Identification of Acetobacter Strains Isolated in Thailand Based on The

R ESEARCH ARTICLE ScienceAsia 38 (2012): 44–53 doi: 10.2306/scienceasia1513-1874.2012.38.044 Identiﬁcation of Acetobacter strains isolated in Thailand based on the phenotypic, chemotaxonomic, and molecular characterizations Jintana Kommaneea, Somboon Tanasupawata,∗, Pattaraporn Yukphanb, Nuttha Thongchulc, Duangtip Moonmangmeed, Yuzo Yamadab,e a Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330 Thailand b BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120 Thailand c The Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok 10330 Thailand d Department of Microbiology, Faculty of Science, King Mongkut’s University of Technology Thonburi, Bangkok 10140 Thailand e JICA Senior Overseas Volunteer, Japan International Cooperation Agency (JICA), Shibuya-ku, Tokyo 151-8558, Japan, Professor Emeritus, Shizuoka University, Shizuoka 422-8529, Japan

∗Corresponding author, e-mail: [email protected] Received 14 Sep 2011 Accepted 14 Dec 2011

ABSTRACT: The restriction analysis of 16S-23S rRNA gene internal transcribed spacer regions (ITS) using TaqI, AluI, HpaII, and AvaII revealed that forty-seven bacterial isolates found in fruits and flowers collected in Thailand belong to the genus Acetobacter. They were identified as follows: Group 1 containing 11 isolates was identified as A. pasteurianus, Group 2 containing nine isolates was identified as A. orientalis, Group 3 containing eight isolates was identified as A. lovaniensis, Group 4 including eight isolates was identified as A. indonesiensis, Group 5 containing three isolates was identified as A. tropicalis, Group 6 containing five isolates was identified as A. ghanensis, and Group 7 containing four isolates was identified as A. orleanensis. The differentiation of Acetobacter species by the 16S-23S rRNA gene ITS restriction analysis was discussed. The phenotypic, chemotaxonomic, and molecular characteristics including 16S rRNA gene sequences supported the above-mentioned identification. All Thai isolates were screened for their alcohol dehydrogenase activity and exhibited an activity of 2.05–7.52 units/mg protein at 30 °C. Isolate PHD-23 produced the highest quantity of 1.50% acetic acid (v/v) from 4.0% ethanol (v/v).

KEYWORDS: acetic acid bacteria, acetic acid production, alcohol dehydrogenase, 16S-23S rRNA gene ITS restriction analysis

INTRODUCTION membrane-bound alcohol dehydrogenase (ADH) and aldehyde dehydrogenase 7. The genus Acetobacter contains 19 species compris- Thailand is located in the tropical area and there ing A. aceti, A. indonesiensis, A. cerevisiae, A. cib- are diverse fruits and flowers from which acetic acid inongensis, A. pasteurianus, A. lovaniensis, A. or- bacteria were isolated 8,9 . In Europe, Acetobacter leanensis, A. estunensis, A. malorum, A. orientalis, strains, the most popular strains for making acetic A. peroxydans, A. pomorum, A. syzygii, A. tropicalis, acid in vinegar factories, are mesophilic, with opti- A. oeni, A. ghanensis, A. nitrogenifigens, A. senegalen- mum temperature for growth at about 30 °C 10. The sis, and A. fabarum 1–5. Acetobacter strains exhibit industrial vinegar production is strictly controlled at the capability of producing acetic acid from ethanol, 30 °C. In many countries, the fermentation rate and oxidize acetate and lactate to CO2 and water, and fermentation efficiency decrease when the tempera- contain the major ubiquinone with nine isoprene units ture increases by 2–3 °C. Therefore, it would be (Q-9) 3,6 . They are well known as vinegar producers desirable to find strains of acetic acid bacteria (AAB) from ethanol by two sequential catalytic reactions of that can work optimally at temperatures above 30 °C. www.scienceasia.org ScienceAsia 38 (2012) 45

