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, unicellular, 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, considerable 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 production 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 aerobically 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 peritrichous 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 produced. M.R, test: Negative. Acetylmethyl carbinol not produced. Neither acid nor gas is produced from glycerol, xylose, glucose, sucrose, lactose and starch in peptone media. According to HUGH and LEIF50N's method, acid, but no gas, is produced aerobically from glycerol, xylose, glucose, and sucrose, but not from lactose and starch. (Variation: Strain No. 9-6 did not produce acid from xylose. Strain No. 9-8 produced acid from starch but not from glycerol.) Anaerobic production of acid from carbohydrates is not so clear. Glucose, gluconate, citrate, and succinate are not utilized as sole carbon sources with ammonical nitrogen. Cellulose not attacked. Blue pigment is not produced on nicotin agar slant. Optimum temperature for growth: 20° to 30°. No growth at 37°. Optimum pH for growth: Between 6.0 and 8.0. No growth at 9.0. 6 IIZUKAand KOMAGATA VOL. 11

Catalase : Positive. Aerobic. Source: Oil-brines in Yabase oil-field, Akita Prefecture, Japan, 1957. 3. Brevzbacterzum sulfureum (BERGEYet al.) BREED Strain: No. 8-3. The strain tested was non-sporef orming, Gram-positive, and motile rods possessing peritrichous flagella, mostly mono-lateral. Pleomorphic changes were not observed. The isolate seemed to be similar to Brev. helvolum, but differed in respect to hydrolysis of starch, production of acid from carbohydrates, and assimilation of carbon and nitrogen compounds. As this species was so concisely reported (11), newly obtained characteristics were

Rods, 0.6 by 1.2 to 1.6 microns. Non-pleomorphic. Motile with peritrichous flagella. Spore not formed. Gram-positive. Metachromatic granule is not observed. Nutrient agar colonies: Large, circular, smooth, entire, raised, glistening, dull yellow, opaque, butyrous. Yeast extract-peptone agar colonies: Circular, smooth, entire, raised, glistening, pale brownish yellow to dull yellow, opaque, butyrous to slightly Photo. 7. Brev. sulfureum No. viscid. 8-3 Cells grown on nutrient agar Nutrient agar slant: Growth slant for 18 hr at 30°. Stained by Toda's method. moderate, filif orm, glistening, dull yellow to dark yellow, opaque. Yeast extract-peptone agar slant: Growth moderate, filif orm, glistening, dull yellow (olive yellow). Glutamate agar slant: Growth scanty, pale yellow. Nutrient broth: Ring, slightly turbid. Glutamate broth: Growth moderate, turbid. Nutrient gelatin stab: Liquefaction. B.C.P. milk: Alkaline. Potato: Growth moderate, yellow. Nitrite is not produced from nitrate in nitrate broth. Nitrate respiration: Negative. Indole not produced. Hydrogen sulfide produced. Starch not hydrolyzed. M.R. test: Negative. Acetylmethyl carbinol not produced. No acid or gas from glycerol, xylose, glucose, sucrose, lactose, and 1965 Microbiological studies on petroleum and natural gas 7 starch in peptone media. According to HUGH and LEIFs0N's method, no acid or gas is produced aerobically or anaerobically from glycerol, xylose, glucose, sucrose, lactose, and starch. Glucose, gluconate, citrate, and succinate are utilized as sole carbon sources with ammonical nitrogen. Cellulose not attacked. Blue pigment is not produced on nicotin agar slant. Optimum temperature for growth: 25° to 30°. Optimum pH for growth: Between 7.0 and 8.0. No growth at 5.0. Catalase: Positive. Aerobic. Source: Oil-brine in Yabase oil-field, Akita Prefecture, Japan, 1957. 4. Arthrobacter ureafaciens (KREBS and EGGLESTON)CLARK Strains: Nos. 113 and 116 The isolates were pleomorphic. Rod-shaped cells were dominant in young cultures ; cells were short and became coccoid in old cultures. Cells swollen at one or both ends, with rudimentary branching, and of large coccoid-shape (cystite) were observed. Ammonical nitrogen was assimilated. In comparison with Arth. ureafaciens ATCC 7562 which is considered to be the type culture (9,12,13), the isolates agree well except hydrolysis of starch. Furthermore, the isolates seemed to be similar to Arth. oxydans SGUROSaccording to the description (9,14), but differed in respect to production of nitrite from nitrate and production of hydrogen sulfide. It was reported that some strains of Arth. oxydans produce blue pigment on nicotin agar slants (9,14). The isolate of strain No. 113 produced blue pigment on nicotin agar slants, but Arth, ureafaciens ATCC 7562 and the isolate of strain No. 116 did not. It is considered that these two species of Arthrobacter are taxonomically similar to each other. The isolates were identified with Arth. ureafaciens because this species was reported prior

