Alicyclobacillus cellulosilyticus sp. nov., a thermophilic, cellulolytic bacterium isolated from steamed Japanese cedar chips from a lumbermill

Abstract

A thermophilic bacterium, strain Sueoka^T, was isolated from steamed Japanese cedar chips from a lumber mill in Gobo, Japan. The strain was able to grow on carboxymethyl cellulose at 60 °C, was Gram-stain-negative, and grew between 40.0 and 67.5 °C (optimum at 55 °C) and between pH 3.5 and 6.5 (optimum at pH 4.8). Comparative analysis of 16S rRNA gene sequences revealed 91.9 , 90.9 , and 90.8 % similarity to Alicyclobacillus macrosporangiidus^T, Alicyclobacillus pomorum^T, and Alicyclobacillus acidocaldarius^T, respectively. The major quinone was MK-7 and the predominant cellular fatty acids were ω-cyclohexane C_19 : 0 and ω-cyclohexane C_17 : 0. The DNA G+C content was 60.8 mol%. Based on the results of this study, strain Sueoka^T is a novel species of the genus Alicyclobacillus, and the namehttp://dx.doi.org/10.1601/nm.5071Alicyclobacillus cellulosilyticus sp. nov. (type strain Sueoka^T = JCM 18487^T = KCTC 33007^T) is proposed.

The genus Alicyclobacillus was proposed in 1992 (Darland & Brock, 1971; Deinhard et al., 1987a, b; Wisotzkey et al., 1992; Dufresne et al., 1996; Nicolaus et al., 1998; Albuquerque et al., 2000; Goto et al., 2002a, 2002b, 2003; Matsubara et al., 2002; Tsuruoka et al., 2003; Simbahan et al., 2004; Karavaiko et al., 2005), and was characterized as comprising moderately thermophilic, acidophilic, strictly aerobic, and endospore-forming bacilli with ω-alicyclic fatty acids (ω-cyclohexane or ω-cycloheptane) as the major membrane lipids. These unique structural polar lipids provide moderate thermal stability (Koga, 2012). Alicyclobacillus sp. mainly inhabits hot springs, soil, and spoiled fruit-based beverages. Some species, in particular Alicyclobacillus acidocaldarius DSM 446^T and Alicyclobacillus sp. A4, are able to grow with the production of thermoacidophilic cellulase (Morana et al., 2008; Bai et al., 2010). In this paper, we describe the characteristics of a novel strain, Sueoka^T, which can grow with the production of thermoacidophilic cellulase and ω-alicyclic fatty acids.

We isolated a moderately thermophilic, cellulolytic, and endospore-forming bacterium from steamed Japanese cedar chips from a lumber mill in Gobo, Wakayama, Japan. These chips were steamed at over 180 °C within 60 min for antiseptic processing of ants. The strain name ‘Sueoka’ originates from the researcher’s name, Mr ‘Ueoka’, and the ‘small’ colony size. Based on the low similarities of the 16S rRNA hypervariable region, the strain was distinct from other species of the genus Alicyclobacillus. The objective of the present study was to establish the taxonomic position of strain Sueoka^T using polyphasic taxonomic approaches. No related strains were isolated, therefore a single Sueoka^T strain was used for this study alongside the reference strain Alicyclobacillus macrosporangiidus (JCM21814).

The characteristics of strain Sueoka^T were determined in 573C medium (modified ATCC 573 medium) containing the following components (l⁻¹): 2.5 g carboxymethyl cellulose sodium salt (CMC), 1.3 g (NH₄)₂SO₄, 0.37 g KH₂PO₄, 1 g glucose, 0.25 g yeast extract (Difco), 0.9 g tryptone (Difco), and 20 g agar for solid medium. After autoclaving, 1 ml of the following metal solutions was added aseptically: 25 % MgSO₄.7H₂O (final concentration: 0.25 g L⁻¹), 7 % CaCl₂.2H₂O (final concentration: 0.07 g l⁻¹), and 2 % FeCl₃.6H₂O (final concentration: 0.02 g l⁻¹). The pH was approximately 6 and the culture temperature was 55 °C.

Colony morphology was examined on 573C plate medium after incubation for one week at 55 °C. Gram staining was performed according to Dussault (1955). Cell morphology was examined using phase-contrast and scanning electron microscopy (Fig. S1 and S2; available in the online Supplementary Material). Motility was checked using phase-contrast microscopy and/or using semi-solid agar stabs (2 g agar l⁻¹).

