Vol. 40, No. 3
INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, July 1990, p. 292-296 OO20-7713/90/030292-05$02.OO/O Copyright 0 1990, International Union of Microbiological Societies
Paracoccus kocurii sp. nov. a TetramethylammoniumAssimilating Bacterium MAR1 OHARA,l YOKO KATAYAMA,l* MASAAKI TSUZAKI,2 SHINYA NAKAMOT0,2 AND HIROSHI KURAISHI' Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183,l and NEC Corporation, Kawasaki, Kanagawa 213,2 Japan A new species of tetramethylammonium-assimilatingbacteria was isolated from an activated sludge which was used for the treatment of tetramethylammoniumhydroxide contained in the wastewater from semiconductor manufacturing processes. Cells of the bacteria were gram-negative,nonmotile, short rods (0.5 to 0.8 pm by 0.7 to 1.1 pm). The major respiratory quinone component of the bacteria was Q-lo. The G+C content was 71 mol% Isolates are mesophilic and assimilate methylated amines such as tetramethylammonium, trimethylamine, dimethylamine, and methylamine under neutral conditions. The isolates resemble Purucoccus species with respect to morphology but were distinguishable from the known species of the genus. We propose Paracoccus kocurii sp. nov. The type strain is strain B (=JCM 7684).
.
Semiconductor manufacturing processes use tetramethylammonium (TMA) hydroxide as a main ingredient in developers for positive photoresists, which are a mixture of phenol resin and naphthoquinone azide. Positive photoresists are used to draw fine patterns on wafers of silicon, gallium arsenide, etc., for integrated circuits. Patterning is done through a process called photolithography, in which the UV-light-exposed part of a positive photoresist on a wafer is removed with a developer and the remaining photoresist on the wafer is utilized as a protective film in the following wafer processing. In order to remove the UVexposed photoresist, the developer must contain an alkali. However, hydroxides of alkali metals, such as sodium hydroxide and potassium hydroxide, are inappropriate because alkali metals easily diffuse into semiconductor wafers and cause malfunction of integrated circuits fabricated in the wafers. TMA hydroxide is, therefore, used as an alkali in positive photoresist developers. TMA is chemically classified among the quaternary ammonium compounds, which are generally used as antibacterial and antifungal agents. TMA, therefore, has been considered to be nearly immune to attack by microorganisms. Biodegradation is potentially the most cost effective means for treating wastewater containing TMA. However, little was known of the bacteria which degrade TMA. Hampton and Zatman (3) isolated TMA-degrading bacteria from soil. Their isolate, strain 5H2, was gram negative, nonmotile, rod shaped, and a facultative methylotroph; however, a detailed taxonomic study has not been provided. Urakami et al. isolated TMA-degrading bacteria from soil which were identified as Pseudomonas aminovorans, Methylobacterium extorquens, and Mycobacterium spp. (T. Urakami, H. Araki, H. Kiga, and H. Kobayashi, Abstr. Annu. Meet. Ferment. Technol., Japan, 1987, abstr. no. 508, p. 154). In this paper, we characterize TMA-degrading bacteria isolated from an activated sludge system which was used for the treatment of TMA and propose Paracoccus kocurii sp. nov .
MATERIALS AND METHODS Isolation and maintenance. Samples for the isolation of the bacteria were obtained from a pilot activated sludge system used for treating wastewater from a semiconductor manufacturing process. The inlet concentration of TMA was around 800 ppm (800 Fg/ml), and TMA was not detected at the outlet of the system. In the following experiments, we used the chloride form of TMA as a growth substrate, because TMA hydroxide was strongly basic. TMAdegrading bacteria were isolated on the mineral medium described by Colby and Zatman (2), which was supplemented with 0.1% (wthol) TMA and 0.01% yeast extract (Difco Laboratories). The isolates were maintained on the mineral medium described by Stanier et al. (10) supplemented with 0.1% (wt/vol) TMA, 0.5 mg of thiamine hydrochloride per liter, and 15 g of agar per liter (ST agar medium). Solutions of TMA were sterilized by membrane filtration (pore diameter, 0.22 pm). Unless otherwise mentioned, cultures were incubated at 30°C. Bacterial strains. The TMA-degrading strains used in this study were A, BT, C, El, E2, and F1. Assimilation of organic substances was examined on these strains, and detailed characterizations were done on strains BT, E l , and F1. Their characteristics were compared with those of the known species of the genus Paracoccus, Paracoccus denitrijicans IAM 12479T, I F 0 12442, and IF0 13301, and Paracoccus halodenitrificans CCM 286T. Since the new isolates were facultative methylotrophs, Pseudomonas aminovorans ATCC 23314T and Methylobacterium extorquens JCM 2802T were selected as reference strains of C1 compound-utilizing bacteria (1, 8). Thiobacillus versutus THI 041T (=IAM 12815T),another facultative methylotroph (ll), also was tested in DNA-DNA homology experiments. Morphological and physiological characteristics. Cells of the isolates grown in ST medium were negatively stained with 1%(wthol) phosphotungstic acid, and their shape and size were examined with a model JEOL 200CX transmission electron microscope at 100 kV. Nitrate reduction and denitrification were examined in nutrient broth containing 1 g of potassium nitrate per liter. Denitrification was determined by the formation of gas in a
* Corresponding author. 292
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TABLE 1. DNA-DNA relatedness among the isolates, Paracoccus species, Pseudomonas aminovorans, and Methylobacterium extorquens % Homology with labeled DNA from?
