ISOLATION OF NEW METHANOL-UTILIZING BACTERIA AND ITS THIAMINE-REQUIREMENT FOR GROWTHS KAGEAKI KOUNO, TOSHIKAZU OKI, HIDEO NOMURA, AND ASAICHIRO OZAKI

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1 J. Gen. Appl. Microbiol., 19, (1973) ISOLATION OF NEW METHANOL-UTILIZING BACTERIA AND ITS THIAMINE-REQUIREMENT FOR GROWTHS KAGEAKI KOUNO, TOSHIKAZU OKI, HIDEO NOMURA, AND ASAICHIRO OZAKI Central Research Laboratory of Sanraku-Ocean Co., Ltd., Fu jisawa 251, Japan (Received July 31, 1972) Non-pigmented methanol-dependent bacteria have been isolated in pure culture from a wide variety of natural sources. They are capable of growing only on methanol among many carbon sources tested, and showed no growth on ordinary nutrient media. These bacteria were identified as a new species, Methanomonas methylovora. The organisms are gram-negative, nonsporeforming rods, 0.4 to 0.7 i by 1.0 to 4.0 i in size, having a single polar flagellum. GC content of DNA is 51.2 to 53.6%. Some of them required a relatively high concentration of thiamine as an essential factor for cell growth, and produced an intracellular yellow pigment on methanol-containing agar medium. Current studies on microbial utilization of methanol have shown the possibility of a new potential source for single cell protein with the advent of large scale production of cheap substrate from natural gas (1-4). Several bacteria capable of utilizing methane as their sole source of carbon and energy are known. Most are gram-negative and pigmented rods with a single polar flagellum. They use only methane or methanol for growth. They have been identified as Met hanomonas methanica (5, 6), Pseudomonas methanica (7-9), and Methanomonas methanooxidans (10, 11). Methylococcus capsulatus (12), which is thermo-tolerant, non-motile, non pigmented, and gram-negative coccus, also oxidizes both methane and methanol. Although a number of papers have been published on the properties of bacteria capable of growing on methanol, such as Pseudomonas sp. M27 (13), Pseudomonas PRL-W4 (14), Pseudomonas AM1 (15, 16), and Vibrio extorquens (17), their identity is not clear so far. Recently, WHITTENBURY et al. (18) isolated more than 100 gram-negative, strictly aerobic, methane utilizers, and classified them into 5 groups and 15 subgroups on the basis of * This paper was presented of Japan held on April 4, 1969, at in the Annual Meeting of Agricultural Chemical Society Fukuoka, Japan. 11

