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Microbiological Studies on Petroleum and Natural Gas Iii J. Gen.V Appl. Microbiol. ol. 11, No. 1, 1965. MICROBIOLOGICAL STUDIES ON PETROLEUM AND NATURAL GAS III. DETERMINATION OF BREVIBACTERIUM, ARTHROBACTER, MICRO CO CC US, SAR CINA, AL CALIGENES, AND A CHROMOBA CTER ISOLATED FROM OIL-BRINES IN JAPAN HIROSHI IIZUKA and KAZUO KOMAGATA* Institute of Applied Microbiology, University of Tokyo, Tokyo Received August 24, 1954 In the previous papers (1, 2, 3), it was briefly reported that about ten million aerobic bacteria were found in one ml of oil-brines raised from the oil-layers of a depth from two hundred to seven hundred meters in Japan. They belonged mainly to the genus Pseudomonas, but a few other bacteria were also isolated. This paper deals with the determinative studies of Brevibacterium, Art hrobacter, Micrococcus, Sarcina, Alcaligenes, and Achro- mobacter isolated from oil-brines in the petroleum zones in Japan. MATERIALS AND METHODS Isolation and Determination of Microorganisms. Methods of isolation and determination of bacteria were mainly those described in the previous papers (3, 4, 5, 6, 7, S). For the tests of assimilation of carbon compounds both liquid media and SIMMON'sagar slants supplemented with ammonium salt as the sole nitrogen source were employed. Glucose, gluconate, citrate, and succinate were used as the sole carbon source. Pro- duction of blue pigment from nicotin was ascertained by testing the growth on nicotin agar slants at 30°. Nicotin agar was composed of nicotin, 4.0 g ; KH2PO4, 2.0 g ; KCI, 5.0 g ; yeast extract, 0.1 g ; trace amounts of MgSO4.7H2O and FeSO4.7H2O; and tap water, 1000 ml; and adjusted pH to 6.8. RESULTS AND DISCUSSION All the brevibacteria isolated from oil-brines belonged to motile species such as Brevibacterium pusillum, Brev. helvolum, and Brev. sulfureum. They were aerobic, Gram-positive, non-sporef orming, non-pleomorphic, uni- cellular, and small rod-shaped bacteria. They were motile with peritrichous flagella, mostly mono-lateral. Only a few motile species of Brevibacterium have been described (7, 9). It is of interest that a considerable number of T Present address: Central Research Laboratories , Ajinomoto Co., Inc., Suzuki•cho, Kawasaki, Japan. 1 2 IIZUKA and KOMAGATA VOL. 11 motile brevibacteria were isolated from oil-brines. In the previous papers (T, 8), the taxonomic position of motile brevibacteria isolated from rice in comparison with other allied genera, such as Corynebacterium, Art hrobacter, Microbacterium, Propionibacterium, Pseudobacterium, etc., was discussed, and the reason why aerobic, Gram-positive, non-sporef orming, non-pleomorphic, unicellular, motile, and small rod-shaped bacteria should be included in the genus Brevibacterium was described. The brevibacteria described in this paper have enough characteristics of the genus Brevibacterium already reported by the present authors. The new species of Brev. pusillum was identified by us. Arthrobacter ureafaciens and Arth. tumescens are non-motile and rudimentary branchings of cells are observed. Cocci isolated from oil-brins were usually yellow-pigmented and belonged to Micrococcus varians, Micr. conglomeratus, and Sarcina lutea. Limited numbers of Alcaligenes and Achromobacter were also isolated from oil-brins. As mentioned above, con- siderable numbers of aerobic bacterial species except Pseudomonas were found in oil-brines, but the numbers of colonies of these, such as Brevi- bacterium, Arthrobacter, Micrococcus, Sarcina, Alcaligenes and Achromobac- ter, were less than those of Pseudomonas which appeared on isolation media. These bacteria were nutritionally more complex than pseudomonads. It is believed that these bacteria may be secondary invaders of oil-brines and are of small significance in microflora of petroleum. DESCRIPTIONS 1. Brevibacterium pusillum nov. sp. Strains: Nos. 100 and 106 The isolates were non-sporeforming, Gram-positive, and motile rods possessing peritrichous flagella, mostly mono-lateral. Pleomorphic changes were not observed. Acid was produced aerobically and anaerobically from carbohydrates. The isolates seemed to be similar to Brev. imperiale (STEINHAUS) BREEDand Brev. helvolum (ZIMMERMANN)LOCHHEAD. In comparison with Brev. imperiale ATCC 8365 which is considered to be the type culture, the isolates differed in respect to characteristics of colonies, hydrolysis of starch, and pro- duction of acid from lactose. Moreover, the isolates were not identical with the below mentioned strains of Brev. helvolum isolated by us in respect to characteristics of colonies, liquefaction of gelatin, and production of hydrogen sulfide. From these facts, the isolates were identified with a new species. Strain No. 100 produced acid more strongly from carbohydrates than strain No. 106. The type culture is Brev. pusillum No. 100, and deposited with the Institute of Applied Microbiology, University of Tokyo. Small rods, 0.4 to 0.6 by 0.8 to 1.0 microns. Non-pleomorphic. Motile with peritrichous flagella, usually mono-lateral. Spore not formed. Gram- positive. Metachromatic granule is not observed. Nutrient agar colonies: Circular, smooth, entire, raised, glistening, dull yellow to pale yellow, opaque, butyrous. 