Method for culturing microorganism

A method for culturing microorganisms belonging to the genus Pseudomonas or the genus Escherichia and having tolerance to an organic solvent such as any one of hydrocarbons, alcohols, ethers, ketones and their derivatives or their mixture in a medium containing the organic solvent in a concentration of 0.3% or more. The present method can be widely utilized in the fields of bioreactor, liquid-waste treatment, protein engineering, etc.

BACKGROUND OF THE INVENTION 
This invention relates to a method for culturing a microorganism having a 
tolerance to an organic solvent. In further detail, this invention relates 
to a method for culturing a microorganism having a tolerance to an organic 
solvent selected from among hydrocarbons, alcohols, ethers, ketones, their 
derivatives and their mixtures. 
As conventional examples of culturing a microorganism in a medium 
containing hydrocarbons or their derivatives, there are such many reports 
as of the growing of Nocardia sp. in a medium containing hexane or 
hexadecane [R. L. Raymond, Appl.. Microbiol., vol. 15, pp. 857.about.865 
(1967)], the growing of Bacterium JOB5 in a medium containing cyclopentane 
or cyclohexane [J. Ooyama and J. W. Foster, Antonie von Leenwenlook, vol. 
31, pp. 45.about.65 (965)], the growing of Fseudomonas sp., Achromobacter 
sp., and Nocardia sp. in a medium containing benzene, ethyl benzene, 
toluene or xylene [D. Cleus and N. Walkes, J. Gen. Microbiol, vol. 36, pp. 
107.about.122 (1964)], etc. can be enumerated. However, these 
microorganisms are cultured by bringing the same into contact with 
hydrocarbons which are concentrated low or in the form of steam in any 
case owing to the fact that hydrocarbons generally show toxicity to 
microorganisms. That is, when the fermentation is carried out by using 
these hydrocarbons as substrates, it is carried out by supplying these 
compounds in the form of steam so that the compounds are not brought into 
contact with microorganisms directly or by maintaining these compounds in 
low concentration (0.2or less) at which a toxic effect is not shown. 
Consequently, in the fermentation using hydrocarbons as substrates, there 
are problems not only of low productivity but also of operation because of 
the difficulty in controlling the substrates to low concentration. 
Furthermore, in case of using a lightly water-soluble substance, there is 
a disadvantage that the productivitY becomes low in &he microbial reaction 
going to the low solubility of the substance. 
SUMMARY OF THE INVENTION 
In order to solve the foregoing problems, the present inventors have made 
an extensive search so as to obtain a microorganism which can grow up in a 
medium containing a solvent such as hYdrocarbon or the like in a high 
concentration, that is, a microorganism having a tolerance to a solvent 
such as hydrocarbon or the like. As a result, the prevent inventors found 
out microorganisms having the aforementioned tolerance and have completed 
the invention of a method for culturing these microorganisms in a medium 
containing a solvent. 
That is, the object of the present invention lies in providing a method for 
culturing a microorganism having a tolerance to an organic solvent. 
Because microorganisms belonging to the genus Pseudomonas or the genus 
Escherichia in the present invention have excellent tolerance to a solvent 
containing hydrocarbons, alcohols, ethers, ketones and their derivatives 
or their mixtures which are commonly used, said microorganisms can be 
prevented from the saprophyte coniamination by culturing the same in the 
presence of the above solvents. In this case, because the heat killing is 
not required, it becomes possible to use thermolabile additives. In case 
of carrying out the culture by using these solvents as substrates, the 
substrates can be supplied at high concentrations. Thus, the improvement 
in productivity can be expected. In addition, in case that the substrate 
concentration is high as described above, the control over the addition of 
the substrate becomes easier. In case of toxic substances to be used for 
the culture by dissolving in these solvents , the concentration control 
also becomes possible. Similarly, in case of slightly water soluble 
substances &o be used also by dissolving in the solvents, they can be used 
at high concentrations. Because of the excellent effects as above, the 
present method for culturing a microorganisms can be utilized widely in 
the fields of bioreactor, liquid-waste treatment, protein engineering, 
etc. 