2 12 KOUNO, OKI, NOMURA, and OZAKI VOL. 19 morphology, fine structure, types of resting stage, and other properties. The present paper describes the isolation of methanol-utilizing bacteria and identification of a new species, Methanomonas methylovora. A forthcoming paper will describe the optimal growth conditions of these bacteria, since little information has been published on the efficiency of conversion of methanol to microbial biomass. MATERIALS AND METHODS Media for isolation and purification. Basal methanol medium, consisting of (NH4)2S04, 3 g ; KH2P04, 2g ; K2HP04i 7 g ; MgS04.7H20, 0.5 g ; yeast extract, 0.1 g ; biotin, 10 pg ; thiamine HCI, 100 ~ig ; Mn2+ (as MnSO4. 5H20), 2 mg ; Fe2+ (as FeSO4.7H2O), 2 mg ; methanol, 20 ml ; and distilled water, 1,000 ml ; ph 6.8 to 7.0, was used for the enrichment and propagation of the methanol-utilizers. For solid medium, 1.5% agar was added to the abovementioned medium. Nutrient agar and other media enriched with peptone were used to detect the presence of bacteria capable of utilizing carbon sources other than methanol. Vitamin and amino acid requirements were determined by using a minimum medium omitting yeast extract, biotin, and thiamine hydrochloride from the basal medium. Enrichment and isolation procedures. Methanol-utilizers were isolated from a wide variety of natural sources by several repetitions of enrichment culture and plating alternately using basal methanol medium. Approximately 0.1 g of soil, activated sludge, sewage, compost, humus, and materials around the petroleum refinery were placed in a Monod-shaking tube containing 10 ml of basal methanol medium. The tube was incubated with reciprocal shaking at 28 to 30 for 3 days. After several subcultures, methanolutilizing bacteria were plated on a basal methanol agar plate and picked up after 5 days' incubation at 28. Identification of isolates. General characteristics of methanol-utilizers were studied mainly according to the routine methods recommended by the Society of American Bacteriologists (19). Methanol-salt medium was a modified Dworkin-Foster's medium (7) ; NaN03, 2.0 g ; MgSO4.7H2O, 0.2 g ; FeSO4.7H2O, 1.0 mg ; Na2HP04, 0.21 g ; KH2P04i 0.09 g ; Cu+2 (as CuSO4 5H20), 50 g; B+3 (as H3B03), 10 pg ; Mn+2 (as MnSO4.5H2O), 10 tg ; Zn+2 (as ZnS04.7H20), 70 g; Mo+6 (as Mo03), 10 pg ; methanol, 10 ml ; and distilled water, 1,000 ml. For assimilation test of carbohydrates, hydrocarbons, amino acids, and organic acids, 0.5 to 1.0% of one of them was added to this medium and cultivated with shaking at 28 for 7 days. Assimilation of gaseous hydrocarbons was carried out by using Thunberg tube in which a mixture of 50% of substrate and 50% air was contained. Toxic substrates were added to the medium at the concentrations of 0.05, 0.1, and 0.5%. Further, assimilation of formate and formaldehyde was tested by growth in the medium added at the concentration of 0.005, 0.01, and 0.02%. Assimila-

3 1973 New Methanol-utilizing Bacteria 13 tions of amides and alkylamines were tested by using Peter Hirsch Conti's ( 20 ) and den Dooren de Jong's solid media ( 21). Modified Heinemann's nitrogen-free agar; glucose was replaced by 1% methanol for detection of nitrogen fixation. For other tests, conventional media with 1% methanol were used. The bacteria were identified with reference to Bergey's Manual of Determinative Bacteriology, 7 th edition (1957) and other papers ( 7-15, 18). DNA extraction from bacterial cells and determination of its Tm. Bacterial DNA was extracted by the method of MARMUR ( 22 ) using sodium lauryl sulfate and chloroform-isoamyl alcohol. Tm of bacterial DNA was measured by the procedure of MARMUR and DoTY ( 23 ), and guanine-cytosine (GC) content of DNA was calculated from their equation. Isolation of methanol-utilizers RESULTS After 3 days of incubation of the first enrichment culture, there appeared a distinct yellowish turbidity. A small amount of this culture (0.1 ml) was then transferred to the same medium and re-incubated under the same conditions. Bacterial growth expressed by the optical density reached 0.4 to 0.6 at 610 nm after 3 to 4 days. Subsequent transfers succeeded to yield significant growth, and a loopful of the broth was streaked on basal methanol agar plate. Small, pale yellowish white, pink, and colorless colonies appeared after incubation for 4 to 5 days at 28. After four alternate repetition of enrichment culture and plating, several methanol-utilizers were obtained and they showed some difference in their morphology, appearance of colony, and physiological properties. The most abundant growth in basal methanol medium was shown by 44 cultures. Properties of isolates Methanol-utilizers isolated were gram-negative, usually occurring singly, but sometimes in pairs or in mass, and non-sporeforming rods, 0.4 to 0.7 tc by 1.0 to 4.0,u. The cells were motile by means of a single polar flagellum. Colonies of 42 out of 44 strains were non-chromogenic, circular, convex, smooth, and glistening on methanol-salt medium. Two other strains, M140-1 and M 170-1, appeared as yellowish colonies on a methanol-containing medium. On the basal methanol-containing agar, some of non-chromogenic strains occasionally had a tinge of pink in old culture (over 2 weeks). Although the colonies had a mucoid appearance, they had a tacky consistency and became difficult to disrupt with a needle after 1 to 2 weeks' incubation. Growth in the shaking culture was either dispersed or clumpy. In stationary cultures, the fresh isolates grew turbid throughout the medium with thin and white pellicle, and with accumulation of sedimented cells as the culture aged.