1965 Microbiological studies on petroleum and natural gas 3 Yeast extract-peptone agar colonies: Small, circular, smooth, entire, raised, slightly dull or glistening, yellowish gray, opaque, butyrous. Nutrient agar slant: Growth moderate, filif orm, glistening, yellowish gray to yellowish brown, opalescent. Photo 1. Brev. pusillum nov. sp. Photo 2. Brev. pusillum nov. sp. No. 100 Cells grown on nutrient agar No. 106 Cells grown on nutrient agar slant for 1.8 hr at 25°. Stained by slant for 18 hr at 25°. Stained by Toda's methods. Toda's method. Yeast extract-peptone agar slant: Growth moderate, filif orm, glistening, yellowish gray, opaque, medium unchanged. Glutamate agar slant: No growth. Nutrient broth : Slightly turbid. Nutrient gelatin stab: No liquefaction. (Variation: Strain No. 106 slowly liquefied gelatin after 20 days.) B.C.P. milk: Slightly acid, not coagulated. Potato: Growth moderate, pale yellow. Nitrite is not produced from nitrate in nitrate broth. Nitrate respiration: Negative. Indole not produced. Hydrogen sulfide not produced. Starch hydrolyzed. M.R. test: Negative. Acetylmethyl carbinol not produced. Slight amounts of acid are produced from glycerol, xylose, glucose, and sucrose, but not from lactose and starch in peptone media after 7 to 10 days. According to HUGH and LEIFsoN's method, acid, but no gas, is aero- bically produced from glycerol, xylose, glucose, and sucrose, but not from lactose and starch. Anaerobically, slight amounts of acid are produced from xylose, glucose, and sucrose, but not from glycerol, lactose, and starch. Glucose, gluconate, citrate, and succinate are not utilized as sole carbon sources with ammonical nitrogen. Cellulose not attacked. 4 IIZUKAand KOMAGATA VOL. 11 Blue pigment is not produced on nicotin agar slant. Optimum temperature for growth: 25° to 30°. Growth scanty at 20° and 37°. Optimum pH for growth: Between 5.0 and 8.0. No growth at 4.0 and 9.0. Catalase: Positive. Aerobic. Source: Oil-brines in Yabase oil-field, Akita Prefecture, Japan, 1957. 2. Brevibacterium helvolum (ZIMMERMANN)LOCHHEAD Strains: Nos. 123, 129, 9-6, and 9-8. The isolates were non-sporef orming, Gram-positive, and motile rods possessing peritrichous flagella. Pleomorphic changes were not observed. Acid was produced from carbohydrates. Strain Nos. 123 and 129 produced Photo 3. Brev. helvolum No. 123 Cells grown on nutrient agar slant for 18 hr at 25°. Stair ed by Toda's method. Photo 4. Brev. helvolum No. 129 Cells grown on nutrient agar slant for 18 or at 30°. Stained by Toda's method. Photo. 5. Brev. helvolum No. 9-6 Cells grown on nutrient agar slant for 18 hr at 3J°. Stained by Toda's method. Photo. 6. Brev. helvolum No. 9-8 Cells grown on nutrient agar slant for 18 hr at 30°. Stained by Toda's method. 1965 Microbiological studies on petroleum and natural gas 5 nitrite from nitrate in nitrate broth, but strains Nos. 9-6 and 9-8 did not produce nitrite from nitrate. This species was so briefly described (10) that newly obtained characteristics were added to the description. As mentioned above, this species seemed to be similar to Brev. pusillum, but differed in respect to characteristics of colonies, liquefaction of gelatin, and production of hydrogen sulfide. Rods, 0.4 by 1.0 to 1.6 microns. Non-pleomorphic. Motile with peri- trichous flagella. Spore not formed. Gram-positive. Metachromatic granule is not observed. Nutrient agar colonies: Circular, smooth, entire, raised to convex, glistening, pale yellow to dull yellow, opaque, butyrous. Yeast extract-peptone agar colonies: Circular, smooth, entire, convex, glistening, dull yellow (citron yellow), opaque, butyrous to slightly viscid. Nutrient agar slant: Growth moderate, filiform, glistening, pale yellow to dark yellow, opaque, medium unchanged. Yeast extract-peptone agar slant: Growth moderate, filiform, glistening, pale olive to citron yellow, medium unchanged. Glutamate agar slant: No growth. Nutrient broth: Ring, moderately turbid. Nutrient gelatin stab: Liquefaction. B.C.P. milk: Almost unchanged. (Variation: Strain No. 9-6 digested casein in alkaline reaction.) Potato: Growth moderate, yellow. (Variation: Strain No. 9-6 showed wrinkled surface.) Nitrite is produced from nitrate in nitrate broth. (Variation: Strain Nos. 9-6 and 9-8 did not produce nitrite from nitrate in nitrate broth.) Nitrate respiration: Negative. Indole not produced. Hydrogen sulfide
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