts prepared from organism SP1 (tentatively identified as a

species ofPseudomonas) grown on (a) dipotassium

orthophosphate, (b) sodium diethyl dithiophosphate and (c)

sodium diethyl dithiophosphate in soluble oil. The release of ethanol

(), aldehyde () and phosphate (r) by cell extracts in the

absence of the substrate is also shown. All results are expressed as

mmol product mg protein1


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64 R . E. SH ER BU R N AN D P. J. LAR GE

tatively assigned to the genera Aeromonas (FM2), Bacillus

(CL1), Flavobacterium(AV1) and Pseudomonas(SP1).

Formation of products from sodium diethyl

dithiophosphate

Extracts of all the organisms studied produced alcohol, alde-hyde and phosphate in excess of those produced in the

controls. No products were found in the control experiments

where boiled extracts were used or the extract was omitted.

Neither the alcohol nor aldehyde methods are specific for

ethanol and acetaldehyde, but it seems improbable that other

products could be formed. Extracts from cells grown on

sodium diethyl dithiophosphate yielded higher measure-

ments of products than extracts from cells grown on di-

potassium orthophosphate (Figs 1 and 2). This shows that

prior acclimatization to NaDDP increased the capacity of cell

extracts to degrade NaDDP. The highest measured quantities

of ethanol occurred before the subsequent accumulation of

aldehyde and phosphate. In all cases, ethanol was present in

lower detectable quantities than aldehyde or phosphate.

Phosphate production by extracts from cells cultured on

sodium diethyl dithiophosphate (Fig. 1b, 2b) was similar to,

or lower than phosphate production by extracts from cells

cultured on sodium diethyl dithiophosphate and soluble oil

(Fig. 1c, 2c). There was a reduction in aldehyde and ethanol

production when soluble oil was present in the original

growth medium.

Of the four organisms studied, Flavobacterium AV1

appeared to catabolize NaDDP most rapidly. However, only

10% of the total phosphate of the NaDDP molecule was

released during the 2 h study period. No hydrogen sulphideproduction by any of the four organisms was detected under

the assay conditions used.

Phosphodiesterase activity

No alkaline phosphodiesterase activity was detected. There

were elevated levels of acid phosphodiesterase activity in all

four organisms grown on sodium diethyl dithiophosphate

compared with extracts from cells grown on dipotassium

orthophosphate (Table 1). Phosphatase activity (acid and

Table 1 Acid phosphodiesterase activity in the isolates

Organism

Phosphorus source

and growth Activity (units g protein1)

conditions FlavobacteriumAV1 BacillusCL1 AeromonasFM2 PseudomonasSP1

K2HPO4 00402 0002 00252 0001 00072 0002 00222 0005

NaDDP 03212 0054 01722 0032 00482 0006 01552 0025

NaDDP plus soluble oil 02142 0032 00712 0009 00362 0002 01242 0029


alkaline)was present at significant levels but was notincreased

during growth on NaDDP.

D I S C U S S I O N

The isolation of bacteria from contaminated cutting oils con-firms the observations of Cook etal. (1978) that diethyl dithio-

phosphate can serve as a phosphorus source for environ-

mental bacteria both in defined medium and (in our case) in

a soluble oil emulsion. The data reported here suggest that

the breakdown of NaDDP involves an acid phosphodiesterase

attack leading to the liberation of ethanol. The biodegradation

of mono- and dithiophosphates has been known for some

years to begin with the hydrolytic removal of the esterified

groups (Matsumura and Boush 1966; Helling etal. 1971;

Mulbry and Karns 1989). Indeed, the first reported hydro-

lytic attack on a dialkyl dithiophosphate is the work of Forrest

(1955), who showed that potassium diisopropyl dithiophos-phate is slowly hydrolysed to isopropanol by Clarase (Taka-

diastase, a crude enzyme preparation fromAspergillus oryzae).

In our system, the ethanol released must then be further

metabolized by oxidation to account for the formation of

aldehyde.

The decrease in detectable phosphate in extracts from

cells cultured on soluble oil (Fig. 1c) may possibly be due to

interaction with small oil micelles within the extract.

The lack of hydrogen sulphide can be attributed to the

properties of NaDDP itself, which is used to remove sulphide

from mineral ores and would be expected to form disulphide

complexes with sulphide ions.

As yet we do not know what phosphate derivatives liebetween the de-ethylated NaDDP and orthophosphate, to

what extent the transformations of the products may be non-

enzymic (Burn etal. 1990), or how soluble oil and NaDDP

interact. Kertesz etal. (1994) postulate that the degradation

of diethyl monothiophosphate (EtO)2P(:S)OH, the product

of phosphotriesterase activity on parathion, proceeds via

monoethyl monothiophosphate EtOP(:S)(OH)2 and thio-

phosphoric acid (HO)3P:S, or alternatively, via di- and mono-

ethyl hydrogen phosphates.


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D I TH I OPH OSPH ATE BR EAKD OWN 65

A C K N O W L E D G E M E N T S

RES thanks the Biotechnology and Biological Sciences

Research Council and Castrol International for support via a

CASE Studentship. The authors thank Michael Wright and

Diana Ciccognani of Castrol International Technology Cen-

tre for advice and co-operation.

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