4 14 KOUNO, OKI, NOMURA, and OZAKI VOL. 19 Fig. 1. Electron micrograph of Strain M16-8 was negatively stained Methanomonas methylovora. with 1% phosphotungstic acid. Physiological characteristics of all isolates were tested. They were nonphotosynthetic, aerobic bacteria which had catalase, urease, and oxidase, and could reduce nitrate to nitrite. However, they could neither digest starch, cellulose, gelatin, nor casein. These strains were capable of growing only on media containing less than 5 % methanol, and could not grow on ordinary nutrient media at all. Methane, formaldehyde, and formate of C1 compounds, gaseous hydrocarbons such as ethane, propane, and butane, 15 sugars, amino acids, alkylamines, alcohols such as ethanol, propanol, and butanol, and organic acids such as acetate, pyruvate, oxalate, lactate, propionate, malonate, malate, fumarate, succinate, citrate, and gluconate were not assimilated. Bacterial DNA was extracted and its GC content was calculated from Tm value. It fell in the range of 51.2 to 53.6%. Thiamine requirement for bacterial growth Although three strains, M 8-5 (ATCC 21370), M 8-6, and M 14-1, out of 42 non-chromogenic bacteria developed fairly well on the basal methanol medium containing 0.01% of yeast extract, 10 pg biotin per liter, and 100 pg thiamine hydrochloride per liter, these organisms grew very poorly on the methanol-salt medium and methanol-containing minimum medium, and did not grow in the case of small inoculum. Omission and addition tests for vitamins and vitamin-free casamino acids showed that the growth factor

5 1973 New Methanol-utilizing Bacteria 15 Fig. 2. Effect of thiamine hydrochloride concentration on the maximum growth of Methanomonas methylovora M8-5. The medium consisting of 2% methanol, 0.3% (NH4)2SO4i 0.2% KHZPO4i 0.7% K2HPO4, 0.05% MgSO4 7H20, and 2 ppm each of Mn2+ and Fez, ph 7.0, was supplemented with thiamine hydrochloride as indicated. A small amount of washed cells was inoculated in 10 ml of this medium in a Monod-shaking tube, and incubated reciprocally at 28' for 48 hr. Two percent of methanol was fed twice during the cultivation according to the amount consumed, and ph was maintained at 7.5 by supplying 25% urea solution. -0-, M8-5; - -, M16-8. can be provided by the addition of yeast extract, but not by casamino acid or casein hydrolysate. A survey of known vitamins demonstrated that the concentration of thiamine hydrochloride required for maximum growth of strain M 8-5 was considerably higher, 100 ug/liter or more, while the growth of strain M 16-8 was not stimulated by thiamine hydrochloride as shown in Fig. 2. DISCUSSION From the characteristics of the isolates, they closely resemble the methane-

6 16 KOUNO, OKI, NOMURA, and OzAKI VOL. 19 Table 1. Comparison of various bacteria or methanol-assimilating bacteria which were reported previously, as summarized in Table 1. From the viewpoint of morphological characteristics, Methylomonas capsulatus (12) is quite different from the isolates in being diplococcoid form, but other methane- or methanol-utilizers listed in Table 1 are not much different from rod-shaped and polar flagellated isolates in major properties. By comparing their physiological characteristics, especially their assimilability of the carbon source, bacteria that grow on methane or methanol can be separated into two groups, one that grows on methane and/or methanol only but cannot grow on sugar and/or ordinary nutrients such as peptone,