To determine the growth characteristics bacteria were grown in 573M medium [without CMC, yeast extract and tryptone, and with 1 g malt extract broth l⁻¹ (Difco) added]. The pH range for growth was assayed at pH 2.5–8.0 at intervals of 0.5. The temperature range for growth was determined at 20–70 °C at intervals of 2.5 °C. NaCl tolerance was tested at 0–10 % (w/v) at intervals of 0.5 % (w/v). Utilization of single or complex carbon sources was assessed in 573 basal salt medium (573BS) containing the salt components of 573C medium and 1 ml vitamin 10 mixture l⁻¹ (Janssen et al., 1997), supplemented with 0.2 % (w/v) of the carbon source.

Acid formation from sugar was tested using API 50 CH (Sysmex) supplemented with 0.015 mg bromophenol blue l⁻¹ as an indicator. Tests for catalase, oxidase, urease, aesculin hydrase (β-glucosidase), β-galactosidase, nitrate reduction, and indole production from tryptophan were performed using API 20NE (Sysmex). Other enzyme activities were assessed using API ZYME (Sysmex) or general plate methods, as described by Hudson et al. (1986). Cell suspensions for the API tests were prepared in 573C basal salt medium (pH 4.8). The incubation temperature for the tests was 55 °C, and the incubation times were: 24 h for API ZYME; 48 h for API 20 NE and seven days for API 50 CH.

H₂S formation was determined from a black sulfide precipitate in medium containing 0.05 g sodium thiosulfate l⁻¹ or 0.2 g l-cysteine l⁻¹ (Smibert & Krieg, 1994). The Voges–Proskauer reaction was performed as described previously (Smibert and Krieg, 1981). Anaerobic growth was tested on 573E medium supplemented with elemental sulfur (5.0 g l⁻¹), ferrous iron (FeCl₂.4H₂O; 0.07 g l⁻¹), potassium tetrathionate (3.0 g l⁻¹), or sodium thiosulfate (0.15 g l⁻¹).

Detailed results are included in the species description and differences between the Sueoka^T strain and related species are highlighted in Table 1.

Table 1. Characteristics differentiating strain Sueoka^T from species of the genus Alicyclobacillus.

Strains: 1, Sueoka^T; 2, A. aeris ZJ-6^T; 3, A. ferrooxydans TC-34^T; 4, A. pomorum 3A^T; 5, A. contaminans 3-A191^T; 6, A. tolerans K1^T; 7, A. hesperidum DSM 12489^T; 8, A. macrosporangiidus DSM 17980^T; 9, A. acidoterrestris DSM 3922^T; 10, A. sacchari DSM 17974^T; 11, A. cycloheptanicus DSM 4006^T; 12, A. acidiphilus TA-67^T; 13, A. fastidiosus DSM 17978^T; 14, A. kakegawensis DSM 17979^T; 15, A. shizuokensis DSM 17981^T; 16, A. herbarius DSM 13609^T; 17, A. disulfidooxidans DSM 12064^T; 18, A. vulcanalis CsHg2^T; 19, A. sendaiensis JCM 11817^T; 20, A. pohliae MP4^T; 21, A. acidocaldarius subsp.acidocaldarius DSM 446^T. +, Positive; −negative; nd, not determined; w, weakly positive; v, variable between strains. Data for columns 2-21 are from Guo et al. (2009) and the present study.