This study identifies Acetobacter isolates isolated CGCGGCTGCTG-30; positions 536–519), 920F (50- in Thailand by 16S-23S rRNA gene internal tran- AAACTCAAATGAATTGACGG-30; positions 907– scribed spacer region (ITS) restriction analysis. It 926), and 920R (50-CCGTCAATTCATTTGAGTTT- includes phenotypic and chemotaxonomic character- 30; positions 926–907). Multiple sequence align- izations and 16S rRNA gene sequence analysis, along ments were performed with the program CLUSTAL with screening for acetic acid production by isolates X (version 1.83) 19. Gaps and ambiguous bases were using ADH activity. eliminated from the calculations. Distance matrices for the aligned sequences were calculated by the two- MATERIALS AND METHODS parameter method of Kimura 20. A phylogenetic tree Isolation of acetic acid producing bacteria based on 16S rRNA gene sequences was constructed by the neighbour-joining method 21 with the program The forty-seven isolates were isolated from fruits 22 and flowers collected in Thailand by an enrichment MEGA 4 . The confidence values at individual branches in the phylogenetic tree were determined by culture approach using glucose/ethanol/yeast extract 23 (GEY) medium 8. A sample source was incubated using the bootstrap analysis of Felsenstein based on at pH 4.5 and 30 °C for 3–5 days in a liquid 1000 replications. medium. When microbial growth was found, the Restriction analysis of 16S-23S rRNA gene ITS culture was streaked onto a GEY-agar plate containing 11 regions: The 16S-23S rRNA gene ITS PCR am- 0.3% CaCO3 (w/v) . AAB were selected as acid- plification was made with two primers 24, which producing bacterial strains that form a clear zone were 50-TGCGG(C/T)TGGATCACCTCCT-30 (posi- around colonies growing on the agar plate. tion 1522–1540 on 16S rRNA by the Escherichia 18 0 Identification methods coli numbering system) and 5 -GTGCC(A/T)AG- GCATCCACCG-30 (position 38–22 on 23S rRNA). Phenotypic and chemotaxonomic characteriza- The purified PCR products for Acetobacter isolates tions: Phenotypic characterizations were carried out were separately digested with restriction endonucle- by incubating test strains at 30 °C and pH 6.8 for ases TaqI, AluI, HpaII, and AvaII (New England Bi- two days on glucose/yeast extract/peptone/glycerol oLabs, Beverly, Massachusetts, USA) according to (GYPG) agar 8. For Gram staining of bacterial cells, 12 the manufacturer’s instructions. The resulting reaction the Hucker-Conn modified method was used. Physi- products were analysed by 2.5% (w/v) agarose gel ological and biochemical characterizations were made electrophoresis 8. by the methods of Asai et al 13 and Gossele´ et al 14. The selected isolates were grown in GYPG media Acetic acid production on a rotary shaker (150–200 rpm) at 30 °C for 24 h. Ubiquinone was extracted and purified, as reported by Alcohol dehydrogenase activity assay: ADH ac- Yamada et al 6. The purified ubiquinone preparation tivity was measured colourimetrically with potassium ferricyanide as an electron acceptor, as described by was distinguished from its homologues by reversed- 25 phase paper chromatography 6 and by high perfor- Adachi et al . Acetobacter isolates were inocu- mance liquid chromatography 15. DNA was extracted lated into 200 ml of potato medium, which contained by the method described by Saito and Miura 16. DNA 0.5% D-glucose, 1% yeast extract, 1% peptone, 2% base composition was determined by the method of glycerol, 10% potato extract at 30 °C for 48 h on a Tamaoka and Komagata 17. rotary shaker (200 rpm). Cells were harvested by centrifugation at 8000g for 10 min and washed twice 16S rRNA gene sequencing and phylogenetic anal- with cooled 5 mM K3PO4 buffer, pH 6.0. The washed yses: The extracted DNA of an isolate was amplified cells were re-suspended in the same buffer and placed for 16S rRNA genes with two primers, 20F (50-GAG- in a sonicator at 16 000 lb/min for 10 min. Cell debris TTTGATCCTGGCTCAG-30; positions 9–27 by the was removed by centrifugation at 8000g for 10 min, Escherichia coli numbering system, accession number and then the supernatant was used for ADH assay. The V00348) 18 and 1500R (50-GTTACCTTGTTACGA- reaction mixture (1 ml) contained enzyme solution, CTT-30; positions 1509–1492). The amplified 16S McIlvaine buffer (McB), pH 5.0, substrate (100 µl rRNA genes were sequenced with an ABI PRISM of 1 M ethanol) and 100 µl of 0.1 M ferricyanide BigDye Terminator V3.1 8. For the sequencing of solution. After 5 min, 500 µl of Dupanol reagent 16S rRNA genes, the following six primers were was added, and incubated for 20 min. Then, 3.5 ml 0 used: 20F, 1500R, 520F (5 -CAGCAGCCGCGGTA- of dH2O was added and mixed well. The absorbance ATAC-30; positions 519–536), 520R (50-GTATTAC- at 660 nm was measured on a UV spectrophotometer.

www.scienceasia.org 46 ScienceAsia 38 (2012)

