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Bacillus asahii sp. nov., a novel bacterium isolated from soil with the ability to deodorize the bad smell generated from short-chain fatty acids

¹ Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan
² Department of Bioscience and Technology, School of Engineering, Hokkaido Tokai University, Minaminosawa, Minami-ku, Sapporo 005-8601, Japan
³ Laboratory of Electron Microscopy, School of Dentistry, Hokkaido University, Kita-ku, Sapporo 060-8586, Japan
⁴ Asahi Vet Japan Co., Ltd, 724-1 Midorimachi, Shiraoi-cho, Hokkaido 059-0931, Japan

Correspondence
Isao Yumoto
i.yumoto{at}aist.go.jp

	ABSTRACT

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In a screening campaign to isolate strains with the ability to remove the bad smell associated with animal faeces, strain MA001^T was isolated from a soil sample obtained from Shizuoka prefecture, Japan. The isolate grew at pH 6–9 but not at pH 10. Cells were Gram-positive, straight rods with peritrichous flagella and produced ellipsoidal spores. The isolate was positive for catalase and oxidase tests but negative for indole production, deamination of phenylalanine and H₂S production. The isolate did not produce acid from any carbohydrates tested and could not grow in more than 2 % NaCl. The DNA G+C content was 39·4 mol%. The cellular fatty acids profile consisted of significant amount of C₁₅ branched-chain fatty acids, iso-C_{15 : 0} and anteiso-C_{15 : 0}. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain MA001^T was closely related to Bacillus simplex and Bacillus psychrosaccharolyticus. DNA–DNA hybridization revealed a low relatedness of the isolate to several phylogenetically close neighbours (less than 9 %). On the basis of the phenotypic characteristics observed, phylogenetic data based on 16S rRNA gene sequencing and DNA–DNA relatedness data, it is concluded that the isolate should be classified as representing a novel species, for which the name Bacillus asahii is proposed. The type strain is MA001^T (=JCM 12112^T=NCIMB 13969^T).

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain MA001^T is AB109209.

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Recently, offensive smells emanating from faeces in limited spaces where a large number of livestock and poultry are kept have become a serious environmental problem in Japan. The smell is caused by short-chain fatty acids, ammonia, indole and trimethylamine, etc., contained in the animal faeces. The use of micro-organisms with the ability to deodorize animal faeces has been considered as a possible solution to the problem. Strain MA001^T had been screened and isolated with the aim to remove the bad smell from animal faeces (Aoshima, 1996, 1998a, b). The isolate was able to reduce levels of the compounds causing the bad smell and was tentatively identified as Bacillus badius based only on phenotypic characterization at the time. Therefore, the application of more informative classification procedures had been necessary for a more precise identification of the isolate.

In the present study, taxonomic studies were performed on the isolated micro-organism, which exhibits a deodorizing effect on animal faeces. Physiological and biochemical characterization, phylogenetic analysis based on 16S rRNA gene sequences and DNA–DNA hybridization on the isolate and phylogenetic neighbours showed that the isolate should be classified as a novel species belonging to the genus Bacillus.

The strain examined was Bacillus sp. MA001^T isolated from soil obtained from Tagata-gun, Shizuoka, Japan. The isolate was cultured in PY-1 medium (pH 7·9) containing 8 g peptone (Kyokuto), 3 g yeast extract (Merck) and 15 g agar (if required) in 1 litre of distilled water. Cells for chemotaxonomic analysis were harvested in the late-exponential phase after cultivation with reciprocal shaking (140 r.p.m.) in PY-1 broth at 27 °C. In addition to the isolate, Bacillus psychrosaccharolyticus ATCC 23296^T, Bacillus flexus ATCC 49095^T, Bacillus simplex ATCC 49097^T and Bacillus badius IAM 11059^T were used as reference for phenotypic characteristics, utilization of butyrate and DNA–DNA hybridization with the isolate. These micro-organisms were cultivated under the same conditions.

For phenotypic characterization, PY-1 was used as the basal medium. The culture was incubated at 27 °C for 2 weeks and the phenotypic characterization experiment was performed three times. Acid production from carbohydrates was determined by the method of Hugh & Leifson (1953). The growth experiment at pH 5–10 was performed using PY-1 medium containing 100 mM acetate buffer, 100 mM NaH₂PO₄/Na₂HPO₄ buffer and 100 mM NaHCO₃/Na₂CO₃ buffer, at pH 5, 6–8 and 9–10, respectively. Requirement for and tolerance of NaCl was determined using a medium (pH 7·7) containing 2 g glucose, 1 g peptone (Difco), 0·1 g yeast extract (Difco) and 0–20 % NaCl at 1 % intervals in 1 litre of distilled water. Other physiological and biochemical characteristics were examined according to the methods as described in Barrow & Feltham (1993). Enzymic activity was determined by using API ZYM (bioMérieux).

For observation of negatively stained cells under a transmission electron microscope (TEM) and platinum- and palladium-coated cells under a scanning electron microscope (SEM), cells were grown on a PY-1 agar slant. The procedure for TEM and SEM preparations and observations were performed as described by Yumoto et al. (2002).

Analysis of whole-cell fatty acids was performed as described by Yumoto et al. (2001, 2002).

Bacterial DNA was prepared according to the method of Marmur (1961). The DNA base composition was determined by the HPLC method of Tamaoka & Komagata (1984). The level of DNA–DNA relatedness was determined fluorometrically by the method of Ezaki et al. (1989) using photobiotin-labelled DNA probes and black microplates.