Characteristic	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Growth temperature (○C)	40-67.5	25-35	17-40	30-60	35-60	20-55	35-60	35-60	35-55	30-55	40-53	20-50	20-55	40-60	35-60	35-65	Apr-40	35-65	40-65	42-60	45-70
Growth pH range	3.5-6.0	2.0-6.0	2.0-6.0	3.0-6.0	3.0-6.0	1.5-5.0	2.0-6.0	3.0-6.5	2.2-5.8	2.0-6.5	3.0-5.5	2.5-5.5	2.0-5.5	3.0-6.5	3.0-6.5	3.0-6.5	0.5-6.0	2.0-6.0	2.5-6.5	4.5-7.5	2.0-6.0
G+C content (mol%)	60.8	51.2	48.6	53.1	60.3	48.7±0.6	53.3	62.5	52.2	56.6	55.6	54.1	53.9	61.5±0.2	60.5	56.2	53	62	62.3	55.1	60.3
Oxidase	-	-	+	+	-	w	-	-	-	-	+	-	-	-	-	-	-	nd	-	-	-
Catalase	-	-	+	+	-	w	-	w	+	-	+	+	+	w	+	+	-	-	-	-	+
Hydrolysis of:
Gelatin	-	+	-	+	+	-	+	-	+	+	-	-	+	-	-	-	-	nd	nd	nd	+
Starch	-	-	+	+	-	+	+	-	-	+	-	-	-	-	-	-	+	+	nd	nd	+
Carboxymethyl cellulose	+	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	+
Nitrate reduction to Nitrite	-	+	-	-	-	-	-	-	-	-	-	-	-	-	-	+	-	nd	+	nd	-
Growth in 5 % NaCl	-	-	-	-	-	nd	-	+	+	-	+	-	-	-	+	+	nd	-	nd	-	-
Acid production from:
Indole production	-	-	+	nd	-	nd	nd	-	nd	-	nd	-	-	-	-	-	nd	nd	nd	nd	-
Voges-Proskauer reaction	-	-	-	nd	-	nd	nd	-	nd	-	nd	w	-	-	-	-	nd	nd	+	nd	w
Glycerol	-	+	-	+	+	w	+	+	+	+	-	-	-	-	-	+	+	nd	+	-	+
D-Arabinose	-	-	-	-	-	w	-	+	-	-	+	+	+	+	-	+	+	nd	+	+	-
L-Arabinose	+	+	+	-	+	w	+	+	+	+	+	+	+	+	+	+	+	nd	+	+	+
D-Xylose	+	+	-	-	+	+	+	+	-	+	+	+	+	+	+	+	+	nd	nd	+	+
L-Xylose	+	+	-	-	-	+	-	+	-	-	+	-	-	+	+	-	+	nd	nd	+	-
Methyl β-xyloside	w	-	+	-	-	+	-	-	-	+	-	-	+	-	-	-	nd	-	nd	-	-
D-Galactose	+	+	-	-	+	+	+	+	+	+	-	+	+	+	+	+	-	nd	+	+	+
L-Sorbose	-	-	-	+	v	+	-	+	-	-	+	+	-	+	-	-	+	nd	nd	+	-
Rhamnose	-	+	-	-	v	-	-	+	+	+	+	-	+	+	-	+	-	-	-	+	+
Mannitol	-	+	-	+	+	+	+	-	+	+	+	-	+	+	+	+	+	+	nd	-	+
Sorbitol	-	+	+	-	-	-	-	+	+	-	+	+	-	+	-	-	-	-	-	-	-
Methyl α-D-mannoside	-	-	+	-	-	+	-	+	-	-	-	-	-	+	-	+	nd	nd	nd	-	-
Amygdalin	+	-	-	+	-	+	-	-	-	-	-	-	-	+	-	+	+	nd	-	-	-
Arbutin	+	-	-	-	+	-	+	-	-	+	-	+	-	+	+	+	-	w	+	w	+
Aesculin	+	+	+	+	+	+	+	+	-	-	+	+	-	+	+	+	+	nd	-	+	+
Salicin	+	-	-	+	v	-	-	+	-	+	-	+	-	+	+	+	-	-	+	w	+
Cellobiose	+	-	-	-	+	-	+	-	+	+	-	+	-	+	+	+	+	nd	+	+	+
Maltose	+	-	-	+	+	w	+	+	+	+	-	+	-	+	+	+	+	nd	+	+	+
Lactose	+	-	-	-	v	w	-	+	+	+	-	+	-	+	-	+	-	-	+	+	+
Melibiose	+	-	-	-	-	-	-	-	w	+	-	-	+	-	+	+	+	+	-	+	+
Sucrose	+	-	-	+	+	+	+	+	+	+	-	+	-	+	+	+	+	+	+	+	+
Trehalose	-	-	+	+	+	+	+	+	+	+	w	+	+	+	+	+	-	+	+	w	+
β-Gentiobiose	+	-	-	-	v	nd	w	+	-	+	-	+	-	+	+	+	nd	-	nd	+	-
Turanose	+	-	+	+	-	w	+	-	-	+	-	+	-	+	-	+	-	+	+	+	-
D-Lyxose	-	-	+	-	-	nd	-	+	-	-	+	-	+	+	-	-	nd	nd	nd	+	-
D-Tagatose	-	-	+	+	v	w	-	-	-	-	+	-	+	v	-	-	+	nd	nd	+	-
5-Ketogluconate	+	+	+	+	-	+	+	-	-	-	+	-	-	-	-	+	+	w	-	+	-