One unit of enzyme activity was defined as the quan- Table 1 Identification and ADH activity of isolates assigned tity of enzymes catalysing the oxidation of 1 µmol of to the genus Acetobacter. ethanol per min under the operating conditions. The Isolate Source Location ADH activity* specific activity was expressed as units per mg protein, (unit/mg) and the protein content was determined by the Lowry Group 1 or A. pasteurianus 26 method with bovine serum albumin as standard. PHD-23 Little Yellow Star1 Rayong 7.01 ± 0.01 PHD-24 Little Yellow Star1 Rayong 5.89 ± 0.03 Effect of ethanol and initial acetic acid concentra- PHD-32 Mango2 Khon Kaen 4.63 ± 0.04 tion on acetic acid production: The selected isolate PHD-33 Mango2 Khon Kaen 5.08 ± 0.02 that showed the highest ADH activity was inoculated PHD-56 Peach2 Bangkok 5.68 ± 0.01 and cultivated in 100 ml potato medium at 30 °C on a PHD-57 Peach2 Bangkok 5.06 ± 0.01 2 rotary shaker (200 rpm) for 24 h. PHD-70 Red Grape Rayong 4.96 ± 0.02 PHD-71 Red Grape2 Rayong 5.33 ± 0.01 Effects of initial ethanol concentration: 10 ml of 2 the culture mentioned before was transferred to 90 ml PHD-76 Salas Rayong 4.89 ± 0.03 PHD-77 Salas2 Rayong 4.75 ± 0.04 YE medium, which contained 0.3% yeast extract PHD-78 Salas2 Rayong 3.89 ± 0.03 and 0–12% ethanol in a 500 ml embossed flask and Group 2 or A. orientalis cultivated at 30 °C on a rotary shaker (200 rpm) for PHD-12 Jujube2 Trad 3.16 ± 0.02 three days. In the YE medium, ethanol concentration PHD-34 Mango2 Bangkok 3.89 ± 0.01 was varied as 0, 2, 4, 6, 8, 10, and 12% (v/v). PHD-35 Mango2 Bangkok 3.66 ± 0.01 Effects of initial acetic acid concentration: 10 ml PHD-37 Mango2 Nontaburi 3.12 ± 0.02 of the culture mentioned before was transferred to PHD-38 Mango2 Nontaburi 2.89 ± 0.01 90 ml YEA medium, which contained 0.3% yeast PHD-51 Orange2 Khon Kaen 3.45 ± 0.04 2 extract, 4% ethanol, and 1–3% acetic acid in a 500 ml PHD-73 Rumbutan Khon Kaen 3.33 ± 0.01 2 embossed flask and cultivated at 30 °C on a rotary PHD-74 Rumbutan Khon Kaen 4.56 ± 0.03 2 ± shaker at 200 rpm for three days. In the YEA medium, PHD-75 Rumbutan Khon Kaen 4.12 0.02 acetic acid concentration was varied as 0, 1, 1.5, 2, Group 3 or A. lovaniensis PHD-16 Makrut2 Changmai 4.05 ± 0.01 2.5, and 3% (v/v). Samples were taken for biomass PHD-17 Makrut2 Saraburi 4.45 ± 0.02 evolution and acetic acid analysed as described below. PHD-18 Makrut2 Saraburi 3.66 ± 0.01 PHD-25 Longan2 Rayong 2.58 ± 0.03 Analytical methods PHD-26 Longan2 Rayong 2.65 ± 0.02 PHD-63 Pineapple2 Chantaburi 3.12 ± 0.01 The total biomass evolution was determined by the 2 turbidimetric method (absorbance at 600 nm mea- PHD-91 Tamarind Chantaburi 3.48 ± 0.04 PHD-92 Tamarind2 Chantaburi 2.89 ± 0.02 sured on a spectrophotometer). Acetic acid determi- A. indonesiensis nation using 1–10% absolute ethanol as standard was Group 4 or PHD-3 Guava2 Kanchanaburi 2.05 ± 0.02 carried out by a gas chromatograph equipped with a PHD-5 Guava2 Kanchanaburi 2.54 ± 0.01 capillary column and an FID detector. All the assays PHD-7 Guava2 Ubon 2.28 ± 0.03 were performed in triplicate to obtain valid statistical PHD-8 Hog Plum2 Nongkhai 2.72 ± 0.02 evaluation of results, expressed as mean ± s.e.m. PHD-9 Ixoria/Ixora1 Rayong 2.58 ± 0.01 PHD-13 Jujube2 Trad 2.75 ± 0.04 RESULTS AND DISCUSSION PHD-44 Musk-melon2 Saraburi 2.99 ± 0.01 PHD-45 Musk-melon2 Saraburi 2.56 ± 0.01 Strain identification Group 5 or A. tropicalis All isolates had characteristics of Gram-negative, aer- PHD-4 Guava2 Kanchanaburi 2.24 ± 0.02 obic, and rod-shaped. They produced catalase but not PHD-6 Guava2 Ubon 3.14 ± 0.01 PHD-42 Musk-melon2 Bangkok 2.34 ± 0.02 oxidase and showed clear zones on GEY/CaCO3 agar plates. All isolates oxidized acetate and lactate and (Continues on next page.) showed a major ubiquinone of Q-9. Therefore, they were assigned to the genus Acetobacter 6, 13 (Table 1 and Table 2). Group 1 included 11 isolates: PHD-23, PHD-24, The Acetobacter isolates were divided into seven PHD-32, PHD-33, PHD-56, PHD-57, PHD-70, groups based on the restriction analysis using the PHD-71, PHD-76, PHD-77, and PHD-78 (Table 1). four restriction endonucleases; TaqI, AluI, HpaII, and They produced acid from L-arabinose, meso-erythri- AvaII. tol, D-fructose, D-galactose, D-glucose, D-mannose, www.scienceasia.org ScienceAsia 38 (2012) 47

