Abstract:
New microorganisms belonging to Pseudomonas putida or Pseudomonas sp., which are isolated from soil and have tolerance to one or more of hydrocarbons, alcohols, ethers, ketones and their derivatives or their mixture. These new microorganisms can be used in the fields of bioreactor, liquid-waste treatment, protein engineering, etc.

Description:
BACKGROUND OF THE INVENTION 
     This invention relates to biologically pure cultures of microorganisms and, in particular, to biologically pure cultures of microorganisms tolerant to hydrocarbons, alcohols, ethers, ketones or mixtures thereof. 
     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. Microbial., vol. 15, pp. 857˜865 (1967], the growing of Bacterium JOB5 in a medium contining cyclopentane or cyclohexane [J. Ooyama, J. W. Foster, Antonie von Leenwenlook, vol. 31, pp. 45˜65 (1965)], the growing of Pseudomonas sp., Achromobacter sp., and Nocardia sp. in a medium containing benzene, ethyl benzene, toluene or xylene [D. Cleus and N. Walkes, J, Gen. Microbial, vol. 36, pp. 107˜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 of the above cases because the hydrocarbons generally show toxicity to microorganisms. That is, when the fermentation is carried out by using these hydrocarbons as substrates, it is carried by supplying these compounds in the form of steam so that the compounds may not be brought into contact with microorganisms directly or by maintaining the compounds at low concentration (0.2% or 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 adjusting the substrates to low concentration. Furthermore, in case of using slightly water-insoluble substances, there is a disadvantage that the productivity becomes low in the microbial reaction owing to the low solubility of the substances. 
     SUMMARY OF THE INVENTION 
     In order to solve the foregoing problems, the present inventors have extensively searched soil 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 present inventors found out microorganisms having the aforementioned tolerance and completed the present invention. 
     That is, the object of the present invention lies in providing new microorganisms belonging to Pseudomonas putida and Pseudomonas sp. and having toIerance to one or more of aliphatic hydrocarbons, alicyclic hydrocarbons, aromatic hydrocarbons, alcohols, ethers, ketones and their derivatives or their mixture. 
     Because microorganisms belonging to Pseudomonas putida and Pseudomonas sp. according to 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 contamination 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 solvent, the concentration control also becomes possible. Similarly, in case of slightly water-soluble substances to be used also by dissolving in the solvents, they can be used at high concentrations. Thus, the present microorganisms can also contribute to the improvement in productivity in these respects. 
     Furthermore, the present strains are useful as supply sources of resistance genes, and by &amp;he use of the present strains, the cell fusion of solvent tolerant microorganisms producing useful substances and the breeding of said microorganisms according to the gene engineering technology become possible. The present strains have such excellent effects as above, so that they can be utilized widely in the fields of bioreactor, liquid-waste treatment, protein engineering, etc. 
     DETAILED DESCRIPTION OF THE INVENTION 
     As specific examples of the present microorganisms, Pseudomonas putida STM-603, Pseudomonas sp. STM 801 and Pseudomonas sp. STM-904 can be enumerated. These strains 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 the improvement of productivity and the control over the substrate concentration become easier and the prevention of 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 hydrocarbons. Still furthermore, these microorganisms enable the breeding of solvent-tolerant microorganisms producing useful substances by the application of the cell fusion and the gene recombination technology, and are also useful as supply sources of resistance genes. 
     These strains, 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.% 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 aromatic hydrocarbons; 1-heptanol, 1-octanol, 1-decanol, etc. as alcohols; n-hexyl ether, n-butyl phenyl ether, diphenyl ehter, dibenzyl ether, methoxytoluene, etc. as ethers; and 2-pentanone, 2-hexanone, 2-heptanone, cyclohexanone, etc. as kentones can be enumerated. 
     Hereinafter, the bacteriological properties of the present strains, i.e., STM-603, STM-80and STM-904 and the identification results of the same will be given. 
     