Source of cold DNA"
G+C content (mol%)
Strain B
Paracoccus denitriJicans IAM 12479T
Paracoccus halodenitrificans - CCM 286T
Strain B Strain E l Strain F1 Paracoccus denitrijicans IAM 12479T Paracoccus denitrijicans IF0 12442 Paracoccus denitrijicans IF0 13301 Paracoccus halodenitrificans CCM 286T Pseudomonas aminovorans ATCC 23314T Methy lobacterium extorquens JCM 2802= Thiobacillus versutus IAM 1281ST Micrococcus luteus IAM 1056T
71 71 71 67 67 67 64 62 67 67' 73
100 87 80 30 15 26 3 4 3 2 10
31 10 34 100 43 61 5 12 8 4 6
10 5 0 0 0 8 100 1 0 0 1
Pseudomonas aminovorans ATCC 23314T
23 11 11 13 7 11 9
100 18 1 12
Methylobacterium extorquens JCM 2802T
16 14 2 1 5 12 3 3 100 0 9
" ATCC, American Type Culture Collection, Rockville, Md. ; CCM, Czechoslovak Collection of Microorganisms, J. E. Jurkyne University, Brno, Czechoslovakia; IAM, Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan; IFO, Institute for Fermentation, Osaka, Japan; JCM, Japan Collection of Microorganisms, Saitama, Japan. Data from reference 6.
'
stab culture of ST medium containing 0.1% (wt/vol) soft agar. The nitrogen source was examined in a medium containing (grams per liter): KH2P04, 1; MgSO, 7H@, 0.5; KC1, 0.2; thiamine hydrochloride, 0.0005; and acetic acid (pH 7.2), 5 , to which potassium nitrate (1 g/liter), ammonium sulfate (0.78 g/liter), or sodium glutamate (0.15 g/liter) was added. Urease activity was examined in Christensen medium (Nissui Seiyaku, Tokyo, Japan) supplemented with 20 g of urea per liter. Salt tolerance was determined in medium B (12) supplemented with 0.15% sodium acetate instead of methanol. Utilization of thiosulfate for growth was examined in SM-basal salts medium (6) with 0.5% (wthol) Na,S,03 - 5H,O and 0.5 mg of thiamine hydrochloride per liter. Unless otherwise indicated, results of these tests were obtained after incubation for 1 week at 30°C. Assimilation of organic compounds. Assimilation of organic compounds was examined in medium B supplemented with 25 mg of yeast extract per liter and one of the compounds shown in Table 2 as a growth substance. Assimilation of formate, methanol, ethanol, and oxalate was tested at concentrations of 5, 10, and 20 mM. Determination of quinone system. Quinone fractions were extracted with chloroform-methanol (2:1, vol/vol) from the lyophilized cells and subsequently purified with preparative thin-layer chromatography developed with petroleumbenzine-diethyl ether (9:1, vol/vol). The fractions were then subjected to high-performance liquid chromatography in an LC-3A chromatograph (Shimadzu Corporation, Kyoto, Japan) equipped with a Zorbax-ODS prepacked column (4.6 by 250 mm). Ubiquinones were detected with a Shimadzu SPD-2A spectrophotometric detector at 275 nm. Methanolisopropylether (4:1, voYvol) was used as the eluent. A quinone homolog consisting of more than 90% of the ubiquinone fractions was considered to be the major ubiquinone component. Determination of cellular fatty acids. Cellular fatty acids were extracted with n-hexane after methylation with 5% HCI-methanol and then analyzed by gas-liquid chromatography with a Shimadzu GC-6AM apparatus. Fatty acid methyl esters were identified by capillary-column chromatography (Advance-DS and Silicone OV-101; inside diameter, 0.24 mm by 25 m; Chromato Packing Center, Tokyo, Japan) after fractionation of the esters to nonpolar, 2-OH and 3-OH fatty
acids by thin-layer chromatography (silica gel; Merck & Co., Inc., Rahway, N.J.); the solvent system used was n-hexanediethyl ether (85:15, volhol). G+C content of DNA. DNA was prepared according to the procedure described by Saito and Miura (9). G+C content of DNA was determined by using high-performance liquid chromatography (Shimadzu LC-3A equipped with a ZorbaxODS prepacked column [4.6 by 250 mm]) after digestion of the DNA to deoxyribonucleotides with nuclease P1 (5). The mobile phase used was 20 mM KH,PO, (pH 3.5)-methanol (60: 1, volhol). DNA relatedness. DNAs were labeled by nick translation by using a commercial kit (N 5000; Amersham International Ltd., Amersham, United Kingdom), and DNA-DNA hybridization was carried out at 65°C as previously described (4). Micrococcus luteus IAM 1056 was used as the reference strain.