Cellular fatty acids and quinones were extracted as described by Komagata & Suzuki (1987). Analysis of cellular fatty acids was performed using the Sherlock Microbial Identification System Version 4.5 (MIDI, Delaware) with the MIS Standard Libraries TSBA40 and using gas chromatography-mass spectrometry (GC/MS-QP2010, Shimadzu Co., Kyoto, Japan) with Inert Cap WAX column (GL science, Tokyo, Japan). Fragment ions at m/z 83, 199 and 227 in the GC/MS spectra (Supplementary Fig. S5(a and b)) could be attributed to ω-alicyclic fatty acids (Oshima et al., 1975). Cellular fatty acid components were analysed by the Technosuruga Laboratory Co. Ltd (Shizuoka, Japan) using cells grown in 573C medium for six days. The major fatty acids of strain Sueoka^T were ω-cyclohexane C_19 : 0 (45.7 %), ω-cyclohexane C_17 : 0 (43.9 %), C_17 : 0 (2.3 %), iso-C_17 : 0 (1.9 %), C_16 : 0 (1.8 %), anteiso-C_17 : 0 (1.3 %) and iso-C16 : 0 (0.9 %). ω-Cycloheptane fatty acids were not detected. A comparison of the fatty acid profiles of various species of the genus Alicyclobacillus is provided in Table S1. Quinones were extracted and analysed as described by Tamaoka (1986). The major quinone in the Sueoka^T strain was menaquinone-7 (MK-7).

Genomic DNA was extracted using a DNeasy Blood and Tissue kit (Qiagen) and purified using a QIAquick Gel Extraction kit (Qiagen). The DNA G+C content was determined by standard methods (Tamaoka & Komagata, 1984; Katayama-Fujimura et al., 1984) using a Prominence UFLC HPLC system (SHIMADZU) with a cosmosil 5C₁₈-AR-II column (φ4.6×250 mm) (Nacalai Tesque). The DNA G+C content of strain Sueoka^T was 60.8 mol%, within the range of species of the genus Alicyclobacillus (48.7–62.7 mol%) (Karavaiko et al., 2005). The 16S rRNA gene of strain Sueoka^T was amplified with the primers 27f(5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r(5′-GGCTACCTTGTTACGACTT-3′) and the following PCR conditions: an initial step at 95 °C for 2.5 min, 50 °C for 2 min, 72 °C for 1.5 min, followed by 28 cycles of 95 °C for 0.5 min, 50 °C for 2 min, 72 °C for 1.5 min and a final step at 72 °C for 10 min. PCR products were purified with a QIAquik PCR purification kit (Qiagen). Sequencing of the 16S rRNA gene, including the hypervariable region (Goto et al., 2002c), was conducted with the following six primers: 27f(5′-AGAGTTTGATCCTGGCTCAG-3′), 341r(5′-CTGCTGCCTCCCGTAGG-3′), 785r(5′-CTACCAGGGTATCTAATCC-3′), 1100r(5′-AGGGTTGCGCTCGTTG-3′), 1114f(5′-GGTTAAGTCCCGCAACG-3′) and 1492r(5′-GGCTACCTTGTTACGACTT-3′) (Lane, 1991) using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems).

The 16S rRNA gene sequence of strain Sueoka^T (1503 bp) and related taxa obtained from GenBank were aligned by clustal_x ver. 2.1 (Larkin et al., 2007). Gaps and ambiguous nucleotides were removed from the alignment using GENETYX ver. 10 (GENETYX Corporation) and then the 1379 bp of the resultant alignment were used for phylogenetic analysis. A phylogenetic tree was reconstructed using the neighbour-joining (NJ) (Fig. 1) and maximum-likelihood (ML) methods with GTRCAT or GTRGAMMAI (Fig. S3 and S4), and evaluated with bootstrap sampling and with values expressed as percentages of 1000 replicates. The ML (GTRGAMMA) method generally showed low bootstrap values, however it is often more accurate at inferring the correct tree than other methods (Kuhner & Felenstein, 1994; Huelsenbeck, 1995).

Fig. 1.

Phylogenetic tree reconstructed from 16S rRNA gene sequences showing the position of strain Sueoka^T among related strains. The tree was reconstructed by the Neighbour-Joining (NJ) method. Bootstrap values >60 % (1000 replicates) are shown. Bar, 0.01 sequence divergence.

From comparisons of 16S rRNA gene sequences strain Sueoka^T was closely related phylogenetically to species of the genus Alicyclobacillus: A. macrosporangiidus DSM 17980^T (91.9 %), A. pomorum 3A^T (90.9 %), and A. acidocaldarius DSM 446^T (90.8 %). All trees (Fig. 1) also supported the conclusion that strain Sueoka^T belongs to the genus Alicyclobacillus and represents a novel species.

Phenotypic, chemotaxonomic, and phylogenetic data based on 16S rRNA gene sequence comparisons support the classification of strain Sueoka^T as a novel species of the genus Alicyclobacillus. We propose strain Sueoka^T to be the novel species Alicylobacillus cellulosilyticus sp. nov.