Photo. 8. Arth. ureafaciens No. Photo. 9. Arth. ureafaciens No. 113 Cells grown on nutrient agar 116 Cells grown on nutrient agar slant for 2 days at 30°. Gram stain. slant for 2 days at 30°. Cell wall strain by Bisset and Hale's method. 8 IIZUKAand KOMAGATA VOL. 11 to Arth. oxydans. As this species was so concisely reported (7,14,15), the newly observed characteristics were added to the description. Rods, 0.6 to 0.8 by 1.4 to 2.0 microns. Curved, swollen, club-shaped cells and rudimentary branching of cells are observed. Pleomorphic. Non- motile. Spore not formed. Gram-positive. Metachromatic granule is not observed. Nutrient agar colonies : Circular, smooth, entire, raised, yellowish gray, opaque, butyrous. Yeast extract-peptone agar colonies: Circular, smooth, entire, raised, glistening, pale yellow opaque, butyrous medium slightly brown. Glutamate agar colonies: Circular, smooth, entire, covex, pale yellow, opaque, butyrous. Nutrient agar slant: Growth moderate, filiform, glistening, smooth, grayish yellow, opaque. Yeast extract-peptone agar slant: Growth moderate, filiform, glistening, dull yellow, opaque. Glutamate agar slant: Growth moderate, filiform, glistening, smooth, yellow, opaque, medium unchanged. Nutrient broth: Very slightly turbid. Nutrient gelatin stab : Liquefaction. Milk: Becoming pink color after 20 days, and peptonized after 35 days. B.C.P. milk: Alkaline, and peptonized after 35 days. Potato: Dirty yellow. Nitrite is not produced from nitrate in nitrate broth. Nitrate respiration: Negative. Indole not produced. Hydrogen sulfide produced. Starch hydrolyzed. M.R. test: Negative. Acetylmethyl carbinol not produced. No acid or gas from glycerol, xylose, glucose, sucrose, lactose, and starch in peptone media. According to HUGH and LEIFs0N's method, acid, but no gas, is aerobically produced from glycerol, xylose, glucose, and sucrose, but not from lactose and starch. (Variation: Strain No. 116 did not produce acid from xylose.) Anaerobically, no acid or gas is produced from the carbohydrates mentioned above. Glucose, gluconate, citrate, and succinate are utilized as sole carbon sources with ammonical nitrogen. Blue pigment is produced on nicotin agar slants by strain No. 113, but not by strain No. 116. Color of this pigment turns from blue to brown. Optimum temperature for growth: 20° to 30°. No growth at 37°. Optimum pH for growth: Between 6.0 and 8.0. Catalase : Positive. 1965 Microbiological studies on petroleum and natural gas 9

Aerobic. Source: Oil-brine in Yabase oil-field, Akita Prefecture, Japan, 1957. 5. Arthrobacter tumescens (JENSEN) CONN and DIMMICK Strains: Nos. S-1 and 8-2. The strains tested were Gram-positive and pleomorphic. In comparison with Arth. tumescens ATCC 6947 which is considered to be the type culture, the isolates agree well with it. In BERGEY's Manual, 7th edition (9), Arth. tumescens was described as follows: "unable to utilize nitrates or ammonium salts as nitrogen source, or citrate as sole carbon source ", and "starch weakly hydrolyzed " . In the paper of CoNN and DIMMICK(15), this species was described as having " no diastatic action" and "it apparently utilizes urea and NO3 nitrogen ". The description of CONN and DIMMICK was based upon a single strain obtained from JENSEN. However, in the original description, JENSEN(16) described it as follows: "nitrate, ammonia and asparagin are but very slightly ulilized " and "starch is hydrolyzed faintly by strain B ". In JENSEN'S paper, it was reported that one of the three strains hydrolyzed starch. According to our tests, Arth. tumescens ATCC 6947 could utilize ammonium salt as the sole source of nitrogen, and weakly hydrolyzed starch. From these descriptions and the results obtained, it is considered that Arth. tumescens can utilize inorganic nitrogen as the sole source of nitrogen, and hydrolysis of starch varies from strain to strain. It is described in BERGEY'sManual, 7th edition that Arth. tumescens is catalase-negative, but the strain of Arth. tumescens ATCC 6947 and the isolates are catalase-positive. From these points of view, the isolates were identified with Arth. tumescens. Rods, 0.6 by 1.2 to 1.6 microns. Swollen cells, snapping cells, large coccoid (cystite) and rudimentary branching of cells are observed. Pleomorphic. Non-motile. Spore not formed. Gram- positive. Metachromatic granule is not observed. Nutrient agar colonies: Circular, Photo . 10. Arth. tumescens No. smooth or slightly rough, entire, raised, 8-1 Cells grown on nutrient agar dull, brownish white or yellowish slant for 18 hr at 30°. Gram stain. gray, opaque, butyrous. Yeast extract-peptone agar colonies: Circular, smooth, entire, raised, glistening, pale yellowish brown or yellowish gray, opaque, slightly viscid. Nutrient agar slant: Growth moderate, filif orm, dull, pale olive to light olive, opaque. Yeast extract-peptone agar slant: Growth moderate, filif orm glistening, yellowish gray, opaque. 10 IIZUKA and KOMAGATA VOL. 11