RESULTS AND DISCUSSION Isolation. Samples were taken from an activated sludge in which TMA was being treated; they were diluted and spread on agar medium containing TMA as the sole energy source. Strains A, BT, C, E l , E2, and F1 were isolated in pure culture by streak plating. The isolates grew in mineral salts medium supplemented with TMA and small amounts of yeast extract but were unable to grow in the absence of either substrate. TMA-degrading bacteria were considered to belong to the genus Paracoccus because of their cell shape, lack of motility, facultatively anaerobic growth, quinone system, and nonpolar fatty acid composition. However, tolerance to NaCl, assimilation of organic compounds, hydroxy fatty acid composition, and other phenotypic characteristics indicate that the isolates are distinct species in the genus Paracoccus. Results on DNA relatedness supported this conclusion (Table 1).Therefore, we propose a new species, Paracoccus kocurii, for TMA-degrading bacteria. Description of Puracoccus kocun'i sp. nov. (k0.cu'ri.i. N. L. Gen. n. kocurii of Kocur; named for Miloslav Kocur, Czechoslovakian bacteriologist). The colonial appearance and assimilation of organic compounds of all of the isolates were quite similar. Further characterization was carried out on strains BT, E l , and F1. The isolates are gram-negative,
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OHARA ET AL.
nonmotile, short rods (0.5 to 0.8 by 0.7 to 1.1 pm) (Fig. 1). Colonies are pinhead size, circular, smooth, convex, entire, transparent, and yellowish white on nutrient agar and pinhead size, circular, entire, convex, transparent, and brownish white on TMA agar after 10 days of incubation. Production of pigment was not observed. Optimum temperature for the growth is between 25 and 30°C; no growth occurs at 20 or 40°C. Optimum pH for the growth is between pH 6.6 and pH 8.2; no growth occurs below pH 6.1 or above pH 8.8, which distinguishes the isolates from Paracoccus alkaliphilus (13). The isolate is facultatively anaerobic, uses nitrate as a terminal electron acceptor, and evolves gas. Growth occurs in the absence of NaCl, but no growth is produced in 3% NaCl (Table 2), which is distinct from Paracoccus halodenitriJcans, which grows optimally in the presence of 4.4 to 8.8% NaCl(7). The isolates use ammonium salt as a nitrogen source but are unable to grow on nitrate or glutamate. Oxidase and catalase activities are produced, but urease activity is not. Cells require thiamine for growth. Thiosulfate does not support their growth. Assimilation of organic compounds by TMA degraders is restricted to a narrow range of substrates, as shown in Table 3. The isolates are unable to use the amino acids or sugars examined, These assimilations are different from those of Paracoccus denitrijicans, which has versatile abilities on amino acids and sugars. The isolate grows on TMA, trimethylamine, trimethylamine-N-oxide, dimethylamine, and methylamine. The major respiratory quinone is Q-lo. The major cellular fatty acids are C18:1 and ClgcYc,as nonpolar acids, and 3-OH Cloz0and 3-OH C12:o,as hydroxy acids (Table 4). The base composition of DNA of the type strain is 71 mol% G+C* The type strain is strain B (=JCM 7684). The organism
FIG. 1. Electron micrograph of Paracoccus kocurii BT. The cells were stained with phosphotungstic acid. Bar = 1 pm.
TABLE 2. Differential characteristics of Paracoccus species, Pseudomonas aminovorans, and Methylobacterium extorquensa Characteristic
Cell shape Cell size (pm) Width Length Motility Growth in: 0% NaCl 3% NaCl 6% NaCl 12% NaCl Nitrate reduction Denitrification Nitrogen source: KNO, (NH,),SO, Glutamate Urease Optimum pH Optimum temp Vitamin requirement
Paracoccus kocurii (3 strains)
Paracoccus denitrificans ( 3 strains)
Paracoccus halodenitrificans CCM 286T
Short rod
Short rod
Short rod
0.5-0.8 0.7-1.1
0.6-0.8 0.7-1.0 -
-
+
-
W -
6.6-8.2 2530°C Thiamine
+ + + +
Coccoid or short rod
Rod
Rod
o.fs1.0
0.5-0.7 1.&1.4
1.1-1.3 2.2-3.0
+ +
-
-
+ +
Methylobacterium extorquens JCM 2802T
0.8-2.0
+ + + +" +"
-
alkaliphilusb
Pseudomonas aminovorans ATCC 23314T
Paracoccus
+
-
W
+ + -
+ -
+ +
W
+
W
-
8-9
30°C -
25-30°C' Thiamine"
Biotin ~~
~~
Symbols: +, all strains positive; W, weak positive; -, all strains negative. All strains are gram negative, nonpigmented, and oxidase and catalase positive, accumulate poly-P-hydroxybutyrate, and fail to grow in 15% NaCl or to hydrolyze starch. Data from reference 13. Data from reference 7. a
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TABLE 3. Assimilation of organic compounds by Paracoccus species, Pseudomonas aminovorans, and Methylobacterium extorquensa Growth of? Concn
Substance
(mM)
