Cell morphology was observed under a light microscope (BH-2, Olympus, Japan) and by scanning electron microscopy (SU 8010, Hitachi, Japan) after incubation on PCA plates for 72 h (Ivanova et al. 1998). Gram staining was performed as described by Gerhardt et al (Gerhardt et al. 1994). In order to determine the optimal growth conditions, cultures of WLY-B-L8T were inoculated in PCA for 3 days at 4, 10, 15, 20, 25, 30, 37, 40, 42, 45, and 50°C. The pH and NaCl (0-5%) ranges for growth were measured at 37°C buffered with NaH2PO4/Na2HPO4 to pH 3.0-12.0 at 1.0 pH unit intervals. Growth at different temperature, pH and NaCl was detected after 72 h, when the strain was in the exponential growth phase. Colonies after 72 h of incubation on Tryptose Soya Agar (TSA) medium are circular, convex and beige with a dry and opaque appearance, and an average diameter of 2 to 3 mm. The strain was strictly aerobic, Gram-stain-positive with rod shape cells 0.7-1.3 μm wide and 1.6-3.4 μm long, arranged singly or in pairs (Fig. 4). WLY-B-L8T was able to grow at 15-42 °C, with optimal growth at 40 °C. Strain WLY-B-L8T was able to grow at pH 5.0-10.0, and the optimal pH for growth was 8.0-9.0. Optimal growth was with 1% (w/v) NaCl, and no growth occurred with greater than 3.0% (w/v) NaCl for WLY-B-L8T.
Acid production from carbon sources was tested by using API 50CH strips according to the manufacturer’s instructions (BioMérieux, France). Enzyme activities were characterized using API ZYM strips. API 20E and API 20NE were used to perform physiological and biochemical tests, including carbon substrate utilization, nitrate reduction, β-galactosidase, arginine dihydrolase, citrate utilization, H2S production, Voges-Proskauer test and hydrolysis of gelatin, aesculin and urea. These tests were performed at 37°C for 72 h. Anaerobic cultivation was performed on TSA using the Oxoid AnaeroGen system with anaerobic bag (Mitsubishi Gas Chemical, Japan) for 1 week (Park et al. 2011). Motilities were tested by the hanging-drop technique using semi-solid TSA medium containing 0.5% agar. Oxidase activity was determined by reagent colour change using API 55635 kits (BioMérieux, France) and catalase activity was determined by bubble production in 3% (v/v) H2O2.
WLY-B-L8T was positive for gelatin hydrolysis, alkaline phosphatase, leucine arylamidase, chymotrypsin, catalase, acid phosphatase, α-glucosidase, β-glucosidase, esterase (C4), lipoid esterase (C8), naphthol AS-BI-phosphate hydrolase, N-acetylglucosaminase, nitrate reduction and aesculin. but negative for citric acid utilization, valine arylamidase, cystine arylamidase, oxidase, arginine dihydro-lase, Voges-Proskauer (VP) test, glucose assimilation, trypsin, α-galactosidase, esterase (C14), β-galactosidase, β-glucuronidase, α-mannosidase and β-fucosidase. It also can use N-acetylglucosamine, D-maltose, glucose, malic acid and gelatin as sole carbon sources. Detailed physiological and biochemical characteristics of WLY-B-L8T and the reference strains are shown in Table 1.
Chemotaxonomic analyses indicated that ribose, xylose, arabinose, mannose, glucose and galactose were the major cell-wall sugar. MK-7 was the predominant respiratory quinone, which were typical of the large majority of members of the genus Bacillus (Xie et al. 2020). The meso-diaminopimelic acid (meso-DAP) was the diagnostic amino acid. The main polar lipids of WLY-B-L8T included diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), that were characteristic lipids presented in the species of genus Bacillus (Xie et al. 2020). In addition, unidentified aminolipids (UAL 1-2), unidentified aminophospholipid (UAPL), unidentified aminoglycolipid (UAGL) and unidentified lipid (UL) were also detected. Total fatty acids profile (Table 2) revealed that iso-C15:0 (23.0%) was the major component and branched fatty acids accounted for 69% in strain WLY-B-L8T (iso-C15:0 23.0%, iso-C17:0 8.9%, iso-C13:0 7.9%, iso-C17:1ω5c 7.0%, anteiso-C13:0 6.3%,iso-C12:0 4.8%, C18:3ω6c 4.3%, C16:0 3.9% and iso-C16:03.5%).
Volatile flavor compounds produced by the strain in the baobaoqu cultures for 3 days in 30°C were determined using the headspace solid phase microextraction combined with gas chromatography-mass spectrometer (Luo et al. 2023). Wuliangye baobaoqu cultures is prepared with pure wheat. Before incubation, the raw wheat is ground and its moisture content is adjusted to about 38% accordingly (Zheng et al. 2018). The results showed that 2-pipendinone (22.0 mg/L), phenylethyl alcohol (19.1 mg/L), hydrocinnamic acid (18.6 mg/L), and acetoin (7.6 mg/L) were the major volatile flavor compounds of WLY-B-L8T (Table 1).
Therefore, based on phenotypic, genotypic, and chemotaxonomic characteristics and phylogenetic analysis, the strain WLY-B-L8T (CICC 25210T = JCM 36284T) is suggested to represent a novel species of the genus Bacillus, for which the name Bacillus multifaciens sp. nov. is proposed.