Description of Alicylobacillus cellulosilyticus sp. nov.

http://dx.doi.org/10.1601/nm.5071Alicyclobacillus cellulosilyticus (cel.lu.lo.si.ly′ti.cus. N.L. n. cellulosum cellulose; N.L. part. adj. lyticus from Gr. adj. lutikos dissolving; N.L. masc. part. adj. cellulosilyticus cellulose-dissolving).

Cells are Gram-stain-negative, aerobic, non-motile rods (0.5–0.8×2.0–6.0 µm) during the exponential growth phase. Colonies on modified bacillus acidcaldarius medium (BAM)-CMC plates are white with diameters of 0.5–1.0 mm after incubation for one week; the colour gradually changes to grey from the centre to the rim after an additional week (diameter 1.2–1.8 mm). Cells grow in BAM-MEB medium over a temperature range of 40.0–67.5 °C (optimum at 55 °C) and a pH range of 3.5–6.5 (optimum at pH 4.8). Sensitive to NaCl and grows in the presence of 0.5 % (w/v) NaCl (weakly), but growth is completely inhibited by 1 % (w/v) NaCl.

Oxidase, catalase, nitrate reduction, indole production, and the Voges–Proskauer reaction are negative. H₂S is not produced from either sodium thiosulfate or cysteine. Urease is positive. Aesculin and carboxymethyl cellulose are hydrolysed, but casein, gelatin, starch, and Tween 80 are not. A. acidocaldarius DSM 446^T hydrolysed carboxymethyl cellulose, however results from other species were unidentified.

Alkaline phosphatase, acid phosphatase, esterase (C4), esterase lipase (C8), naphthol-AS-BI-phosphohydrolase, α-galactosidase, α-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase are positive. Arginine dehydrolase, leucine arylamidase, valine arylamidase, cystine arylamidase, lipase (C14) trypsin, and chymotrypsin are absent.

The following substrates are utilized as single or complex carbon sources: d-cellobiose, d-galactose, d-glucose, d-maltose, d-mannose, d-raffinose (weakly), d-xylose, l-arabinose, lactulose, ribose (weakly), sodium l-lactate, sucrose, malt extract, and malt extract broth. The following substrates are not utilized: d-fructose, d-mannitol, d-sorbitol, d-trehalose, glycerol, glycine, glucosamine hydrochloride, meso-erythritol, myo-inositol, ribitol, sodium acetate, sodium citrate, sodium fumarate, sodium l-malate, sodium propionate, sodium pyruvate, sodium succinate, sodium α-ketoglutarate, α-l-rhamnose, ϵ-amino-n-caproic acid, casamino acids , neopeptone (peptone yeast extract, l-alanine, l-arginine chloride, l-asparagine, sodium l-aspartate, l-glutamine, sodium l-glutamate, l-lysine chloride, l-methionine, l-serine, l-sorbose, and l-threonine.

Acid is produced from aesculin, l-arabinose, 5-keto-gluconate, amygdalin, arbutin, d-cellobiose, d-galactose, d-glucose, d-lactose, d-maltose, d-mannose, d-melezitose, d-melibiose, d-methyl-β-d-xylopyranoside, d-raffinose, d-ribose, d-sucrose, d-turanose, d-xylose, aesculin ferric citrate, gentiobiose, methyl-α-d-glucopyranoside, and salicin. Acid is not produced from 2-keto-gluconate, ribitol, d-arabinose, d-arabitol, d-fructose, d-fucose, d-lyxose, d-mannitol, d-sorbitol, d-tagatose, d-trehalose, dulcitol, erythritol, gluconate, glycerol, glycogen, inositol, inulin, lactulose, l-arabitol, l-fucose, l-rhamnose, l-sorbose, l-xylose, methyl-α-d-mannopyranoside, N-acetylglucosamine, sodium acetate, sodium citrate, sodium fumarate, sodium l-malate, sodium propionate, sodium pyruvate, sodium succinate, sodium α-ketoglutarate, starch, xylitol, α-l-rhamnose, and ϵ-amino-n-caproic acid. It can grow chemoautotrophically using ferrous iron as an electron donor, but not using elemental sulfur, potassium tetrathionate, or sodium thiosulfate. The major cellular fatty acids are ω-cyclohexane C_19 : 0, and ω-cyclohexane C_17 : 0. The major quinone is MK-7.

The type strain Sueoka^T (JCM 18487^T = KCTC 33007^T) was isolated from steamed Japanese cedar chips from a lumber mill in Gobo, Wakayama, Japan. The DNA G+C content is 60.8 mol%.