         __________________________________________________________________________(1)  Bacteriological Properties of STM-603A.  Morphological Properties (24-hr Culture in a  Bouillon Liquid Medium):a.  Shape and size of the cell:        Rod, 0.7˜1.0 × 2˜4μb.  Polymorphism of the cell:                       -c.  Motility:               +d.  Sporulation:            -e.  Gram&#39;s stain:           NegativeB.  State of Growth in Various Media (24-hr culture  at 30° C.):a.  Bouillon agar plate culture:        Circular colonies of 0.5 to        1 mm with lustrously flesh-        colored surface.b.  Bouillon agar slant culture:        Grown on the surface of the        medium.c.  Bouillon liquid culture:        Grownd.  Bouillon gelatin stab culture:        No gelatin liquefaction takes        place.C.  Physiological Propertiesa.  Nitrate reduction:      Negativeb.  Starch hydrolysis:      Negativec.  Poly-β-hydroxybutyrate hydrolysis:                       Negatived.  Tween 80 hydrolysis:    Negativee.  Arginine hydrolysis:    Positivef.  Pigment formation (King B medium):        Yellowish green, water-soluble        fluorochrome is formed.g.  Oxydase:                Positiveh.  Catalase:               Positivei.  Growth range:   pH:    5.0˜9.5   Temperature:          Not grown at 41° C.j.  Behavior toward oxygen: Aerobick.  O-F test:               Oxidativel.  Citrate utilization:    Positivem.  Levan production from sucrose:                       Negativen.  DNase production:       Negativeo.  Acylamidase production: Negativep.  Assimilation:   D-glucose           +   D-fructose          +   D-xylose            +   D-maltose           -   Sucrose             -   Lactose             -   D-trehalose         -   Mannitol            -   2-ketogluconic acid +   L-valine            +   β-alanine      +   DL-arginine         +   Acetoamido          -   Meso-inositol       -   Benzylamine         +   Geraniol            -__________________________________________________________________________ 
    
     On the basis of the above bacteriological properties, the screening was carried out according to Bergey&#39;s Manual of Determinative Bacteriology (8th ed., 1975). As a result, these properties were compatible with those of Pseudomonas putida. However, Pseudomonas putida is intolerant to hydrocarbons. The tolerance of a standard strain of Pseudomonas putida and the present strain STM-603 to various solvents were examined. The results were given in Table 1. 
     
                       TABLE 1______________________________________        Pseudomonas putidaSolvent      lFO 3738      STM-603______________________________________Toluene      --            +P-xylene     --            +Styrene      --            +______________________________________ 
    
     From the facts that the strain STM-603 and Pseudomonas putida had morphological, physiological and bacteriological properties in common but differed from one another in the behavior toward the solvent tolerance as described above, the present strain was recognized as a new strain belonging to Pseudomonas putida and designated as Pseudomonas putida var. STM-603. The present strain has been deposited in Fermentation Research Institute of Agency of Industrial Science and Technology with the accession number FERM BP-1751 (Bikoken-kinki No. 9228). 
     