Methylamine Dimethylamine Trimeth ylamine Trimeth ylamine-N-oxide TMA Methanol Methanol Methanol Ethanol n-Butanol Glycerol Cyclohexanol G1ycine L- Alanine L-Valine L-Leucine L-Isoleucine L-Threonine L-Cysteine L-Methionine L-Phen ylalanine L-Tyrosine L-Histidine L-Tryptophan L-Aspartate L-Asparagine L-Glutamate L-Glutamine L-Lysine L- Arginine L-Ornithine L- Arabinose D-Ribose D-Xylose D-Glucose D-Galactose D-Fructose Sucrose Trehalose Lactose Maltose L-Cellobiose Mannitol D-Sorbitol Inositol Formate Propionate n-Buty late Lactate Pyruvate Gluconate m ma late Succinate a-Ketoglutarate Adipate Tartarate Citrate p-H ydrox ybenzoate p -Aminobenzoate DL-Mandelate ~
10 10 10 10 10 5 10 20 5, 10, 20 20 20 10 20 20 20 20 20 20 20 20 10 20 5 5 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 5 , 10, 20 20 20 20 20 20 20 20 20 20 20 20 10 10 10 ~
Paracoccus kocurii (6 strains)
Paracoccus denitriJicans (3 strains)
+ +
Paracoccus alkaliphilus'
+
W -
-
+ D(2/3) D(2/3)
+ +
D(2/3) -
+ +
-
+ -
+
+ + +
W -
+ +
W W -
W W
D(1/3) D( 1/3) -
Methy lobacterium extorquens JCM 2802T
-
+
+ + + + + + + + + + + + W + + D(2/3) + W + + + + + W + W + + + + + + + + + + +-
Pseudomonas aminovorans ATCC 23314T
+
+ + + + + + + W + + W + + + W + + + + + + + + + + + W + + + + + W + W + + + W W W
+
W -
W ~
~
All tested strains assimilated 40 rnM L-serine, 20 rnM L-proline, and 20 mM acetate. In addition, acetate was reported to be assimilated by Paracoccus alkaliphilus (13). No tested strains assimilated 5 mM L-tryptophan; 5, 10, or 20 rnM oxalate; 20 mM glutarate; 20 mM pimerate; 10 mM benzoate; or 10 rnM p-aminobenzoate. Symbols: +, all strains positive; D, different reactions among strains; W, weak positive; -, all strains negative. Values within parentheses are the number of positive strains/nurnber of strains tested. Data from reference 13. a
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OHARA ETAL.
was isolated from activated sludge for the treatment of TMA hydroxide. Differential characteristics among species of Paracoccus are given in Tables 2, 3, and 4. Since taxonomic studies on microorganisms using the quinone system show that differentiation of taxa is at the genus level, the fact that Paracoccus halodenitrificans contained Q-9 as the major quinone system may indicate that further taxonomic studies are required on Paracoccus halodenitrijicans. ACKNOWLEDGMENTS
c,
c,
f
We thank M. Kocur, Czechoslovak Collection of Microorganisms, Bmo, Czechoslovakia; Japan Collection of Microorganisms, Riken, Saitama, Japan; and Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan, for supplying the strains. We also thank K. Komagata, Tokyo Agriculture University, and J. Sugiyama, Institute of Applied Microbiology, University of Tokyo, for their helpful discussions and I. Hirata, Institute of Applied Microbiology, University of Tokyo, for technical assistance with electron microscopy. LITERATURE CITED
4 0
-3 c Y
5iE 3;
1. Bousfield, I. J., and P. N. Green. 1985. Reclassification of Dacteria of the genus Protomonas Urakami and Komagata 1984 in the genus Methylobacterium (Patt, Cole, and Hanson) emend. Green and Bousfield 1983. Int. J. Syst. Bacteriol. 35209. 2. Colby, J., and L. J. Zatman. 1973. Trimethylamine metabolism in obligate and facultative methylotrophs. Biochem. J. 132: 101-112. 3. Hampton, D., and L. J. Zatman. 1973. The metabolism of tetramethylammonium chloride by bacterium 5H2. Biochem. SOC.Trans. 1:667-668. 4. Katayama-Fujimura, Y., Y. Enokizono, T. Kaneko, and H. Kuraishi. 1983. Deoxyribonucleic acid homologies among species of the genus Thiobacillus. J. Gen. Appl. Microbiol. 29: 287-295. 5. Katayama-Fujimura, Y., Y. Komatsu, H. Kuraishi, and T. Kaneko. 1984. Estimation of DNA base composition by high performance liquid chromatography of its nuclease P1 hydrolysate. Agric. Biol. Chem. 483169-3172. 6. Katayama-Fqiimura, Y., N. Tsuzaki, and H. Kuraishi. 1982. Ubiquinone, fatty acid and DNA base composition determination as a guide to the taxonomy of the genus Thiobacillus. J. Gen. Microbiol. 128:1599-1611. 7. Kocur, M. 1984. Genus Paracoccus Davis 1969, p. 399402. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 8. Palleroni, N. J. 1984. Genus Pseudomonas Migula 1894, p. 141-199. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 9. Saito, H., and K. Miura. 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72:619-629. 10. Stanier, R Y., N. J. Palleroni, and M. Doudoroff. 1966. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43:159-271. 11. Taylor, B. F., and D. S. Hoare. 1969. New facultative Thiobacillus and a reevaluation of the heterotrophic potential of Thiobacillusnovellus. J. Bacteriol. 1W487-497. 12. Urakami, T., and K. Komagata. 1984. Protomonas, a new genus of facultatively methylotrophic bacteria. Int. J. Syst. Bacteriol. 34:188-201. 13. Urakami, T., J. Tamaoka, K. Suzuki, and K. Komagata. 1989. Paracoccus alkaliphilus sp. nov., an alkaliphilic and facultatively methylotrophic bacterium. Int. J. Syst. Bacteriol. 39: 116-121.
INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, July 1990, p. 292-296 OO20-7713/90/030292-05$02.OO/O Copyright 0 1990, International Union of Microbiological Societies
Paracoccus kocurii sp. nov. a TetramethylammoniumAssimilating Bacterium MAR1 OHARA,l YOKO KATAYAMA,l* MASAAKI TSUZAKI,2 SHINYA NAKAMOT0,2 AND HIROSHI KURAISHI' Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183,l and NEC Corporation, Kawasaki, Kanagawa 213,2 Japan A new species of tetramethylammonium-assimilatingbacteria was isolated from an activated sludge which was used for the treatment of tetramethylammoniumhydroxide contained in the wastewater from semiconductor manufacturing processes. Cells of the bacteria were gram-negative,nonmotile, short rods (0.5 to 0.8 pm by 0.7 to 1.1 pm). The major respiratory quinone component of the bacteria was Q-lo. The G+C content was 71 mol% Isolates are mesophilic and assimilate methylated amines such as tetramethylammonium, trimethylamine, dimethylamine, and methylamine under neutral conditions. The isolates resemble Purucoccus species with respect to morphology but were distinguishable from the known species of the genus. We propose Paracoccus kocurii sp. nov. The type strain is strain B (=JCM 7684).
.
Semiconductor manufacturing processes use tetramethylammonium (TMA) hydroxide as a main ingredient in developers for positive photoresists, which are a mixture of phenol resin and naphthoquinone azide. Positive photoresists are used to draw fine patterns on wafers of silicon, gallium arsenide, etc., for integrated circuits. Patterning is done through a process called photolithography, in which the UV-light-exposed part of a positive photoresist on a wafer is removed with a developer and the remaining photoresist on the wafer is utilized as a protective film in the following wafer processing. In order to remove the UVexposed photoresist, the developer must contain an alkali. However, hydroxides of alkali metals, such as sodium hydroxide and potassium hydroxide, are inappropriate because alkali metals easily diffuse into semiconductor wafers and cause malfunction of integrated circuits fabricated in the wafers. TMA hydroxide is, therefore, used as an alkali in positive photoresist developers. TMA is chemically classified among the quaternary ammonium compounds, which are generally used as antibacterial and antifungal agents. TMA, therefore, has been considered to be nearly immune to attack by microorganisms. Biodegradation is potentially the most cost effective means for treating wastewater containing TMA. However, little was known of the bacteria which degrade TMA. Hampton and Zatman (3) isolated TMA-degrading bacteria from soil. Their isolate, strain 5H2, was gram negative, nonmotile, rod shaped, and a facultative methylotroph; however, a detailed taxonomic study has not been provided. Urakami et al. isolated TMA-degrading bacteria from soil which were identified as Pseudomonas aminovorans, Methylobacterium extorquens, and Mycobacterium spp. (T. Urakami, H. Araki, H. Kiga, and H. Kobayashi, Abstr. Annu. Meet. Ferment. Technol., Japan, 1987, abstr. no. 508, p. 154). In this paper, we characterize TMA-degrading bacteria isolated from an activated sludge system which was used for the treatment of TMA and propose Paracoccus kocurii sp. nov .
MATERIALS AND METHODS Isolation and maintenance. Samples for the isolation of the bacteria were obtained from a pilot activated sludge system used for treating wastewater from a semiconductor manufacturing process. The inlet concentration of TMA was around 800 ppm (800 Fg/ml), and TMA was not detected at the outlet of the system. In the following experiments, we used the chloride form of TMA as a growth substrate, because TMA hydroxide was strongly basic. TMAdegrading bacteria were isolated on the mineral medium described by Colby and Zatman (2), which was supplemented with 0.1% (wthol) TMA and 0.01% yeast extract (Difco Laboratories). The isolates were maintained on the mineral medium described by Stanier et al. (10) supplemented with 0.1% (wt/vol) TMA, 0.5 mg of thiamine hydrochloride per liter, and 15 g of agar per liter (ST agar medium). Solutions of TMA were sterilized by membrane filtration (pore diameter, 0.22 pm). Unless otherwise mentioned, cultures were incubated at 30°C. Bacterial strains. The TMA-degrading strains used in this study were A, BT, C, El, E2, and F1. Assimilation of organic substances was examined on these strains, and detailed characterizations were done on strains BT, E l , and F1. Their characteristics were compared with those of the known species of the genus Paracoccus, Paracoccus denitrijicans IAM 12479T, I F 0 12442, and IF0 13301, and Paracoccus halodenitrificans CCM 286T. Since the new isolates were facultative methylotrophs, Pseudomonas aminovorans ATCC 23314T and Methylobacterium extorquens JCM 2802T were selected as reference strains of C1 compound-utilizing bacteria (1, 8). Thiobacillus versutus THI 041T (=IAM 12815T),another facultative methylotroph (ll), also was tested in DNA-DNA homology experiments. Morphological and physiological characteristics. Cells of the isolates grown in ST medium were negatively stained with 1%(wthol) phosphotungstic acid, and their shape and size were examined with a model JEOL 200CX transmission electron microscope at 100 kV. Nitrate reduction and denitrification were examined in nutrient broth containing 1 g of potassium nitrate per liter. Denitrification was determined by the formation of gas in a
* Corresponding author. 292
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293
TABLE 1. DNA-DNA relatedness among the isolates, Paracoccus species, Pseudomonas aminovorans, and Methylobacterium extorquens % Homology with labeled DNA from?