Table 1 (Cont.) PHD-3 (AB619752) PHD-5 (AB619753) NRIC 0313T (AB032356) * A. indonesiensis Isolate Source Location ADH activity 100 PHD-8 (AB619754) PHD-7 (AB619755) Group 4 (unit/mg) PHD-9 (AB619756) PHD-13 (AB619757) Group 6 or A. ghanensis PHD-44 (AB619758) 2 PHD-45 (AB619759) PHD-14 Jujube Trad 2.12 ± 0.01 A. senegalensis LMG 23690T (AY883036) 2 PHD-4 (AB619760) PHD-15 Makrut Changmai 3.02 ± 0.03 85 PHD-42 (AB619762) Group 5 2 98 A. tropicalis NRIC 0321T (AB032354) PHD-61 Pineapple Chantaburi 2.88 ± 0.02 PHD-6 (AB619761) 2 A. cerevisiae LMG 1625T (AJ419843) PHD-62 Pineapple Chantaburi 3.12 ± 0.01 LMG 1746T (AJ419844) 100 A. malorum PHD-72 Rose apple2 Ubon 2.11 ± 0.02 PHD-84 (AB619770) 57 64 PHD-85 (AB619771) 96 PHD-86 (AB619768) Group 7 Group 7 or A. orleanensis T A. orleanensis IFO 13752 (AB032350) 2 PHD-87 (AB619769) PHD-86 Strawberry Trad 3.11 ± 0.04 T A. cibinongensis 4H-1 (AB052710) 2 T PHD-87 Strawberry Trad 2.14 ± 0.01 A. orientalis 21F-2 (AB052706) 2 PHD-12 (AB619735) PHD-84 Star fruit Chantaburi 3.16 ± 0.03 77 PHD-34 (AB619736) 2 PHD-35 (AB619737) PHD-85 Star fruit Chantaburi 2.88 ± 0.01 PHD-37 (AB619738) Group 2 99 PHD-38 (AB619739) 1 2 PHD-51 (AB619740) Flower, Fruit PHD-73 (AB619741) * PHD-74 (AB619742) Values are the mean of three determinations. PHD-75 (AB619743) PHD-23 (AB618996) PHD-24 (AB619725) PHD-32 (AB619726) A. pasteurianus LMD22.1T (X71863) PHD-33 (AB619727) 100 PHD-56 (AB619728) Group 1 D-melibiose, or D-xylose, and weakly from D-arab- PHD-57 (AB619729) PHD-70 (AB619730) inose, glycerol, D-sorbitol, or sucrose. Some strains 100 PHD-71 (AB619731) PHD-76 (AB619732) produced acid weakly from dulcitol or rafﬁnose but 75 PHD-77 (AB619733) PHD-78 (AB619734) not from lactose, maltose, D-mannitol, L-rhamnose, A. pomorum LTH 2458T (AJ001632) A. peroxydans IFO 13755T (AB032352) or L-sorbose. They grew on meso-erythritol but not PHD-14 (AB619763) PHD-15 (AB619764) 94 T on D-arabitol, L-arabitol, or meso-ribitol. They did A. ghanensis LMG 23848 (EF030713) 53 PHD-61 (AB619765) Group 6 not produce 2-keto-D-gluconate from D-glucose. All 82 PHD-62 (AB619766) PHD-72 (AB619767) isolates grew at 37 °C. They showed almost the same A. syzygii 9H-2T (AB052712) PHD-16 (AB619744) phenotypic characteristics as the type strain of A. pas- 99 PHD-17 (AB619745) PHD-18 (AB619746) 2 PHD-25 (AB619747) teurianus (Table 2). All the isolates were located PHD-26 (AB619748) Group 3 99 PHD-63 (AB619749) within the cluster of A. pasteurianus in the phyloge- PHD-91 (AB619750) A. lovaniensis IFO 13753T (AB032351) netic tree based on 16S rRNA gene sequences (Fig. 1) PHD-92 (AB619751) A. oeni B13T (AY829472) and had 100% pairwise 16S rRNA gene sequence 51 A. aceti NBRC 14818T (X74066) 63 A. nitrogenifigens RG1T (AY669513) similarity to the type strain of A. pasteurianus.A A. estunensis IFO 13751T (AB032349) T K G. oxydans NBRC14819 X73820 representative isolate PHD-23 showed the restriction nuc 0.005 patterns that coincided with those of the type strain of

A. pasteurianus when digested with TaqI and AluI. In Fig. 1 Phylogenetic relationships of isolates of Groups 1–7 TaqI digestion, the isolate showed the same restric- based on 16S rRNA gene sequences. The phylogenetic tree tion pattern as the type strains of A. orientalis and was constructed by the neighbour-joining method. Numbers A. pasteurianus, but differed from the type strain of at nodes indicate bootstrap percentages derived from 1000 A. orientalis when digested with AluI(Fig. 2). DNA replications. G+C content of isolate PHD-23 was 53.3 mol%. From the data obtained above, all isolates accommodated to Group 1 were identiﬁed as A. pasteurianus 2. the same phenotypic characteristics as the type strain Group 2 included nine isolates: PHD-12, of A. orientalis 4 (Table 2). They were located within PHD-34, PHD-35, PHD-37, PHD-38, PHD-51, the cluster of A. orientalis in the phylogenetic tree PHD-73, PHD-74, and PHD-75 (Table 1). They pro- based on 16S rRNA gene sequences (Fig. 1) and had duced acid from L-arabinose, D-glucose, D-mannose, 100% pairwise 16S rRNA gene sequence similarity rafﬁnose, or D-xylose but not from D-arabinose, to the type strain of A. orientalis. A representative dulcitol, meso-erythritol, D-galactose, D-fructose, isolate PHD-12 showed the same restriction pattern as glycerol, lactose, maltose, D-mannitol, melibiose, the type strains of A. orientalis and A. pasteurianus L-rhamnose, L-sorbose, D-sorbitol, or sucrose. The when digested with TaqI, but differed from the type isolates did not grow on meso-erythritol, D-arabitol, strain of A. pasteurianus when digested with AluI L-arabitol, or meso-ribitol. They produced 2-keto- (Fig. 2). Isolate PHD-12 had DNA G+C content D-gluconate from D-glucose. They showed almost of 52.2 mol%. From the data obtained above, all