         __________________________________________________________________________(2)  Bacteriological Properties of STM-801A.  Morphological Properties (24-hr Culture in a  Bouillon Liquid Medium)a.  Shape and size of the cell:        Rod, 0.7˜1.0 × 2˜15μb.  Polymorphism of the cell:                       -c.  Motility:               + or -d.  Sporulation:            -e.  Gram&#39;s stain:           NegativeB.  State of Growth in Various Media (24-hr culture  at 30° C.):a.  Bouillon agar plate culture:        Circular colonies of 0.5 to        l mm with lustrously flesh-        colored surface.b.  Bouillon agar slant culture:        Grown on the surface of the        medium.c.  Bouillon liquid culture:        Grownd.  Bouillon gelatin stab culture:        No gelatin liquefaction takes        place.C.  Physiological Properties:a.  Nitrate reduction:      Negativeb.  Starch hydrolysis:      Negativec.  Poly-β-hydroxybutyrate hydrolysis:                       Negatived.  Tween 80 hydrolysis:    Negativee.  Arginine hydrolysis:    Positivef.  Pigment formation (King B medium):        Yellowish green, water-soluble        fluorochrome is formed.g.  Oxydase:                Positiveh.  Catalase:               Positivei.  Growth range:   pH:    5.0˜9.0   Temperature:          Not grown at 41° C.j   Behavior toward oxygen: Aerobick.  O-F test:               Oxidativel.  Citrate utilization:    Positivem.  Levan production from sucrose:                       Negativen.  DNase production:       Negativeo.  Acylamidase production: Negativep.  Assimilation:   D-glucose           +   D-fructose          +   D-xylose            +   D-maltose           -   Sucrose             -   Lactose             -   D-trehalose         -   Mannitol            -   2-ketogluconic acid +   L-valine            +   β-alanine      +   DL-arginine         +   Acetoamido          -   Meso-inositol       -   Benzylamine         +   Geraniol            -(3)  Bacteriological properties of STM-904A.  Morphological Properties (24-hr. Culture in a  Bouillon Liquid Medium):a.  Shape and size of the cell:        Rod, 0.7˜1.O × 3˜15μb.  Polymorphism of the cell:                       -c.  Motility:               + or -d.  Sporulation:            -e.  Gram&#39;s stain:           NegativeB.  State of Growth in Various Media (24-hr. culture  at 30):a.  Bouillon agar plate culture:        Circular colonies of 0.5 to        l mm with lustrously flesh-        colored surface.b.  Bouillon agar slant culture:        Grown on the surface of the        medium.c.  Bouillon liquid culture:        Grownd.  Bouillon gelatin stab culture:        No gelatin liquefaction takes        place.C.  Physiological Properties:a.  Nitrate reduction:      Negativeb.  Starch hydrolysis:      Negativec.  Poly-β-hydroxybutyrate hydrolysis:                       Negatived.  Tween 80 hydrolysis:    Negativee.  Arginine hydrolysis:    Positivef.  Pigment formation (King B medium):        Not formed.g.  Oxydase:                Positiveh.  Catalase:               Positivei.  Growth range:   pH:    5.0˜9.0   Temperature:          Not grown at 41° C.j.  Behavior toward oxygen: Aerobick.  O-F test:               Oxidative1.  Citrate utilization:    Positivem.  Levan production from sucrose:                       Negativen.  DNase production:       Negativeo.  Acylamidase production: Negativep.  Assimilation:   D-glucose           +   D-fructose          +   D-xylose            +   D-maltose           -   Sucrose             -   Lactose             -   D-trehalose         -   Mannitol            -   2-ketogluconic acid +   L-valine            +   β-alanine      +   DL-arginine         +   Acetoamido          -   Meso-inositol       -   Benzylamine         +   Geraniol            -__________________________________________________________________________ 
    
     On the basis of bacteriological properties as given in above (2) and (3), the screening was carried out according to Bergey&#39;s Manual of Determinative Bacteriology (8th ed., 1975). As a result, these strains STM-801 and STM-904 were found to be similar to Pseudomonas putida as a known strain. Thus, the present inventors have made a further detailed comparison between the present strains STM-801 and STM-904 and a standard strain of Pseudomonas putida IFO 3738 with respect to their bacteriological properties, thereby obtaining the following results (see Table 2 also): 
     (I) The cell size of Pseudomonas putida is 0.7 to I.0μ by 2 to 4μ, while that of STM-801 and STM-904 are respectively 0.7 to 1.0μ by 2 to 15μ and 0.7 to 1.0μ by 2 to 15μ. That is, the size of the prestrains is 3 to 4 times that of Pseudomonas putida. 
     (2) In case of Pseudomonas putida, all the cells show the motility. However, in case of STM-801 and STM-904, some show the motility but the other do not. 
     (3) In case of Pseudomonas putida, yellowish green water-soluble fluorochrome is formed. 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 recoganized 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 strains STM-801 and STM-904 as Pseudomonas sp. STM-801 and Pseudomonas sp. STM-904 respectively. 
     
                                           TABLE 2__________________________________________________________________________       Pseudomonas putida       IFO 3738  STM-801  STM-904__________________________________________________________________________(1)  Size of Cell       0.7˜1.0 × 2˜4μ                 0.7˜1.0 × 2˜15μ                          0.7˜1.0 × 3˜15μ(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 sp. STM-801 and Pseudomonas sp. STM-904 were deposited in Fermentation Research Institute of Agency of lndustrial Science and Technology respectively with the accession numbers FERM BP-1749 (Bikoken-kinki No. 9226) and FERM BP 1750 (Bikoken kinki No. 9227). 
     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 those 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, peptone, meat extract, corn steep liquor, casamino acid, 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. 
     The culture was carried out at pH 5 to 9 at 20° to 40° C. under aerobic conditions. 
    