Source of cold DNA"
G+C content (mol%)
Strain B
Paracoccus denitriJicans IAM 12479T
Paracoccus halodenitrificans - CCM 286T
Strain B Strain E l Strain F1 Paracoccus denitrijicans IAM 12479T Paracoccus denitrijicans IF0 12442 Paracoccus denitrijicans IF0 13301 Paracoccus halodenitrificans CCM 286T Pseudomonas aminovorans ATCC 23314T Methy lobacterium extorquens JCM 2802= Thiobacillus versutus IAM 1281ST Micrococcus luteus IAM 1056T
71 71 71 67 67 67 64 62 67 67' 73
100 87 80 30 15 26 3 4 3 2 10
31 10 34 100 43 61 5 12 8 4 6
10 5 0 0 0 8 100 1 0 0 1
Pseudomonas aminovorans ATCC 23314T
23 11 11 13 7 11 9
100 18 1 12
Methylobacterium extorquens JCM 2802T
16 14 2 1 5 12 3 3 100 0 9
" ATCC, American Type Culture Collection, Rockville, Md. ; CCM, Czechoslovak Collection of Microorganisms, J. E. Jurkyne University, Brno, Czechoslovakia; IAM, Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan; IFO, Institute for Fermentation, Osaka, Japan; JCM, Japan Collection of Microorganisms, Saitama, Japan. Data from reference 6.
'
stab culture of ST medium containing 0.1% (wt/vol) soft agar. The nitrogen source was examined in a medium containing (grams per liter): KH2P04, 1; MgSO, 7H@, 0.5; KC1, 0.2; thiamine hydrochloride, 0.0005; and acetic acid (pH 7.2), 5 , to which potassium nitrate (1 g/liter), ammonium sulfate (0.78 g/liter), or sodium glutamate (0.15 g/liter) was added. Urease activity was examined in Christensen medium (Nissui Seiyaku, Tokyo, Japan) supplemented with 20 g of urea per liter. Salt tolerance was determined in medium B (12) supplemented with 0.15% sodium acetate instead of methanol. Utilization of thiosulfate for growth was examined in SM-basal salts medium (6) with 0.5% (wthol) Na,S,03 - 5H,O and 0.5 mg of thiamine hydrochloride per liter. Unless otherwise indicated, results of these tests were obtained after incubation for 1 week at 30°C. Assimilation of organic compounds. Assimilation of organic compounds was examined in medium B supplemented with 25 mg of yeast extract per liter and one of the compounds shown in Table 2 as a growth substance. Assimilation of formate, methanol, ethanol, and oxalate was tested at concentrations of 5, 10, and 20 mM. Determination of quinone system. Quinone fractions were extracted with chloroform-methanol (2:1, vol/vol) from the lyophilized cells and subsequently purified with preparative thin-layer chromatography developed with petroleumbenzine-diethyl ether (9:1, vol/vol). The fractions were then subjected to high-performance liquid chromatography in an LC-3A chromatograph (Shimadzu Corporation, Kyoto, Japan) equipped with a Zorbax-ODS prepacked column (4.6 by 250 mm). Ubiquinones were detected with a Shimadzu SPD-2A spectrophotometric detector at 275 nm. Methanolisopropylether (4:1, voYvol) was used as the eluent. A quinone homolog consisting of more than 90% of the ubiquinone fractions was considered to be the major ubiquinone component. Determination of cellular fatty acids. Cellular fatty acids were extracted with n-hexane after methylation with 5% HCI-methanol and then analyzed by gas-liquid chromatography with a Shimadzu GC-6AM apparatus. Fatty acid methyl esters were identified by capillary-column chromatography (Advance-DS and Silicone OV-101; inside diameter, 0.24 mm by 25 m; Chromato Packing Center, Tokyo, Japan) after fractionation of the esters to nonpolar, 2-OH and 3-OH fatty
acids by thin-layer chromatography (silica gel; Merck & Co., Inc., Rahway, N.J.); the solvent system used was n-hexanediethyl ether (85:15, volhol). G+C content of DNA. DNA was prepared according to the procedure described by Saito and Miura (9). G+C content of DNA was determined by using high-performance liquid chromatography (Shimadzu LC-3A equipped with a ZorbaxODS prepacked column [4.6 by 250 mm]) after digestion of the DNA to deoxyribonucleotides with nuclease P1 (5). The mobile phase used was 20 mM KH,PO, (pH 3.5)-methanol (60: 1, volhol). DNA relatedness. DNAs were labeled by nick translation by using a commercial kit (N 5000; Amersham International Ltd., Amersham, United Kingdom), and DNA-DNA hybridization was carried out at 65°C as previously described (4). Micrococcus luteus IAM 1056 was used as the reference strain.