www.scienceasia.org 48 ScienceAsia 38 (2012)

Table 2 Differential characteristics of isolates assigned to the genus Acetobacter.

Characteristic G1 Ap G2 Aori G3 Al G4 Ai G5 At G6 Ag G7 Aorl Oxidation of Acetate ++++++++++++++ Lactate ++++++++++++++ Growth at 37 °C +(w4) w −(w2) − −(w3) − − − − − − − − − Growth at 40 °C −(w2) −−−−−−−−−−−−− Growth on mannitol agar −(w3) − −(w2) − −(w2) − −(w1) − − − −(w3) w w − Production of 2-keto gluconic acid − − + + + + + + + + − − + + 5-keto gluconic acid −−−−−−−−−−−−−− Major quinone Q-9 Q-9 Q-9 Q-9 Q-9 Q-9 Q-9 Q-9 Q-9 Q-9 Q-9 Q-9 Q-9 Q-9 Acid production from meso-Erythritol +(w4) + −(w2) − − − −(w1) − − − − − − − Maltose − − − − − − w − − w − − − − Rafﬁnose w w −(w4) − − − − − w w − − − − DNA G+C (mol%) 53.3 52.7* 52.2 52.3* 58.3 58.6* 54.2 53.7* 56.0 55.9* 57.1 57.3† 55.8 56.5* Ap, A. pasteurianus TISTR 1056T; G1, Group 1 (11 isolates); Aori, A. orientalis NBRC 16606T; G2, Group 2 (9 isolates); Al, A. lovaniensis NBRC 13753T; G3, Group 3 (8 isolates); Ai, A. indonesiensis NBRC 16471T; G4, Group 4 (8 isolates); At, A. tropicalis NBRC 16470T; G5, Group 5 (3 isolates); Ag, A. ghanensis LMG 23848T; G6, Group 6 (5 isolates); Aorl, A. orleanensis NBRC 13752T; G7, Group 7 (4 isolates); +, positive; w, weakly positive; −, negative; numbers in parentheses indicate the isolates showing the reaction. *Data cited from Lisdiyanti et al 4; †Data cited from Cleenwerck et al 1.

(a) M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 17 18 M

bp bp (a) M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 17 18 M erythritol, D-galactose, D-fructose, glycerol, lac- 500 500 250bp tose, maltose, D-mannitol, D-melibiose, L-rhamnose, bp 100 250 500 L-sorbose, D-sorbitol, or sucrose. They did not grow 100 500 250 100 250 on meso-erythritol, D-arabitol, L-arabitol, or meso- 100 ribitol. They showed almost the same phenotypic 3 (b) M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 17 18 M characteristics as A. lovaniensis (Table 2). They

bp bp were located within the cluster of A. lovaniensis (b) M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 17 18 M 500 500 in the phylogenetic tree based on 16S rRNA gene bp 250bp 250 100 sequences (Fig. 1) and had 100% 16S rRNA gene 100 500 500 250 sequence similarities, respectively, to the type strain 250 100 Fig.100 2 Restriction analysis of 16S-23S rRNA gene ITS PCR of A. lovaniensis. A representative isolate PHD-16 Fig.products 2 of isolates of Group 1 and Group 2 by digestion showed the same restriction patterns as the type strain with (a) TaqI and (b) AluI. 1, A. orleanensis NBRC 13752T; of A. lovaniensis when digested with TaqI, HpaII, and Fig. 2 2, A. cerevisiae LMG 1625T; 3, A. syzygii NBRC 16604T; AvaII (Fig. 3). In TaqI digestion, the representative 4, A. ghanensis LMG 23848T; 5, A. cibinongensis NBRC isolate also showed the same restriction pattern as 16605T; 6, A. estunensis NBRC 13751T; 7, A. peroxydans the type strains of A. lovaniensis, A. syzygii, and NBRC 13755T; 8, A. senegalensis LMG 23690T; 9, A. trop- A. ghanensis, but differed from the type strains of icalis NBRC 16470T; 10, A. indonesiensis NBRC 16471T; A. ghanensis and A. syzygii when digested with HpaII 11, A. lovaniensis NBRC 13753T; 12, A. malorum LMG and AvaII. Isolate PHD-16 had 58.3 mol% G+C. From 1746T; 13, A. nitrogenifigens LMG 23498T; 14, A. orientalis the data obtained above, all isolates accommodated to 3 NBRC 16606T; 15, A. pasteurianus TISTR 1056T; 16, Group 3 were identified as A. lovaniensis . isolate PHD-23 (Group 1); 17, A. aceti IFO 14818T; 18, Group 4 included eight isolates: PHD-3, PHD-5, isolate PHD-12 (Group 2); M, 50 bp DNA markers. PHD-7, PHD-8, PHD-9, PHD-13, PHD-44, and PHD-45 (Table 1). They produced acid from L-arabinose, D-glucose, D-xylose, D-galactose, or isolates accommodated to Group 2 were identified as D-mannose. Some isolates produced acids from A. orientalis 4. D-fructose, D-mannose, D-melibiose, and D-xylose Group 3 included eight isolates: PHD-16, but not from meso-erythritol, dulcitol, lactose, mal- PHD-17, PHD-18, PHD-25, PHD-26, PHD-63, tose, D-mannitol, L-rhamnose, raffinose, L-sorbose,