DETAILED DESCRIPTION OF THE INVENTION 
A method for culturing a microorganism according to the present invention 
is characterized by culturing a microorganism belonging to the genus 
Pseudomonas or the genus Escherichia and having a tolerance to an organic 
solvent in a medium containing 0.3% or more organic solvent. 
As a microorganism belonging &o the genus Pseudomonas, Pseudomonas 
aeruminosa, pseudomonas fluorescens, Pseudomonas putida, Pseudomonas sp. 
STM-801, Pseudomonas sp. STM 904 can be enumerated. As a microorganism 
belonging to the genus Escherichia, Escherichia coli can be enumerated. 
As an organic solvent to which these microorganisms show tolerance, any one 
of aliphatic hydrocarbons, alicyclic hydrocarbons, aromatic hydrocarbons, 
alcohols, ethers, ketones and their derivatives or their mixture can be 
enumerated. 
The microorganisms belonging to the genus Pseudomonas or the genus 
Escherichia can grow in a medium containing aliphatic hydrocarbons, 
alicyclic hydrocarbons, alcohols, ethers, ketones, aromatic hydrocarbons 
which are particularly highly toxic and their derivatives in a 
concentration as high as 0.3% or more. In addition, these strains can grow 
even in a medium containing the above compounds in a concentration as high 
as 50% or more. Thus, in the culture of these microorganisms, a substrate 
can be supplied in large quantities, Whereby &he improvement of 
productivity and the control over the substrate concentration become 
easier and the prevention of the saprophyte contamination becomes 
possible. Furthermore, the improvement of productivity in the microbial 
reaction and the control over the concentration of toxic substances become 
possible by dissolving slightly water soluble substances in various 
hydrocarbon. 
The strains belonging to the genus Pseudomonas, i.e., STM-603, STM-801 and 
STM 904 were obtained by culturing soil which the present inventors 
collected from all over the country in media containing 0.1 % glucose, 
0.25% yeast extract, 0.5 % peptone and 50% solvent (aliphatic 
hydrocarbons. alicyclic hydrocarbons, aromatic hYdrocarbons, alcohols, 
ethers and ketones) and then isolating colonies formed. 
As specific examples of solvents, pentane, hexane, heptane, octane, 
isooctane, nonane decane, 1- or 2-hexene, 1-octene, 1-dodecene, 
1,3-pentadiene, 1,5-hexadiene, 1,7-octadiene, etc. as aliphatic 
hydrocarbons; cyclopentane, cyclohexane, methyl cyclopentane, methyl 
cyclohexane, etc. as alicyclic hydrocarbons; toluene, xylene, styrene, 
ethyl benzene, chlorobenzene. etc. as aroma&ic hydrocarbons; 1-heptanol, 
1-octanol, 1-decanol, etc. as alcohols; n-hexyl ether, n-butyl phenyl 
ether, diphenyl ether, dibenzyl ether, methoxytoluene, etc. as ethers; and 
2-pentanone, 2-hexanone, 2-heptanone, cyclohexanone, etc. as kentones can 
be enumerated. 
These strains, STM-603, STM-801 and STM-904 have the following 
bacteriological properties respectively: 
TABLE 1 
__________________________________________________________________________ 
Morphological and Physiological Properties of 
Isoltes STM-603, STM-801 and STM-904 
STM-603 STM-801 STM-904 
__________________________________________________________________________ 
A. Morphological Properties 
(24-hr. culture in a 
bouillon liquid medium) 
a. Shape and Size of Cell 
rod rod rod 
0.7.about.1.0 .mu.m .times. 2.about.4 .mu.m 
0.7.about.1.0 .mu.m .times. 2.about.15 
0.7.about.1.0 .mu.m .times. 