RESULTS AND DISCUSSION Isolation. Samples were taken from an activated sludge in which TMA was being treated; they were diluted and spread on agar medium containing TMA as the sole energy source. Strains A, BT, C, E l , E2, and F1 were isolated in pure culture by streak plating. The isolates grew in mineral salts medium supplemented with TMA and small amounts of yeast extract but were unable to grow in the absence of either substrate. TMA-degrading bacteria were considered to belong to the genus Paracoccus because of their cell shape, lack of motility, facultatively anaerobic growth, quinone system, and nonpolar fatty acid composition. However, tolerance to NaCl, assimilation of organic compounds, hydroxy fatty acid composition, and other phenotypic characteristics indicate that the isolates are distinct species in the genus Paracoccus. Results on DNA relatedness supported this conclusion (Table 1).Therefore, we propose a new species, Paracoccus kocurii, for TMA-degrading bacteria. Description of Puracoccus kocun'i sp. nov. (k0.cu'ri.i. N. L. Gen. n. kocurii of Kocur; named for Miloslav Kocur, Czechoslovakian bacteriologist). The colonial appearance and assimilation of organic compounds of all of the isolates were quite similar. Further characterization was carried out on strains BT, E l , and F1. The isolates are gram-negative,
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OHARA ET AL.
nonmotile, short rods (0.5 to 0.8 by 0.7 to 1.1 pm) (Fig. 1). Colonies are pinhead size, circular, smooth, convex, entire, transparent, and yellowish white on nutrient agar and pinhead size, circular, entire, convex, transparent, and brownish white on TMA agar after 10 days of incubation. Production of pigment was not observed. Optimum temperature for the growth is between 25 and 30°C; no growth occurs at 20 or 40°C. Optimum pH for the growth is between pH 6.6 and pH 8.2; no growth occurs below pH 6.1 or above pH 8.8, which distinguishes the isolates from Paracoccus alkaliphilus (13). The isolate is facultatively anaerobic, uses nitrate as a terminal electron acceptor, and evolves gas. Growth occurs in the absence of NaCl, but no growth is produced in 3% NaCl (Table 2), which is distinct from Paracoccus halodenitriJcans, which grows optimally in the presence of 4.4 to 8.8% NaCl(7). The isolates use ammonium salt as a nitrogen source but are unable to grow on nitrate or glutamate. Oxidase and catalase activities are produced, but urease activity is not. Cells require thiamine for growth. Thiosulfate does not support their growth. Assimilation of organic compounds by TMA degraders is restricted to a narrow range of substrates, as shown in Table 3. The isolates are unable to use the amino acids or sugars examined, These assimilations are different from those of Paracoccus denitrijicans, which has versatile abilities on amino acids and sugars. The isolate grows on TMA, trimethylamine, trimethylamine-N-oxide, dimethylamine, and methylamine. The major respiratory quinone is Q-lo. The major cellular fatty acids are C18:1 and ClgcYc,as nonpolar acids, and 3-OH Cloz0and 3-OH C12:o,as hydroxy acids (Table 4). The base composition of DNA of the type strain is 71 mol% G+C* The type strain is strain B (=JCM 7684). The organism
FIG. 1. Electron micrograph of Paracoccus kocurii BT. The cells were stained with phosphotungstic acid. Bar = 1 pm.
TABLE 2. Differential characteristics of Paracoccus species, Pseudomonas aminovorans, and Methylobacterium extorquensa Characteristic
Cell shape Cell size (pm) Width Length Motility Growth in: 0% NaCl 3% NaCl 6% NaCl 12% NaCl Nitrate reduction Denitrification Nitrogen source: KNO, (NH,),SO, Glutamate Urease Optimum pH Optimum temp Vitamin requirement
Paracoccus kocurii (3 strains)
Paracoccus denitrificans ( 3 strains)
Paracoccus halodenitrificans CCM 286T
Short rod
Short rod
Short rod
0.5-0.8 0.7-1.1
0.6-0.8 0.7-1.0 -
-
+
-
W -
6.6-8.2 2530°C Thiamine
+ + + +
Coccoid or short rod
Rod
Rod
o.fs1.0
0.5-0.7 1.&1.4
1.1-1.3 2.2-3.0
+ +
-
-
+ +
Methylobacterium extorquens JCM 2802T
0.8-2.0
+ + + +" +"
-
alkaliphilusb
Pseudomonas aminovorans ATCC 23314T
Paracoccus
+
-
W
+ + -
+ -
+ +
W
+
W
-
8-9
30°C -
25-30°C' Thiamine"
Biotin ~~
~~
Symbols: +, all strains positive; W, weak positive; -, all strains negative. All strains are gram negative, nonpigmented, and oxidase and catalase positive, accumulate poly-P-hydroxybutyrate, and fail to grow in 15% NaCl or to hydrolyze starch. Data from reference 13. Data from reference 7. a
VOL. 40, 1990
P. KOCURZZ SP. NOV., A TMA-ASSIMILATING BACTERIUM
295
TABLE 3. Assimilation of organic compounds by Paracoccus species, Pseudomonas aminovorans, and Methylobacterium extorquensa Growth of? Concn
Substance
(mM)