Glutamate agar slant : Growth moderate, filif orm, glistening, yellowish gray, medium unchanged. Nutrient broth : Slightly turbid. Glutamate broth : Slightly turbid, viscid sediment. Nutrient gelatin stab : Liquefaction. B.C.P. milk: Almost unchanged or slightly alkaline, not peptonized. Potato : Growth moderate, cinnamon brown. Nitrite is produced from nitrate in nitrate broth and succinate-nitrate broth. Nitrate respiration : Negative. Indole not produced. Hydrogen sulfide produced. Starch hydrolyzed. M.R. test: Negative. Acetylmethyl carbinol not produced. No acid or gas from glycerol, xylose, glucose, sucrose, lactose, and starch in peptone media. According to HUGH and LEIFs0N'S method, acid, but no gas, is produced aerobically from glycerol, glucose, and sucrose, but not from xylose, lactose and starch. Anaerobically, no acid or gas is produced from the carbohydrates mentioned above. Glucose, gluconate, citrate, and succinate are utilized as sole carbon sources with ammonical nitrogen. Cellulose not attacked. Blue pigment is not produced on nicotin agar slant. Optimum temperature for growth: 20° to 30°. No growth at 37°. Optimum pH for growth: Between 7.0 and 8.0. No growth at 5.0. Catalase : Positive. Aerobic. Source: Oil-brine in Yabase oil-field, Akita Prefecture, Japan, 1957. 6. Micrococcus varians MIGULA Strains: Nos. 3-1 and 6-1 The isolates were Gram-positive cocci, and 0.8 to 1.2 microns in diameter. The colonies on nutrient agar were circular, smooth, rasied, and yellowish white to pale yellow. Margin was entire and texture was butyrous. Fre- quently colonies showing umbonate elevation were found. Growth on HUCKER's medium and change of indicator were recognized. Scant growth was found on glutamate agar. Slight turbidity was observed in nutrient broth. B.C.P. milk was slightly alkaline, but peptonization and coagulation were not observed. Gelatin was not liquefied. Nitrite was produced from nitrate in nitrate broth. No production of indole and hydrogen sulfide was observed. Urea was not decomposed. Acid was produced from glucose, but not from glycerol, xylose, sucrose, lactose, and starch in peptone media. They were isolated from oil-brines in Yabase oil-field, Japan, in 1957. 7. Micrococcus con glomeratus MIGULA Strains: Nos. 5-3, 5-7, and 6-3. 1965 Microbiological studies on petroleum and natural gas 11