3.about.15 .mu.m 
b. Polymorphism of Cell 
- + or - + or - 
c. Motility - - - 
d. Sporulation 
- - - 
d. Gram's Stain 
- - - 
B. Growth on various media 
(24-hr. culture at 30.degree. C.) 
a. Bouillon agar plate 
Circular colonies 
Circular colonies 
Circular colonies 
culture of 0.5 to 1 mm with 
of 0.5 to 1 mm with 
of 0.5 to 1 mm with 
lustrously flesh- 
lustrously flesh- 
lustrously flesh- 
colored surface 
colored surface 
colored surface 
b. Bouillon agar slant 
Grown on the sur- 
Grown on the sur- 
Grown on the sur- 
culture face of the medium 
face of the medium 
face of the medium 
c. Bouillon liquid medium 
Grown Grown Grown 
d. Bouillon gelatin stab 
No gelatin lique- 
No gelatin lique- 
No gelatin lique- 
culture faction faction faction 
C. Physiological Properties 
a. Nitrate reduction 
- - - 
b. Starch hydrolysis 
- - - 
c. Poly-.beta.-hydroxybutyrate 
- - - 
hydrolysis 
d. Tween 80 hydrolysis 
- - - 
e. Arginine hydrolysis 
+ + + 
f. Pigment formation 
Yellowish green, 
Yellowish green, 
Not formed 
(King B medium) 
water-soluble 
water-soluble 
fluorochrome 
fluorochrome 
g. Oxidase + + + 
h. Catalase + + + 
i. Growth range 
pH 5.0.about.9.5 
5.0.about.9.5 
5.0.about.9.5 
Temperature Not grown at 41.degree. C. 
Not grown at 41.degree. C. 
Not grown at 41.degree. C. 
j. Behavior toward oxygen 
Aerobic Aerobic Aerobic 
k. O-F test Oxidative Oxidative Oxidative 
l. Citrate utilization 
+ + + 
m. Levan production from 
- - - 
sucrose 
n. DNAse production 
- - - 
o. Acylamidase production 
- - - 
p. Assimilation: 
D-glucose + + + 
D-fructose + + + 
D-oxylose + + + 
D-maltose - - - 
Sucrose - - - 
Lactose - - - 
D-trehalose - - - 
Mannitol - - - 
2-ketogluconic acid 
+ + + 
L-valine + + + 
.beta.-alanine 
+ + + 
DL-arginine + + + 
Acetoamido - - - 
Meso-inositol - - - 
Benzyl amine + + + 
Geraniol - - - 
__________________________________________________________________________ 
On the basis of the bacteriological properties given in Table 1, the 
screening of each strain was carried out according to Bergey's Manual of 
Determinative Bacteriology, (8th ed., 1975) was carried out. As a result, 
the bateriological properties of the strain STM-603 were compatible with 
those of Pseudomonas putida. However, Pseudomonas putida is intolerant to 
hydrocarbons. The tolerance of IFO 3738 is a standard strain of 
Pseudomonas putida and the present strain STM-603 to various solvents were 
examined. The results were given in Table 2. 
TABLE 2 
______________________________________ 
Pseudomonas putida 
Solvent IFO 3738 STM-603 
______________________________________ 
Toluene - + 
P-xylene - + 
Styrene - + 
______________________________________ 
From these facts that the strain STM-603 and Pseudo-monas putida had 
morphological, physiological and bateriological properties in common but 
differed from one another in the behavior toward the solvent tolerance, 
the strain STM-603 were recognized as a new strain belonging to 
Pseudo-monas putida and designated as Pseudomonas putida var. STM-603. 
Regarding strains STM-801 and STM-904, Pseudomonas putida can be enumerated 
as their similar known strain. Thus, the present inventors have made a 
further detailed comparison between the present strains STM-801 and 
STM-904 and IFO 3738 as a standard strain of Pseudomonas putida with 
respect to their bacteriological properties, thereby obtaining the 
following results, as Will be also given in Table 3: 
(1) The cell size of Pseudomonas putida is 0.7 to 0.7 to 1.0.mu. by 2 to 
4.mu., while that of STM-801 and STM 904 are respectively 0.7 to 1.0.mu. 
by 2 to 15.mu. and 0.7 to 1.0.mu.by 3 to 15.mu.. That is, the size of the 
present strains is 3 to 4 times that of Pseudomonas putida. 
(2) Regarding the motility, all the cells show the motility in case of 
Pseudomonas putida. However, in case of STM-801 and STM-904, some cells 
show the motility, but others do not. 
(3) Regarding the pigment formation, yellowish green water-soluble 
fluorochrome is formed in case of Pseudomonas putida. However, in case of 
STM-801 and STM-904, the former forms the same pigment as above, but the 
latter does not. 