PHD-91, and PHD-92 (Table 1). They produced acid or sucrose. They produced D-gluconate and 2-keto- from L-arabinose, D-glucose, D-mannose, rafﬁnose, D-gluconate from D-glucose, and dihydroxyacetone or D-xylose but not from D-arabinose, dulcitol, meso- from glycerol. They showed almost the same phe-

www.scienceasia.org ScienceAsia 38 (2012) 49

(a) M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 17 M (a) M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 17 M (a)bp M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 17 M bp M 1 2 3 4 5 6 7 8 9 10 11 M M 12 13 14 15 16 17 M bp bp bp 500bp bp 250 bp 500 500 500250 100250 500 500 500250100 250100 500 250 250 250100 100 100 100 100 (b) M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 17 M (b) M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 17 M bp (b)bp M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 17 M bp bp Fig. 4 Restriction analysis of 16S-23S rRNA gene ITS PCR 500bp bp500 250 250 500 products of isolates of Group 4 by digestion with AluI. 1, 500 100 500250 500250100 T T 250100 A. orleanensis NBRC 13752 ; 2, A. cerevisiae LMG 1625 ; 250100 100 T T 100 3, A. syzygii NBRC 16604 ; 4, A. ghanensis LMG 23848 ; (c) M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 17 M 5, A. cibinongensis NBRC 16605T; 6, A. estunensis NBRC (c) M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 17 M bp bp T T (c) M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 17 M bp 13751 ; 7, A. peroxydans NBRC 13755 ; 8, A. senegalensis bp 500 bp500 bp T T 250500 LMG 23690 ; 9, A. tropicalis NBRC 16470 ; 10, A. indone- 500250 100500250 T 500100 siensis NBRC 16471 ; 11, isolate PHD-3 (Group 4); 12, 250 250 100 250100 T 100 A. lovaniensis NBRC 13753 ; 13, A. malorum LMG 1746; 100 Fig. 3 14, A. nitrogeniﬁgens LMG 23498; 15, A. orientalis NBRC Fig. 3 Restriction analysis of 16S-23S rRNA gene ITS PCR Fig. 3 16606T; 16, A. pasteurianus TISTR 1056T; 17, A. aceti IFO Fig. products 3 of isolates of Group 3 by digestion with (a) TaqI, T (b) HpaII, and (c) AvaII. 1, A. orleanensis NBRC 13752T; 14818 ; M, 50 bp DNA markers.

2, A. cerevisiae LMG 1625T; 3, A. syzygii NBRC 16604T;

A. ghanensis T A. cibinongensis 4, LMG 23848 ; 5, NBRC T T from D-glucose, and dihydroxyacetone from glycerol. 16605 ; 6, A. estunensis NBRC 13751 ; 7, A. peroxydans T T NBRC 13755 ; 8, A. senegalensis LMG 23690 ; 9, A. trop- They showed almost the same phenotypic characteris- T T 3 icalis NBRC 16470 ; 10, A. indonesiensis NBRC 16471 ; tics as the type strain of A. tropicalis (Table 2). All

T A. tropicalis 11, A. lovaniensis NBRC 13753 ; 12, A. malorum LMG isolates were located within the cluster of T 1746; 13, A. nitrogeniﬁgens LMG 23498 ; 14, A. orientalis in the phylogenetic tree based on 16S rRNA gene

NBRC 16606T; 15, A. pasteurianus TISTR 1056T; 16, sequences (Fig. 1) and showed 99.9% pairwise 16S

A. aceti IFO 14818T; 17, isolate PHD-16 (Group 3); M, rDNA sequence similarity with the type strain of

50 bp DNA markers. A. tropicalis. A representative isolate PHD-4 showed the same restriction pattern as the type strain of