The isolates were Gram-positive cocci, and 0.6 to 1.0 microns in diameter. The colonies on nutrient agar were circular, smooth, raised, glistening, opaque and yellowish white to pale yellow. Margin was entire and texture was butyrous. The color of strain No. 6-3 was pale brownish white. Growth was recognized on HucKER's medium and on glutamate agar slant. Slight turbidity was observed in nutrient broth. B.C.P. milk was acidified and coagulated, but peptonization was uncertain. Gelatin was liquefied. Nitrite was produced from nitrate in nitrate broth. Indole and hydrogen sulfide were not produced. Urea was not decomposed. Acid was produced from glucose, but not from glycerol and lactose. Production of acid from xylose, sucrose, and starch was variable from strain to strain. The strains tested were isolated from oil-brines in Yabase oil-field, Akita Prefecture, Japan, in 1957. 8. Micrococcus luteus (SCHROETER)CoHN Strain : No. 119 The isolate was Gram-positive cocci, and 1.0 to 1.5 microns in diameter. The colonies on nutrient agar were circular, smooth, glistening, opaque and pale yellow. Margin was entire and texture was butyrous. Scant growth was observed on HUCKER's medium and glutamate agar. Nutrient broth was slightly turbid. B.C.P. milk was alkaline. Peptonization and coagulation of milk were not recognized. Gelatin was not liquefied. Nitrite was not produced from nitrate in nitrate broth. Indole and hydrogen sulfide were not produced. Urea was not decomposed. No acid was produced from glycerol, xylose, glucose, sucrose, lactose, and starch in peptone media. The strain tested were isolated from oil-brine in Yabase oil-field, Akita Prefecture, Japan, in 1957. 9. Micrococcus candidus CoHN Strains: Nos. 5-5 and 5-8. The isolates were Gram-positive cocci, and 0.7 to 1.2 microns in diameter. The colonies on nutrient agar were circular, smooth, raised, glistening, opaque, and white. Margin was entire. Scant growth was recognized on HUCKER's medium and glutamate agar. Scant growth was observed in nutrient broth. B.C.P. milk was acidified and coagulated, but not peptonized. Gelatin was not liquefied. Nitrite was not produced from nitrate in nitrate broth. No production of indole and hydrogen sulfide was observed. Urea was not decomposed. Acid was produced from glucose, but not from glycerol, xylose, sucrose, lactose, and starch in peptone media. These strains were isolated from oil-brine in Yabase oil-field, Akita Prefecture, in 1957. 10. Sarcina lutea SCHROETER Strain: No. 2-2. The isolate was Gram-positive packet-shaped cocci, and 1.0 to 1.6 microns in diameter. Motility was not observed. The colonies on nutrient agar were circular, smooth, glistening, opaque, and yellow. Margin was entire, and elevation was raised or convex. Growth on nutrient agar slants was 12 IIZUKA and KOMAGATA VOL. 11 abundant, filif orm, and yellow. No growth was observed in glutamate agar slants. Reaction of B.C.P. milk was alkaline, and amorphous precipitate was formed, but peptonization was uncertain. Gelatin was liquefied. Nitrite was not produced from nitrate in nitrate broth. Indole was not produced. hydrogen sulfide was produced. Urea was not decomposed. No acid was produced from glycerol, xylose, glucose, sucrose, lactose, and starch in peptone media. Starch was hydrolyzed. The strain tested was isolated from oil-brine in Yabase oil-field, Akita Prefecture, Japan, in 1957. 11. Sarcina lutea subsp. flava KOCURand MARTINEC Strains : Nos. 1-1, 1-6, 1-9, 2-3, 2-9, 3-3, 3-4, 3-8, 4-1, 4-3, 7-2, and 7-3. Separation of S. lutea SCHROETERand S. flava DE BARY is difficult because these species demonstrate similar characteristics. KOCURand MARTINEC (17) divided S. lutea into one species and one subspecies on the basis of hydrolysis of starch, i.e., the starch hydrolyzing strain was identified with S. lutea, and the strain incapable of hydrolyzing starch was named S. lutea subsp, flava. According to their opinion, isolates which did not hydrolyze starch were identified with S, lutea subsp. flava. These strains exhibited the same characteristics of S. lutea No. 2-2 except hydrolysis of starch. However, about half of the strains (Nos. 1-1, 1-6, 2-3, 2-9, 3-4, 4-3, and 7-3) grew on glutamate agar slants, and urea was converted to ammonia by five strains (Nos. 2-3, 2-9, 3-3, 3-4, and 4-3). Color of colonies on nutrient agar plates of strain No. 7-2 and 7-3 was yellowish white. The strains were isolated from oil-brines in Yabase oil-field, Akita Prefecture, Japan, in 1957. 12. Alcaligenes f aecalis CASTELLANIand CHALMERS Strains: Nos. 56 and 80. The isolates were Gram-negative and motile rods possessing peritrichous flagella. The colonies on nutrient agar were circular, smooth, glistening, opaque, and pale yellowish brown. Evevation of colonies was convex, and texture was butyrous. Scant or no growth was observed on glutamate agar. In nutrient broth fragile pellicles were formed, and moderate turbidity was recognized. Reaction of B.C.P, milk was alkaline. Gelatin was not liquefied. On potato, strain No. 80 grew moderately and showed pale brown coloration, but strain No. 56 failed to grow. Nitrite was produced from nitrate in nitrate broth and succinate-nitrate broth. The isolates grew anaerobically by nitrate respiration. Indole was not produced. Hydrogen sulfide was produced by strain No. 56. Starch was not hydrolyzed. M.R. test was negative and acetylmethyl carbinol not produced. No acid was produced from glycerol, xylose, glucose, sucrose, lactose, and starch in peptone media. According to HUGH and LEIFSON's method, neither aerobic and anaerobic pro- ductions of acid from glucose and lactose were observed. The isolates grew at various temperatures including 20°, 25°, and 30°, but failed to grow at 37°. The isolates grew at pH from 6.0 to 9.0, but not at 4.0. The strains 1965 Microbiological Studies on petroleum and natural gas 13 tested were isolated from oil-brines in Yabase oil-field, Akita Prefecture, Japan, in 1957. 13. Achromobaeter delicatulus (JORDAN)BERGEY et al. Strain: No. 95. The isolate was Gram-negative motile rods, possessing peritrichous flagella. The colonies on nutrient agar were circular, smooth, glistening, and dull yellow. Colonies were raised and texture was butyrous. The