7 1973 New Methanol-utilizing Bacteria 17 capable of methanol assimilation. and another that can grow on both of these substances. The latter methanol-- utilizers, Pseudomonas PRL-W4 (14), Pseudomonas M27 (13), Pseudomonas C (4), Pseudomonas AM-1 (15), Vibrio extorquens (11), and Protaminobacter ruber (25), are not identical with the present isolates, because they grow actively on several carbon sources such as glucose, fructose, glycerol, formate, and peptone, and in most cases grow more vigorously on methane than on methanol. The isolates capable of growing only on methanol among many carbon sources tested belong to the former, so that they are very similar to Pseudomonas methanica (7-9), Methanomonas methanica (6), Methanomonas

8 18 KOUNo, OKI, NOMURA, and OZAKI VOL. 19 methanooxidans (10 ), and four strains of Methylomonas (18 ) which are essentially dependent on methane and/or methanol for growth, being unable to utilize many carbon sources other than C1 compounds. Moreover, when their morphological, cultural, and physiological characters are compared in detail, Methanomonas methanooxidans differs from the present isolates in the point of methane-assimilability, cell morphology, and colony appearance. Methylomonas albus and Methylomonas agile are considered to be similar to the present isolates in several characteristics such as cell morphology and colony color, but their preference for methanol as carbon and energy sources is doubtful. Furthermore, they are not identical with the present isolates in methane-assimilability and formation of a resting stage as immature azotobacter-type cysts. On the other hand, P. methanica coincides closely with the present isolates in major taxonomic features ; cell morphology, colony appearance, flagellation, and assimilability of carbon sources. Strictly speaking, however, there are some differences in pigmentation and methane-assimilation. The non-pigmented methanol-utilizers, occupied the greater part of the isolates which do not attack methane, formate, and formaldehyde at all, are to be distinguished from the pink-pigmented and methane-dependent bacteria described by DWORKIN and FOSTER ( 7 ) and by LEADBETTER and FOSTER ( 9 ), and from a typical pink-pigmented methanol-utilizers reported by HARRINGTON and KALLIO ( 8 ) which does not oxidize methane but utilizes formate and formaldehyde. With reference to Bergey's Manual of Determinative Bacteriology, 7th edition ( 25), the present isolates might be classified into the genus Pseudomonas or Met hanomonas from their cell morphology. Since the organisms of the genus Pseudomonas attacking glucose and other sugars oxidatively are physiologically different from these isolates, it is considered that the genus Methanomonas, capable of securing growth energy by the oxidation of methane, is more adequate for the present isolates from their physiological properties because the pathway of aerobic methane oxidation occurred via methanol. Although DWORKIN and FOSTER (7) classified methane-oxidizer into the genus Pseudomonas only on the basis of cell morphology, it would be more appropriate to re-examine and qualify the identification of methane-oxidizing organisms as a physiological genus. MANDEL (24) reported that the GC content of DNA from P. methanica provided by Foster was 52.1%, and it would therefore be remote from 57 to 70% of DNA base composition of the genus Pseudomonas. The GC content of DNA of the present isolates distributed from 51.9 to 53.6%, and these values are outside the range of the genus Pseudomonas. WHITTENBURY et al. (18) attempted to give the group name "Met hylomonas" as basis for a provisional classification of the methane- or methanol-utilizing bacteria, but it is uncertain whether to recognize the genus Met hylomonas as a valid name so far. Considering the assimilation pathway in the strict