The 16S rRNA gene sequence corresponding to positions 27–1519 in the 16S rRNA gene sequence of Escherichia coli (Brosius et al., 1978) was amplified by PCR. The PCR product (about 1·5 kb) was sequenced directly by the dideoxynucleotide chain-termination method using a DNA sequencer (PRISM 377; Applied Biosystems). Multiple alignments of the sequence were performed and the nucleotide substitution rate (K_nuc value) was calculated. A phylogenetic tree was constructed by the neighbour-joining method (Kimura, 1980; Saitou & Nei, 1987) using the CLUSTAL W program (Thompson et al., 1994). Similarity values for sequences were calculated using the GENETYX computer program (Software Development).

The isolate grew better (OD₆₅₀ 0·145) in 0·3 % sodium butyrate as short-chain fatty acid supplemented medium containing 0·02 % yeast extract (pH 7·9) than in the same medium devoid of the short-chain fatty acid control (OD₆₅₀ 0·001). Utilization of butyrate was also observed in B. simplex ATCC 49097^T, B. flexus ATCC 49095^T and B. badius IAM 11059^T. The OD₆₅₀ ratio in medium containing sodium butyrate versus control was higher (145) than for other tested strains (2·5–13·5). Obvious utilization of butyrate was not observed in B. psychrosaccharolyticus ATCC 23296^T because of its low growth on the medium used in this experiment. Electron microscopic observations by TEM and SEM of strain MA001^T revealed peritrichously flagellated rods of 1·4–3·0x0·4–0·8 µm (Fig. 1).

View larger version (149K): [in this window] [in a new window]   Fig. 1. Negatively stained cell of Bacillus asahii MA001T, showing peritrichous flagellation. Bar, 2 µm.

GLC analysis revealed that the methyl ester derivatives of fatty acids of the strain mainly consisted of iso-C_{15 : 0} (39·0 %), anteiso-C_{15 : 0} (27·8 %), iso-C_{14 : 0} (9·7 %) and C_{16 : 1} (5·4 %). The fatty acid profile further indicated that the isolate belonged to the genus Bacillus.

The 16S rRNA gene DNA of strain MA001^T was sequenced to determine its phylogenetic position. The 16S rRNA gene (1504 bp) of strain MA001^T was compared with the sequence of 18 other species belonging to the genus Bacillus as well as other taxa. The phylogenetic tree, constructed using the neighbour-joining method (Fig. 2), and the 16S rRNA gene similarity values (data not shown) indicated that strain MA001^T was phylogenetically related to other members of the family Bacillaceae. Strain MA001^T was placed in the group 1 Bacillaceae (Ash et al., 1991) in this phylogenetic tree. The highest sequence similarity value was observed with B. simplex (GenBank/EMBL/DDBJ no. AJ439078) (97·2 %) and B. psychrosaccharolyticus (GenBank/EMBL/DDBJ no. AB021195) (95·9 %).

View larger version (16K): [in this window] [in a new window]   Fig. 2. Phylogenetic tree derived from 16S rRNA gene sequence data of Bacillus asahii, members of other Bacillus species and other taxa using the neighbour-joining method. Numbers indicate bootstrap values greater than 500. Bar, 0·01 Knuc unit.

Phenotypic characteristics were examined using the same methods in strain MA001^T and other related strains (Table 1). Although these results differ from those reported by Priest et al. (1988) in several regards, they were reproducible. Strain MA001^T can be differentiated from other relatively closely related species based on its phenotypic characteristics. Although strain MA001^T was very similar to B. badius IAM 11059^T, it was differentiated based on several phenotypic characteristics.

Description of Bacillus asahii sp. nov.
Bacillus asahii (as.a.hi'i. N.L. gen. n. asahii of Asahi; named after Asahi Kasei Co. A researcher working in the company isolated the bacterium).

Cells are Gram-positive peritrichously flagellated straight rods (1·4–3·0x0·4–0·8 µm) and produce terminally or centrally located ellipsoidal spores. Utilizes butyrate as carbon source for growth. Spores do not cause swelling of sporangium. Colonies are circular and white. Catalase and oxidase reactions are positive. Nitrate reduction to nitrite is weakly positive. Negative for indole production, Voges–Proskauer test, methyl red test, growth on MacConkey agar and H₂S production. Trypsin, esterase (C4) and esterase/lipase (C8) are positive. Alkaline phosphatase, valine arylamidase, cystine arylamidase, chymotrypsin, acid phosphatase, -galactosidase, -glucosidase, -glucosidase and N-acetyl--glucosaminidase are negative. Growth occurs at pH 6–9; growth at pH 5 is variable. Growth occurs at 0–1 % NaCl but not at 2 % NaCl. Growth occurs at 15–45 °C, but not above 50 °C. No acid is produced from D-arabinose, D-xylose, L-rhamnose, D-glucose, D-fructose, D-mannose, D-galactose, maltose, sucrose, lactose, trehalose, cellobiose, melibiose, raffinose, glycerol, mannitol, myo-inositol or sorbitol. Hydrolysis of casein, DNA, and Tweens 20, 40 and 60 is observed but hydrolysis of gelatin is not. Hydrolysis of starch is weakly observed. iso-C_{15 : 0} (39·0 %) and anteiso-C_{15 : 0} (27·8 %) represent the main fatty acids produced during growth in PY-1 medium. The DNA G+C content is 39·4 mol%, as determined by HPLC.

The type strain, MA001^T (=JCM 12112^T=NCIMB 13969^T), was isolated from a soil sample obtained from Tagata-gun, Shizuoka, Japan.

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