    
     TEST EXAMPLE 1 
     Pseudomonas putida var. STM-603, Pseudomonas sp. STM-801, Pseudomonas sp. STM-904 and various known strains were respectively inoculated into the prescribed media (pH 7.0) of respective strains. Then, each 5ml of various solvents given in Table 3 was added to each 5ml of said media. After culturing the resulting media at 37° C. for 48 hours, the growth of each strain was compared. The results were given in Table 3. 
     
                                           TABLE 3__________________________________________________________________________Comparison of Solvent Tolerance of Various Strains          Cyclohexane                 Toluene                      P-xylene                           Styrene__________________________________________________________________________Pseudomonas putida var.           +      +    +    +STM-603Pseudomonas sp. STM-801           +      +    +    +Pseudomonas sp. STM-904           +      +    +    +Pseudomonas aruginosa           -      -    -    -IFO-3924Pseudomonas fluorescens           -      -    -    -IFO-3507Pseudomonas putida           +      -    -    -IFO-3738Pseudomonas pseudoalcaligenes           -      -    -    -ATCC-12815Arthrobacter globiformus           -      -    -    -IFO-3062Agrobacterium tumefaciens           -      -    -    -IFO-3058Escherichia coli IFO-3806           -      -    -    -Bacillus cereus IFO-3131           -      -    -    -Bacillus coagulans IFO-3557           -      -    -    -__________________________________________________________________________ -:Not grown +: Grown (O.D.sub.660 &gt;0.50) Bouillon liquid medium: Pseudomonas, Arthrobacter, Agrobacterium and Bacillus LB medium: Escherichia 
    
     EXAMPLE 1 
     A medium prepared by adding 1.0l of distilled water to 1.0 of glucose, 2.5g of yeast extract and 5.0g of peptone and adjusted to pH 7.0 was dispensed in 100ml portions into 500ml ribbed conical flasks, in which Pseudomonas putida var. STM-603 was inoculated without sterilizing said flasks. After adding each 30ml of toluene to the flasks, the culture as carried out at 37° C. for 48 hours. As a result, 1.2mg/ml of Pseudomonas putida var. STM-603 cell mass was obtained, where the contamination and the growth of other microorganisms were not observed. 
     EXAMPLE 2 
     The same medium as &amp;hat of Example 1 was prepared, dispensed in 5ml portions into large test tubes and steamsterilized at 121° C. for 15 minutes. Then, Pseudomonas putida var. STM-603 was inoculated in the large test tubes, to which each 5ml of various solvents given in Table 4 was added. The culture was carried out at 37° C. using a test tube shaker. The state of growth after 48 hours was given in Table 4. The growth was monitored by measuring turbidity (wave length: 660nm, a colorimeter &#34;Spectronic 21&#34; manufactured by Bausch &amp; Lomb Corp.). 
     
                       TABLE 4______________________________________Tolerance of Pseudomonas putida var. STM-603to Various SolventsSolvent          State of Growth______________________________________Aliphatic hydrocarbons:n-pentane        +n-hexane         +n-heptane        +n-octane         +isooctane        +n-nonane         +n-decane         +1- or 2-hexene   +1-octene         +1-dodecene       +1,3-pentadiene   ±1,5-hexadiene    +1,7-octadiene    +Alicyclic hydrocarbons:cyclopentane     +methyl cyclopentane            +cyclohexane      +methyl cyclohexane            +butyl cyclohexane            +cyclooctane      +Aromatic hydrocarbons:toluene          +p-xylene         +o-,m-p-xylene    +chlorobenzene    +o-dichlorobenzene            +1,2,4-trichlorobenzene            +bromobenzene     +ethyl benzene    +propyl benzene   +styrene          +Alcohols:1-heptanol       +1-octanol        +1-decanol        +Ethers:n-hexyl ether    +n-butyl phenyl ether            +diphenyl ether   +dibenzyl ether   +methoxytoluene   +______________________________________ ±: Grown (0&lt;O.D.sub.660 &lt;0.50) +: Grown (O.D.sub.660 &gt;0.50) 
    
     EXAMPLE 3 
     The same medium as that of Example 1 Was prepared, dispensed in 5m L portions into large test tubes and steamsterilized a&amp; 121° for 15 minutes. Then, Pseudomonas putida var. STM-603 was inoculated in the test tubes, to which each 0.25ml of various solvents given in Table 5 was added. The culture was carried out at 37° C. using a test tube shaker. The state of growth after 48 hours was given in Table 5. The growth was monitored by measuring turbidity (wave length: 660nm, a colorimeter &#34;Spectronic 21&#34; manufactured by Bausch &amp; Lomb Corp.). 
     