(4) Regarding the solvent tolerance with respect to toluene, p-xylene and 
styrene, Pseudomonas putida does not show a tolerance at all while STM-801 
and STM-904 show tolerance. 
From these results, it is recognized that the strains STM 801 and STM-904 
correspond to new species because they are obviously different from 
Pseudomonas putida and because there is no known species corresponding to 
these strains. So, the present inventors designated the straina STM-801 
and STM 904 as Pseudomonas sp. STM-801 and Pseudomonas sp. STM-904 
respectively. 
TABLE 3 
__________________________________________________________________________ 
Pseudomonas putida 
IFO 3738 STM-801 STM-904 
__________________________________________________________________________ 
(1) Size of Cell 
0.7.about.1.0 .times. 2.about.4.mu. 
0.7.about.1.0 .times. 2.about.15.mu. 
0.7.about.1.0 .times. 3.about.15.mu. 
(2) Motility 
+ + or - + or - 
(3) Pigment Formation 
Yellowish green, 
Yellowish green, 
Not formed 
water-soluble 
water-soluble 
fluorochrome 
fluorochrome 
(4) Solvent Tolerance: 
Toluene - + + 
P-xylene - + + 
Styrene - + + 
__________________________________________________________________________ 
Said Pseudomonas putida var. STM- 603, pseudomonas sp. STM-801 and 
Pseudomonas sp. STM-04 were deposited in Fermentation Research Institute 
of Agency of Industrial Science and Technology respectively with the 
accession numbers FERM BP-1751 (Bikoken-kinki No. 9228), FERM BP-1749 
(Bikoken-k1nki No. 9226) and FERM BP-1750 (Bikoken-kinki No. 9227). 
As the present microorganisms belonging to the genus Pseudomonas other than 
the aforementioned strains, Pseudomonas aeruginosa IFO 3924, Pseudomonas 
fluorescens IFO-3507, Pseudomonas putida IFO-3738, etc. can be enumerated, 
and as the present microorganism belonging to the genus Escherichia, 
Escherichia coli IFO-3806 can be enumerated. 
As a medium for culturing these strains, an ordinary medium containing a 
carbon source, a nitrogen source, an inorganic ion, etc. is used. 
As a carbon source, any of &hose which can be assimilated, for example, 
sugars such as glucose, fructose, xylose, starch hydrolysate, etc., 
hYdrocarbons such as toluene, p-xylene, etc., alcohols such as methanol, 
ethanol, etc., etc. can be used. As a nitrogen source, yeast extract, dry 
yeast, peptonic, meat extract, corn steep liquor, casamino ac1d, ammonium 
chloride, ammonium sulfate, urea, sodium nitrate, etc. are used. As an 
inorganic ion, phosphoric acid ion, magnesium ion, iron ion, calcium ion, 
potassium ion, copper ion, manganese ion, etc. are used. 
As a solvent, any on.RTM.of aliphatic hYdrocarbons, alicyclic hydrocarbons, 
aromatic hydrocarbons, alcohols, ethers, ketones and their derivatives or 
&heir mixture can be used. As specific examples of these solvents, 
pentane, hexane, heptane, octane, isooctane, nonane, decane, 1- or 2- 
hexene, 1-idene, 1-dodecene, 1,3-pentadiene, i,5-hexadiene, 1,7 octadiene, 
etc. as aliphatic hydrocarbons; cyclopentane, cyclohexane, methyl 
cyclopentane, methyl cyclohexane, etc. as alicyclic hydrocarbons; toluene, 
xylene, styrene, ethyl benzene, chlorobenzene, etc. as aromatic 
hydrocarbons; 1-heptanol, 1-octanol, 1 decanol, etc. as alcohols; n-hexyl 
ether, in butyl phenyl ether, diphenyl ether, dibenzyl ether, 
methoxytoluene, etc. as ethers; and 2 pentanone, 2-hexanone, 2-heptanone, 
cyclohexanone, etc. as kentones can be enumerated. The contents of these 
solvent in a medium is 0.3% or more. Said solvents may be either contained 
in a medium previously or add.RTM.d to a medium afterwards. 
The culture is carried out at pH 5 to 9 at 20 to 40.degree. C. under 
aerobic conditions.