JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 71, No. 3, 194-196. 1991

Estimation of Solvent-Tolerance of Bacteria by the Solvent Parameter Log P

A K I R A INOUE* AND KOKI H O R I K O S H I

The Riken Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-01, Japan

Received 24 October 1990/Accepted 3 December 1990

The solvent-tolerance of Gram-negative and Gram-positive bacteria were determined by the solvent parameter, log P. The solvent-tolerance of bacteria tested were in the following order, Pseudomonas> Escherichia, Serratia, Klebsiella >Proteus, Achromobacter, Acinetobacter > Alteromonas, Aeromonas, Flavobacterium > Alcaligenes, Bacillus > Agrobacterium, Micrococcus, Staphylococcus > Streptococ- cus > Leuconostoc, Lactobacillus > Corynebacterium, Chromobacterium, Brevibacterium, Rhodococcus.

The physiological basis of solvent toxicity is still poor ly unders tood and there is yet no identified physical para- meter by which the relative toxicity of different solvents can be determined. Consequently, the solvent tolerance of microorganisms has not yet been correlated with the organic solvents.

General ly, solvent toxicity is thought to involve incor- pora t ion o f the solvent with membrane lipids which results in d isrupt ion of essential membrane funct ion, a n d / o r , in- act ivat ion or denatura t ion of essential enzymes in the pres- ence of organic solvents.

Recent studies on biocatalysis in water-organic solvent biophasic systems have shown that many enzymes retain their catalytic activities in the presence of high concentra- tions o f organic solvents (1). In such systems, a direct cor- relation has been observed between enzyme activity and the solvent parameter log P. It, therefore, seems that log P can serve as a quanti ta t ive index of the inherent abil i ty of a solvent to distort the funct ional conformat ion of enzymes in a water-solvent environment .

Log P, which is defined as the logar i thm of a solvent 's par t i t ion coefficient in a s tandard octanoh water mixture, was used as a quanti ta t ive index of solvent polar i ty in the study of a quanti tat ive structure-activity relationship analy- sis (2, 3).

In a previous paper on a toluene tolerant bacter ium and its mutants (4), it was shown that the relative toxicities of different solvents were determined by their solvent polar- ity. Limita t ion on the growth of microorganisms by high concentrat ions of various organic solvents was shown to be correlated with the solvent parameter , log P. This indi- cates that the solvent-tolerances of microorganisms are de- termined by a solvent parameter , log P at water-saturated solvent concentrat ions.

In order to investigate the solvent-tolerance propert ies of bacteria, it will be useful to elucidate the mechanisms of solvent-tolerance.

This paper describes the solvent-tolerance limits of bacte- ria in terms of the solvent parameter , log P and compares the solvent-tolerance of Gram-negat ive with Gram-posi - tive bacteria.

Type strains of Gram-negat ive bacteria belonging to

* Corresponding author. Present address: Laboratory of Micro- bial Ecology, The Riken Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-01, Japan.

the genera Pseudomonas, Escherichia, Aeromonas, Alteromonas, Achromobacter, Acinetobacter, Agrobacte- rium, Alcaligenes, Serratia, Proteus, Klebsiella, Flavobac- terium and Chromobacterium as well as Gram-posi t ive bacteria belonging to the genera Bacillus, Micrococcus, Staphylococcus, Streptococcus, Corynebacterium, Brevi- bacterium, Rhodococcus, Leuconostoc and Lacto- bacillus were tested. The growth of the type strains was examined in a modified LB medium consisting of t ryptone, 10 g (Difco, Detroit , Michigan); yeast extract, 5 g (Difco); NaC1, 5 g; glucose, 1 g and MgSO4.7H20, 10 mM in 1 I of deionized water at pH 7.0. The solvent-tolerance of the strains was tested on modified LB agar plates all overlaid with different solvents. Cells o f each strain were streaked on the agar plate medium, directly overlaid with one of the different solvents and incubated at 37°C. The growth was est imated from the development of colonies on the agar plates after 48 h of incubat ion. The limiting log P values for growth were defined as a critical point , at which the sol- vent was polar enough to prevent growth of the strain. Different solvents with log P values ranging from the 2.1 (benzene) to 7.0 (dodecane) were tested. A list of the com- monly used organic solvents with their corresponding log P values is shown in Table 1.