A. tropicalis and differed in this respect from the type notypic characteristics as A. indonesiensis 3 (Table 2). strains of other Acetobacter species when digested Phylogenetically, they were located within the cluster with AluI and HpaII (Fig. 5). DNA G+C content of of A. indonesiensis in the phylogenetic tree based on isolate PHD-4 was 56.0 mol%. From the data obtained 16S rRNA gene sequences (Fig. 1) and had 16S rRNA above, all isolates accommodated to Group 5 were gene sequence similarities around 99.9% to the type identified as A. tropicalis 3. strain of A. indonesiensis. A representative isolate Group 6 included five isolates: PHD-14, PHD-15, PHD-3 showed the same restriction pattern as the type PHD-61, PHD-62, and PHD-7 (Table 1). The iso- strain of A. indonesiensis and differed in this respect lates produced acid from D-arabinose, D-glucose, from the type strains of other Acetobacter species and D-sorbitol. Some isolates produced acids when digested with AluI(Fig. 4). DNA G+C content from L-arabinose, D-fructose, D-mannose, D-mel- of isolate PHD-3 was 54.2 mol%. From the data ibiose, and D-xylose but none did from meso- obtained above, all isolates accommodated to Group 4 erythritol, dulcitol, D-galactose, glycerol, lactose, were identified as A. indonesiensis 3. maltose, D-mannitol, L-rhamnose, raffinose (one Group 5 included three isolates: PHD-4, PHD-6, weakly), L-sorbose, or sucrose. They did not and PHD-42 (Table 1). The isolates produced grow on meso-erythritol, D-arabitol, L-arabitol, and acids from L-arabinose, D-galactose, D-glucose, and meso-ribitol (one weakly). They did not produce D-mannose, and to a less extent from maltose, raffi- 2-keto-D-gluconic acid, 5-keto-D-gluconic acid, or nose, and D-xylose, but not from D-arabinose, dul- 2,5-diketo-D-gluconic acid from D-glucose and grew citol, meso-erythritol, D-fructose, glycerol, lactose, on glycerol weakly but not on maltose or methanol as D-mannitol, D-melibiose, L-rhamnose, L-sorbose, a carbon source. They showed the same phenotypic D-sorbitol, and sucrose. They did not grow on meso- characteristics as the type strain of A. ghanensis 1 erythritol, D-arabitol, L-arabitol, or meso-ribitol. (Table 2). They were located within the cluster of They produced D-gluconate and 2-keto-D-gluconate A. ghanensis in the phylogenetic tree based on 16S

www.scienceasia.org (a)50 M 1 2 3 4 5 6 7 8 9 10 11 M M 12 13 14 15 16 17 M (a) M 1 2 3 4 5 6 7 8 9 10 11 M M ScienceAsia 12 13 14 15 1638 17 (2012) M

M 1 2 3 4 5 6 7 8 9 10 11 M M 12 13 14 15 16 17 M bp bp bp (a)bp bp 500(a) M 1 2 3 4 5 6 7 8 9 10 11 M M 12 13 14 15 16 17 M 500 (a) M 1 2 3 4 5 6 7 8 9 10 11 M M 12 13 14 15 16 17 M 500 bp500 250 250 250 bp 100 250 500100bp 100 bp b500p 100 250 500 500 250 100500 250 250 100500 250 100 100 250 100 100(b) M 1 2 3 4 5 6 7 8 9 10 11 M M 12 13 14 15 16 17 M

(b) M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 17 M (b)bp M 1 2 3 4 5 6 7 8 9 10 11 M M 12 13 14 15 16 17 M bp bp bp M 1 2 3 4 5 6 7 8 9 10 11 M M 12 13 14 15 16 17 M bp (b)bp500 500 M 1 2 3 4 5 6 7 8 9 10 M M 11 12 13 14 15 16 17 M (b) 250 250 500 bp 500 bp 500100 bp 250 500100 250bp 250 100 250 100 500 100500 500 100 500 250 250 250 250 100 100 (100c) M 1 2 3 4 5 6 7 8 9 10 11 M M 12 13 14 15 16 17 M 100 bp Fig. 5 Restriction analysis of 16S-23S rRNA gene ITS PCR bp (c) M 1 2 3 4 5 6 7 8 9 10 11 M M 12 13 14 15 16 17 M products of isolates of Group 5 by (a) digestion with AluI. 1, bp500 500 T T (bpc) M 1 2 3 4 5 6 7 8 9 10 11 M M 12 13 14 15 16 17 M 250 A. orleanensis NBRC 13752 ; 2, A. cerevisiae LMG 1625 ; 250 500100bp T T b500p100 250 3, A. syzygii NBRC 16604 ; 4, A. ghanensis LMG 23848 ; 250 100500 5, A. cibinongensis NBRC 16605T; 6, A. estunensis NBRC 100500 250 Fig.250 6 100 T T 13751 ; 7, A. peroxydans NBRC 13755 ; 8, A. senegalensis 100Fig. 6 Restriction analysis of 16S-23S rRNA gene ITS