Methylamine Dimethylamine Trimeth ylamine Trimeth ylamine-N-oxide TMA Methanol Methanol Methanol Ethanol n-Butanol Glycerol Cyclohexanol G1ycine L- Alanine L-Valine L-Leucine L-Isoleucine L-Threonine L-Cysteine L-Methionine L-Phen ylalanine L-Tyrosine L-Histidine L-Tryptophan L-Aspartate L-Asparagine L-Glutamate L-Glutamine L-Lysine L- Arginine L-Ornithine L- Arabinose D-Ribose D-Xylose D-Glucose D-Galactose D-Fructose Sucrose Trehalose Lactose Maltose L-Cellobiose Mannitol D-Sorbitol Inositol Formate Propionate n-Buty late Lactate Pyruvate Gluconate m ma late Succinate a-Ketoglutarate Adipate Tartarate Citrate p-H ydrox ybenzoate p -Aminobenzoate DL-Mandelate ~
10 10 10 10 10 5 10 20 5, 10, 20 20 20 10 20 20 20 20 20 20 20 20 10 20 5 5 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 5 , 10, 20 20 20 20 20 20 20 20 20 20 20 20 10 10 10 ~
Paracoccus kocurii (6 strains)
Paracoccus denitriJicans (3 strains)
+ +
Paracoccus alkaliphilus'
+
W -
-
+ D(2/3) D(2/3)
+ +
D(2/3) -
+ +
-
+ -
+
+ + +
W -
+ +
W W -
W W
D(1/3) D( 1/3) -
Methy lobacterium extorquens JCM 2802T
-
+
+ + + + + + + + + + + + W + + D(2/3) + W + + + + + W + W + + + + + + + + + + +-
Pseudomonas aminovorans ATCC 23314T
+
+ + + + + + + W + + W + + + W + + + + + + + + + + + W + + + + + W + W + + + W W W
+
W -
W ~
~
All tested strains assimilated 40 rnM L-serine, 20 rnM L-proline, and 20 mM acetate. In addition, acetate was reported to be assimilated by Paracoccus alkaliphilus (13). No tested strains assimilated 5 mM L-tryptophan; 5, 10, or 20 rnM oxalate; 20 mM glutarate; 20 mM pimerate; 10 mM benzoate; or 10 rnM p-aminobenzoate. Symbols: +, all strains positive; D, different reactions among strains; W, weak positive; -, all strains negative. Values within parentheses are the number of positive strains/nurnber of strains tested. Data from reference 13. a
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INT. J. SYST.BACTERIOL.
OHARA ETAL.
was isolated from activated sludge for the treatment of TMA hydroxide. Differential characteristics among species of Paracoccus are given in Tables 2, 3, and 4. Since taxonomic studies on microorganisms using the quinone system show that differentiation of taxa is at the genus level, the fact that Paracoccus halodenitrificans contained Q-9 as the major quinone system may indicate that further taxonomic studies are required on Paracoccus halodenitrijicans. ACKNOWLEDGMENTS
c,
c,
f
We thank M. Kocur, Czechoslovak Collection of Microorganisms, Bmo, Czechoslovakia; Japan Collection of Microorganisms, Riken, Saitama, Japan; and Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan, for supplying the strains. We also thank K. Komagata, Tokyo Agriculture University, and J. Sugiyama, Institute of Applied Microbiology, University of Tokyo, for their helpful discussions and I. Hirata, Institute of Applied Microbiology, University of Tokyo, for technical assistance with electron microscopy. LITERATURE CITED
4 0
-3 c Y
5iE 3;
1. Bousfield, I. J., and P. N. Green. 1985. Reclassification of Dacteria of the genus Protomonas Urakami and Komagata 1984 in the genus Methylobacterium (Patt, Cole, and Hanson) emend. Green and Bousfield 1983. Int. J. Syst. Bacteriol. 35209. 2. Colby, J., and L. J. Zatman. 1973. Trimethylamine metabolism in obligate and facultative methylotrophs. Biochem. J. 132: 101-112. 3. Hampton, D., and L. J. Zatman. 1973. The metabolism of tetramethylammonium chloride by bacterium 5H2. Biochem. SOC.Trans. 1:667-668. 4. Katayama-Fujimura, Y., Y. Enokizono, T. Kaneko, and H. Kuraishi. 1983. Deoxyribonucleic acid homologies among species of the genus Thiobacillus. J. Gen. Appl. Microbiol. 29: 287-295. 5. Katayama-Fujimura, Y., Y. Komatsu, H. Kuraishi, and T. Kaneko. 1984. Estimation of DNA base composition by high performance liquid chromatography of its nuclease P1 hydrolysate. Agric. Biol. Chem. 483169-3172. 6. Katayama-Fqiimura, Y., N. Tsuzaki, and H. Kuraishi. 1982. Ubiquinone, fatty acid and DNA base composition determination as a guide to the taxonomy of the genus Thiobacillus. J. Gen. Microbiol. 128:1599-1611. 7. Kocur, M. 1984. Genus Paracoccus Davis 1969, p. 399402. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 8. Palleroni, N. J. 1984. Genus Pseudomonas Migula 1894, p. 141-199. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 9. Saito, H., and K. Miura. 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72:619-629. 10. Stanier, R Y., N. J. Palleroni, and M. Doudoroff. 1966. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43:159-271. 11. Taylor, B. F., and D. S. Hoare. 1969. New facultative Thiobacillus and a reevaluation of the heterotrophic potential of Thiobacillusnovellus. J. Bacteriol. 1W487-497. 12. Urakami, T., and K. Komagata. 1984. Protomonas, a new genus of facultatively methylotrophic bacteria. Int. J. Syst. Bacteriol. 34:188-201. 13. Urakami, T., J. Tamaoka, K. Suzuki, and K. Komagata. 1989. Paracoccus alkaliphilus sp. nov., an alkaliphilic and facultatively methylotrophic bacterium. Int. J. Syst. Bacteriol. 39: 116-121.