Photo. 11. Al. faecalis No. 80 Photo 12. Ach. delicatulus No. 95 Cells grown on nutrient agar slant Cells grown on nutrient agar slant for 18 hr at 20°C. Stained by Toda's for 18 hr at 20°. Stained by Toda's method. method.

color of colonies was pale yellowish brown (straw color) on yeast extract- peptone agar. The growth on glutamate agar slants was moderate, filiform, glistening, and pale yellow with metallic sheen. In nutrient broth slight turbidity was observed. B.C.P. milk was acidified and coagulated after 20 days. Gelatin was slowly liquefied. Growth on potato was moderate and pale yellowish brown. Nitrite was produced from nitrate in nitrate broth and succinate-nitrate broth. The isolate failed to grow anaerobically by nitrate respiration. Indole was not produced. Hydrogen sulfide was produced. Starch was not hydrolyzed. M.R. test was positive, and acetylmethyl carbinol was not produced. Acid was produced from xylose and glucose, but not from glycerol, sucrose, lactose, and starch in peptone media. Ac- cording to HUGH and LEIFSON's method, acid was produced aerobically or anaerobically from glucose, but not from lactose. The isolate grew at various temperatures including 20°, 25°, and 30°, but did not grow at 37°, Growth was recognized between pH 5.0 and pH 9.0, but not at pH 4.0. The strains tested was isolated from oil-brines in Yabase oil-field, Akita Prefec- ture, Japan, in 1957.

SUMMARY

Determinative studies were carried out with Brevibacterium, Arthro- bacter, Micrococcus, Sarcina, Alcaligenes and Achromobacter isolated from 14 IIZUKA and KOMAGATA VOL. 11 oil-brines obtained in petroleum zones in Japan, and the following species were identified: Brevibacterium pusillum nov. sp., 2 strains; Brev. helvolum (ZIMMERMANN)LOCHHEAD, 4 strains; Brev. sul f ureum (BERGEYet al.) BREED, 1 strain; Arthrobacter ureafaciens (KREBS and EGGLESTON)CLARK, 2 strains; Arth, tumescens (JENSEN) CONN and DIMMICK,2 strains; Micrococcus varians MIGULA, 2 strains; Micr. con glomeratus MIGULA, 3 strains; Micr. luteus (S~HROETER)CORN, 1 strain; Micr. candidus CoHN, 2 strains: Sarcina lutea SCHROETER,1 strain; S. lutea subsp. flava KOCURand MARTINEC,12 strains; Alcaligenes f aecalis CASTELLANIand CHALMERS,2 strains; and Achromobacter delicatulus (JORDAN)BERGEY et al., 1 strain. This paper was read at the 191st Meeting of the Kanto Branch of the Agricultural Chemical Society of Japan held on February 28, 1959. The authors wish to thank Emeritus Professor Dr. K. Sakaguchi, University of Tokyo, for his encouragement throughout this study. The authors are also grateful to Messrs. C. Uchino and C. Sugiyama for their technical assistance, and to Teikoku Sekiyu Co., which afforded sampling facilities.

REFERENCES

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