The solvent-tolerance limits o f the Gram-negat ive and Gram-posi t ive type strains are shown in Table 2 and 3, respectively. As shown in Table 2, the limiting log P value for growth of the genus Pseudomonas varied from 3.1 to 3.4. They could grow in such solvents as p-xylene (log P = 3 . 1 ) and cyclohexane (log P = 3 . 4 ) , but not in solvent having lower log P values, such as styrene (log P = 2 . 9 ) and

TABLE 1. Log P values of commonly used organic solvents

Solvents Log pa Solvents Log pa

Benzene 2.0 Hexane 3.9 Heptanol 2.4 Diphenylether 4.2 Toluene 2.8 Cyclooctane 4.5 Styrene 2.9 Isooctane 4.8 p-Xylene 3.1 Octane 4.9 Et hylbenzene 3.3 Hexylether 5.1 Cyclohexane 3.4 Nonane 5.5 o-Dichlorobenzene 3.6 Decane 6.0 Propylbenzene 3.8 Dodecane 7.0

a Log P values were calculated using the CLOG program version 3.33 (Pomona College, USA).

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TABLE 2. Solvent tolerance of Gram-negative bacteria

NOTES 195

TABLE 3. Solvent tolerance of Gram-positive bacteria

Limiting log P Limiting log P Type strains Type strains values for growth values for growth

Pseudomonas aeruginosa IFO 3924 3.4 I: IFO 3755 3.3

Pseudomonas fluorescens IFO 3507 3.4 a :/ IAM 12022 3.4

Pseudomonas putida IFO 3738 3.1 a :: IFO 1506 3.1

Pseudomonas pseudoalcaligenes ATCC 12815 3.4 Pseudomonas chlororaphis IFO 3904 3.1 Pseudomonas syringae IFO 3310 3.1 Pseudomonas stutzeri IFO 3773 3.4 Escherichia coli IFO 3806 3.8 a

o IFO 3366 3.4 o IFO 3545 3.8 " HB 101 3.4 /t JM 101 3.4 :t JM 109 3.4

Aeromonas hydrophila JCM 1027 4.5 a ~ IFO 3820 4.2

Alteromonas putrefaciens IFO 3908 4.2 Achromobacter delicatulus IAM 1433 3.9 ~ Acinetobacter calcoaceticus IFO 12552 3.9 Agrobacterium tumefaciens IFO 3058 4.8 a Alcaligenesfaecalis JCM 1474 4.5 a Serratia marcescens IFO 3406 3.4

,/ IFO 3736 3.4 Proteus vulgaris IFO 3167 4.2 Proteus mirabilis IFO 3849 3.8 Proteus morganii IFO 3848 4.2 Klebsiellapneumoniae IFO 3317 3.4

:: IFO 3321 3.4 Flavobacterium lutescens IFO 3084 4.2 Flavobacterium suaveolens IFO 3752 5.1 Chromobacterium chocolatum IFO 3758 7.0

Bacillus subtilis AHU 1219 4.9 a :: AHU 1390 4.5 ,/ IFO 3009 5.1

Bacillus brevis IFO 3331 6.0 Bacillus cereus IFO 3131 4.9 Bacillus circulans IFO 3329 7.0 Bacillus macerans IFO 3490 6.0 Bacillus polymyxa IFO 3020 5.1 Bacillus thuringiensis IFO 3951 4.8 Micrococcus luteus IFO 3333 4.8 Micrococcus roseus IFO 3764 4.8 Microcoecus varians IFO 3765 4.8 Staphylococcus aureus IFO 3183 4.8 Staphylococcus epidermMis IFO 3762 4.8 Streptococcus alcalophilus IFO 3531 4.9 Streptococcus faecalis IFO 3826 5. I Streptococcus faecium IFO 3181 5.1 Corynebacterium glutamicum JCM 1318 7.0 a Corynebacterium flavescens IAM 1642 6.0 Corynebacterium herculus ATCC 13868 6.0 Brevibacterium ammoniagenes IFO 12072 7.0 Brevibacterium roseum ATCC 13825 6.0 Brevibacteriumflavum ATCC 13826 7.0 Rhodococcus erythropolis IFO 12320 6.0 Rhodococcus eque IFO 3730 7.0 Leuconostoc mesenteroides IFO 3349 5.1

subsp, dextran Leuconostoc mesenteroides IFO 3832 5.1

subsp, mesente Lactobacillus casei IFO 3425 5.1

subsp, rhamnosus

a Data from ref. 4.