9 1973 New Methanol-utilizing Bacteria 19 methane-methanol bacteria, lack of methane oxidative enzyme in the isolates, and uncertain limitation in the classification currently employed for organisms, it would be assumed that the genus Methanomonas can include in a broader sense both methane- and methanol-utilizing bacteria. Under these circumstances, the present isolates should be suitable to be classified into the genus Methanomonas and identified as a new species, M. met hylovora. In addition, nomenclature problems of the methane-methanol utilizers are still not clear, but we agree with Foster's proposal that these organisms are considered as "methyl" utilizers and the prefix "Methylo-" is suggested as a solution to the problem of generic cognomina. Methanomonas methylovora Strain M 8-5 required relatively high concentration of thiamine hydrochloride for growth. KANEDA and ROXBURGH reported that Pseudomonas PRL-W4 required biotin in very low concentration or one of its analogs as an essential growth factor (14). This is the first report of methane- or methanol-utilizing bacteria which require thiamine as an essential factor for growth. Methanomonas methylovora DESCRIPTION OF THE ISOLATES Strain No.: M16-8, M2-1, M13-A, M7-4, M9-1, M20-8, M , M34-4, M36-1, M45-1, M50-6, 2004, 2006, M193-1, M198-1, M230-1, M80-2, M130-1, M120-1, N15, N21, N23, N22, N28, N41, 805, M140-1, M170-1, M8-6, N16, M8-5 (ATCC 21370), M14-1, N18, 333, N20, N25, N40, N27, N31, N34, N35, N36, N49, and M12-4 (ATCC 21852). Cell morphology : Grown on methanol-salt agar slant for 20 to 24 hr at 28. Vegetative cells (Fig. 1) ; Straight rods, X i, occurring singly or in pairs, not pleomorphic. Motile with single polar flagellum. No spore formation. Gram-negative. Not acid-fast. Cultural characteristics : Methanol-salt agar colonies after 2 to 4 days : Punctiform to circular, convex, entire, smooth, glistening, non-chromogenic or milky white, and translucent or opalesecnt. (Variations : M140-1, and M170-1 are opaque and yellowish white. M8-5, M8-6, and M14-1 grow when 100 jig thiamine hydrochloride per liter added to the medium.) Basal methanol agar : Similar to those of methanol-salt agar colonies, but very slight pinkish white colonies appear in old culture. (Variations : M140-1 and M170-1, opaque in early stage, then change to yellow to pale yellowish white). 1% Methanol-containing nutrient agar colonies : Same as above. Methanol-salt agar stroke after 2 to 3 days : Moderate growth, filiform, smooth, glistening, milky white, opalescent, and butyrous to mucoid. Medium unchanged. (Variations : M140-1 and M170-1 are pale yellow, and M8-5,

10 20 KOUNo, OKI, NOMURA, and OZAKI VOL. 19 M8-6, and M14-1 grow when 100 pg thiamine hydrochloride per liter added to the medium.). Basal methanol agar stroke : The cultural characteristics are almost identical to those on methanol-salt agar. Occasionally, in old culture, chromogenesis is very slightly pinkish white. (Variations : M140-1 and M170-1, dull yellow to yellowish white.) 1% Methanol-containing nutrient agar strokes : Same as above. Modified Heinemann's nitrogen-free methanol agar : No growth. 1% Methanol-containing nutrient gelatin stab for one month at 20 : Best growth at top. 1% Methanol-containing Frazier's gelatin agar : No liquefaction. Methanol-salt broth for 1 to 4 days : Abundant growth, ring or pellicle after 2 to 3 days, and turbid. Sediment exists. These strains cannot grow on ordinary media employed in bacterial diagnosis, but do grow when 1 to 2% methanol is added. Physiological characteristics : Relation to free oxygen : Aerobic Temperature for growth : Optimum growth temperature between 20 and 28. Grow at 10 but not at 37. ph for growth : Optimum ph between 6.5 to 7.5. ph limits for growth,, 5.2 to 9.2. Catalase : Positive Oxidase : Positive Urease : Positive Litmus milk: No reaction Starch hydrolysis : Negative Tween 80 hydrolysis : Negative Nitrate reduction : Positive, but no gas Indole production : None Koser citrate medium : No growth Nutritional requirement : Usually none. (Variations : M8-5 (ATCC21370),. M8-6, and M14-1 require thiamine.) Halotolerance : Growth on a medium containing 2% of NaCI. Inhibition by oxytetracycline (10 pg/ml) : None Acid production from glucose (Hugh and Leifson method) : No growth Utilization of hydrocarbons, carbohydrates, alcohols, amino acids, and organic acids (after 7 to 10 days) : Good growth on mineral salt liquid medium supplemented with 1% methanol as sole carbon source, but no growth with other carbon sources ; glucose, sucrose, fructose, lactose, xylose, mannitol, glycerol, methane, ethane, propane, butane, ethanol, propanol, butanol, formaldehyde, acetaldehyde, acetate, acetone, phenol, benzene, naphthalene, kerosene, formate, pyruvate, oxalate, lactate, propionate, malonate, malate, fumarate,, succinate, gluconate, citrate, DL-isoleucine, DL-glycine, L-alanine, L-leucine,.