                       TABLE 5______________________________________          StrainSolvent        STM-603______________________________________Ketones:2-pentanone    ±2-hexanone     ±2-heptanone    +cyclohexane    ±______________________________________ -: Not grown ±: Grown (0&lt;O.D.sub.660 &lt;0.50) +: Grown (O.D.sub.660 &gt;0.50) 
    
     EXAMPLE 4 
     A medium prepared by adding 1.0l of distilled water to b 1.0g of glucose, 2.5g of yeast extract and 5.0g of peptone and adjusted to pH 7.2 was dispensed in 100ml portions into 500 ml ribbed flasks, in which Pseudomonas sp. STM-801 was inoculated without sterilizing said flasks. After adding each 30ml of toluene to the flasks, the culture was carried out at 37° C. for 48 hours. As a result, 1.1mg/ml of Pseudomonas sp. STM-801 cell mass was obtained, there the contamination and the growth of other microorganisms were not observed. 
     EXAMPLE 5 
     The same medium as that of Example 1 was prepared, dispensed in 5ml portions into large test tubes and steamsterilized at 121° C. minutes. Then, Pseudomonas sp. STM-801 and Pseudomonas sp. STM-904 were respectively inoculated in the large test tubes, to which each 5ml of various solvents given in Table 6 was added. The culture was carried out at 37° C. using a test tube shaker. The state of growth after 48 hours was given in Table 6. The growth was monitored by measuring turbidity (wave length: 660nm, a colorimeter &#34;Spectronic 21&#34; manufactured by Bausch &amp; Lomb Corp.). 
     
                       TABLE 6______________________________________Tolerance of Pseudomonas sp. STM-801 andPseudomonas sp. STM-904 to Various Solvent            State of GrowthSolvent            STM-801  STM-904______________________________________Aliphatic hydrocarbons:n-pentane          +        +n-hexane           +        +n-heptane          +        +n-octane           +        +isooctane          +        +n-nonane           +        +n-decane           +        +1- or 2-hexene     +        +1-octene           +        +1-dodecene         +        +1.3-pentadiene     ±     ±1.5-hexadiene      +        +1,7-octadiene      +        +Alicyclic hydrocarbons:cyclopentane       +        +methyl cyclopentane              +        +cyclohexane        +        +methyl cyclohexane +        +butyl cyclohexane  +        +cyclooctane        +        +Aromatic hydrocarbons:toluene            +        +p-xylene           +        +o-,m-p-xylene      +        +chlorobenzene      +        +o-dichlorobenzene  +        +1,2,4-trichlorobenzene              +        +bromobenzene       +        +ethyl benzene      +        +propyl benzene     +        +styrene            +        +Alcohols:1-heptanol         +        +1-octanol          +        +1-decanol          +        +Ethers:n-hexyl ether      +        +n-butyl phenyl ether              +        +diphenyl ether     +        +dibenzyl ether     +        +methoxytoluene     +        +______________________________________ ±: Grown (0&lt;O.D.sub.660 &lt;0.50) +: Grown (O.D.sub.660 &gt;0.50) 
    
     EXAMPLE 6 
     The same medium as that of Example 1 was prepared, dispensed in 5ml portions into large test tubes and steamsterilized at 121° C. for 15 minutes. Then, Pseudomonas sp. STM-801 and Pseudomonas sp. STM 904 were respectively inoculated in the test tubes, to each of which each 0.25ml of various solvents given in Table 7 was added. The culture was carried out at 37° C. using a test tube shaker. The state of growth after 48 hours was given in Table 7. The growth was monitored by measuring turbidity (wave length: 660nm, a colorimeter &#34;Spectronic 21&#34; manufactured by Bausch &amp; Lomb Corp.). 
     
                       TABLE 7______________________________________         State of GrowthSolvent         STM-801  STM-904______________________________________Ketones:2-pentanone     ±     ±2-hexanone      ±     ±2-heptanone     +        +cyclohexane     ±     ±______________________________________ -: Not grown ±: Grown (0&lt;O.D.sub.660 &lt;0.50) +: Grown (O.D.sub.660 &gt;0.50)