t o l u e n e ( log P = 2 . 8 ) . Pseudomonas putida I F O 3738, Pseudomonas chlororaphis I FO 3904 a n d Pseudomonas syringae IFO 3310 h a d the h ighes t s o l v e n t - t o l e r a n c e a m o n g all t he Pseudomonas species tested. Depending o n the s t ra in , Escherichia coli cou ld g row in the p re sence o f so lven t s h a v i n g log P g rea t e r t h a n 3 .4 or 3.8 ( cyc l ohexane and hexane) , b u t no t in m o r e p o l a r so lven t s . In the same way, o t h e r m i c r o o r g a n i s m s tested had a cha rac t e r i s t i c l imi t ing log P va lue for g r o w t h as s h o w n in T a b l e 2. These b a c t e r i a cou ld g row in so lven t s h a v i n g m o r e t h a n the g r o w t h - l i m i t i n g log P va lue a n d no t in m o r e p o l a r so lven t s w h i c h h a v e lower log P.

As s h o w n in T a b l e 3, the l imi t ing log P va lue for g r o w t h o f Bacillus species r a n g e d f r o m 4.5 to 7.0. T h e y could grow in the presence o f cyclooctane and d o d e c a n e , bu t n o t in so lven t s h a v i n g lower log P values , such as t r i c h l o r o - b e n z e n e (log P = 4.3) a n d h e x a n e (log P = 3.9). Bacillus sub- tilis A H U 1390 h a d the h ighes t s o l v e n t - t o l e r a n c e a m o n g the Bacillus species tested. Also as s h o w n in T a b l e 3, o t h e r G r a m - p o s i t i v e b a c t e r i a h a v e cha rac t e r i s t i c l imi t ing log P va lues for g r o w t h .

Bacillus h a d a wide r a n g e o f g r o w t h - l i m i t i n g log P va lues w h e n c o m p a r e d wi th o t h e r m i c r o o r g a n i s m s tested. T h e r e were s o m e s o l v e n t - t o l e r a n t as well as so lven t - sens i - t ive s t r a in s w i t h i n the genus . Th i s sugges ted t h a t the p ro - per t ies o f the Bacillus ce l l - sur face were m o r e va r i ed t h a n t h o s e o f the o t h e r b a c t e r i a genera .

T h e G r a m - n e g a t i v e b a c t e r i a s h o w e d re la t ively h ighe r sol- v e n t - t o l e r a n c e t h a n the G r a m - p o s i t i v e ones . A m o n g all t he m i c r o o r g a n i s m s tes ted , Pseudomonas g r o u p s h o w e d the h ighes t s o l v e n t - t o l e r a n c e . O n t he o t h e r h a n d , the t o l e r a n c e

o f Chromobacterium, some species w i t h i n the Bacillus, Corynebacterium, B r e v i b a c t e r i u m a n d Rhodococcus to the so lven ts was low. O t h e r g e n e r a s h o w e d m o d e r a t e sol- v e n t - t o l e r a n c e as indicated by the i r l imi t ing log P va lues w h i c h r a n g e d f r o m 3.4 ( cyc looc tane ) to 5.1 (hexyle ther ) .

F r o m these resul ts , the s o l v e n t - t o l e r a n c e o f G r a m - n e g a - t ive a n d G r a m - p o s i t i v e bacteria tested were in the fo l low- ing o r d e r , Pseudomonas> Escherichia, Serratia, Kleb- siella ~ A chromobacter, A cinetobacter, Proteus ~ A ltero- monas, Aeromonas, Flavobacterium > Alcaligenes, Bacil- lus > Agrobacterium, Micrococcus, Staphylococcus'~ Streptococcus > Leuconostoc, Lactobacillus > Corynebac- terium, Chromobacterium, Brevibacterium, Rhodococcus.

These resul t s sugges t t h a t the p o l a r i t y p r o p e r t i e s o f so lven ts affect the ce l l - sur face characteristics and thus the g r o w t h o f the b a c t e r i a s t ra ins . It may , t h e r e f o r e , be possi- b le t h a t ce r t a in u n i q u e ce l l - sur face p r o p e r t i e s c o n t r o l the g r o w t h o f some s t r a ins in w a t e r - s a t u r a t e d o rgan i c so lven t s .

We thank Dr. H. Tanaka of Tsukuba University for reading the manuscript and for many helpful discussions.

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