11 1973 New Methanol-utilizing Bacteria 21 DL-methionine, L-arginine, DL-aspartic acid, L-glutamic acid, DL-tryptophan, L-lysine, formamide, and acetamide. Monomethylamine, dimethylamine, and monoethylamine are not used as carbon and nitrogen sources. No photosynthesis Source : Soil, sewage, and mud GC content of DNA by Tm method : Strain M12-4, 51.9%; M16-8,.52.4%; M8-5, 53.6%; and M140-1, 51.2% The type strain of this species, M12-4 (ATCC 21852), has been deposited with ATCC. 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) 18) 19) 20) 21) 22) 23) 24) 25) REFERENCES H. ASTHANA, A.E. HUMPHREY, and V. MORITZ, Biotech. Bioeng., 13, 923 (1971). L. HAGGSTROM, Biotech. Bioeng., 11, 1043 (1969). P.S. VARY and M.J. JOHNSON, Appl. Microbiol., 15, 1473 (1967). Y. CHALFAN and RI. MATELES, Appl. Microbiol., 23, 135 (1972). S. ORLA-JENSEN, Zentr. Bakteriol. Parasitenk., Abst. II, 22, 305 (1909). N.L. SOHNGEN, Zentr. Bakteriol. Parasitenk., Abst. II, 15, 513 (1906). M. DWORKIN and J.W. FOSTER, J. Bakteriol., 72, 646 (1959). A.A. HARRINGTON and R.E. KALLIO, Can. J. Microbiol., 6, 1 (1960). E.R. LEADBETTER and J.W. FOSTER, Arch. Mikrobiol., 30, 91 (1958). L.R. BROWN, R.J. STRAWINSKI, and CS. MCCLESKEY, Can. J. Microbiol., 10, 791 (1927). P.K. STOCKS and C.S. MCCLESKEY, J. Bacteriol., 88, 1065 (1964). J.W. FOSTER and R.H. DAVIS, J. Bacteriol., 91, 1924 (1966). C. ANTHONY and L.J. ZATMAN, Biochem. J., 92, 609 (1964). T. KANEDA and J.M. ROXBURGH, Can. J. Microbiol., 5, 87 (1959). D. PEEL and JR. QUAYLE, Biochem. J., 81, 465 (1961). J.R. QUAYLE and D. PEEL, Biochem. J., (1960). P.K. STOCKS and CS. MCCLESKEY, J. Bakteriol., 88, 1065 (1964). R. WHITTENBURY, K.C. PHILLIPS, and J.F. WILKINSON, J. Gen. Microbiol., 61, 205 (1970). Society of American Bacteriologists, Manual of Microbiological Methods, McGraw- Hill Book Co., Inc., New York (1957). P. HIRSCH and S.F. CONTI, Arch. Mikrobiol., 48, 358 (1964). L.E. DEN DOOREN DE JONG, Zentr. Bakteriol. Parasitenk., Abst. II, 71, 193 (1927). J. MARMUR, J. Mol. Biol., 3, 208 (1961). J. MARMUR and P. DOTY, J. Mol. Biol., 5, 109 (1962). M. MANDEL, J. Gen. Microbiol., 43, 273 (1966). R.S. BREED, E.G.D. MURRAY, and N.R. SMITH, Bergey's Manual of Determinative Bacteriology, 7th ed., The Williams and Wilkins Co., Baltimore, 1957.