LMG 23690T; 9, A. tropicalis NBRC 16470T; 10, A. indone- Fig.PCR 6 product of isolates of Group 6 by digestion with siensis NBRC 16471T; 11, isolate PHD-4 (Group 5); 12, Fig.(a) 6 TaqI, (b) HpaII, and (c) AvaII. 1, A. orleanensis T T A. lovaniensis NBRC 13753T; 13, A. malorum LMG 1746; NBRC 13752 ; 2, A. cerevisiae LMG 1625 ; 3, A. syzygii T T 14, A. nitrogenifigens LMG 23498T; 15, A. orientalis NBRC NBRC 16604 ; 4, A. ghanensis LMG 23848 ; 5, isolate T 16606T; 16, A. pasteurianus TISTR 1056T; 17, A. aceti IFO PHD-14 (Group 6); 6, A. cibinongensis NBRC 16605 ; T 14818T. (b) Digestion with HpaII. 1, A. orleanensis NBRC 7, A. estunensis NBRC 13751 ; 8, A. peroxydans NBRC T T 13752T; 2, A. cerevisiae LMG 1625T; 3, A. syzygii NBRC 13755 ; 9, A. senegalensis LMG 23690 ; 10, A. tropicalis T T 16604T; 4, A. ghanensis LMG 23848T; 5, A. cibinongensis NBRC 16470 ; 11, A. indonesiensis NBRC 16471 ; 12, T T NBRC 16605T; 6, A. estunensis NBRC 13751T; 7, A. per- A. lovaniensis NBRC 13753 ; 13, A. malorum LMG 1746 ; T oxydans NBRC 13755T; 8, A. senegalensis LMG 23690T; 14, A. nitrogenifigens LMG 23498 ; 15, A. orientalis NBRC T T 9, A. tropicalis NBRC 16470T; 10, A. indonesiensis NBRC 16606 ; 16, A. pasteurianus TISTR 1056 ; 17, A. aceti IFO T 16471T; 11, A. lovaniensis NBRC 13753T; 12, A. malorum 14818 ; M, 50 bp DNA markers. LMG 1746; 13, A. nitrogenifigens LMG 23498T; 14, A. ori-

entalis NBRC 16606T; 15, A. pasteurianus TISTR 1056T;

16, A. aceti IFO 14818T; 17, isolate PHD-4 (Group 5); M, D-arabitol, L-arabitol, or meso-ribitol. They produced 50 bp DNA markers. D-gluconic acid from D-glucose but did not produce 5-keto-D-gluconic acid from D-glucose. They showed the same phenotypic characteristics as A. orleanensis 3 rRNA gene sequences (Fig. 1) and had 16S rRNA (Table 2). All isolates were located within the cluster gene sequence similarities around 99.9% to the type of A. orleanensis in the phylogenetic tree based on strain of A. ghanensis. A representative isolate 16S rRNA gene sequences (Fig. 1) and showed 99.9% PHD-14 showed the same restriction patterns as the pairwise 16S rRNA gene sequence similarities to the type strain of A. ghanensis when digested with TaqI, type strain of A. orleanensis. A representative isolate HpaII, and AvaII. It was discriminated from the type PHD-85 gave the restriction patterns that coincided strains of A. syzygii and A. lovaniensis when digestion with those of the type strain of A. orleanensis when with HpaII and AvaII, respectively (Fig. 6). DNA digested with HpaII and AvaII (Fig. 7). DNA G+C G+C content of isolate PHD-14 was 57.1 mol%. From content of isolate PHD-85 was 55.8 mol%. From the data obtained above, all isolates accommodated to the data obtained above, all isolates accommodated to Group 6 were identified as A. ghanensis 1. Group 7 were identified as A. orleanensis 3. Group 7 included four isolates: PHD-84, In the restriction analysis of 16S-23S rRNA gene PHD-85, PHD-86, and PHD-87 (Table 1). They ITS PCR products, the four restriction endonucleases, produced acid from L-arabinose, D-glucose, maltose, TaqI, AluI, HpaII, and AvaII were useful for differ- but not from D-arabinose, dulcitol, meso-erythri- entiating the Acetobacter strains at the species level. tol, D-fructose, D-galactose, glycerol, lactose, mal- The type strains of A. orleanensis, A. cibinongensis, tose, D-mannitol, D-melibiose, L-rhamnose, raffi- A. estunensis, A. peroxydans, A. senegalensis, A. trop- nose, L-sorbose, D-sorbitol, sucrose, and D-xylose. icalis, A. indonesiensis, A. nitrogenifigens, A. orien- They grew on meso-erythritol, but did not grow on talis, A. pasteurianus, and A. aceti were distinguished

www.scienceasia.org ScienceAsia 38 (2012) 51

(a) M 1 2 3 4 5 6 7 8 9 10 11 M M 12 13 14 15 16 17 M

M 1 2 3 4 5 6 7 8 9 10 11 M M 12 13 14 15 16 17 M bp 1.60 0.60 (a)bp (a) 1.50

bp bp 500 1.40 500 0.50 250 250 100500 500100 1.20 250 250 100 1.02 0.40 100 1.00

M 1 2 3 4 5 6 7 8 9 10 11 M M 12 13 14 15 16 17 M (b) 0.80 0.75 0.30 bp (b)bp M 1 2 3 4 5 6 7 8 9 10 11 M M 12 13 14 15 16 17 M 0.56 OD600 (nm)

bp acid Acetic (%) 0.60 500 0.20 b500p 250 0.35 250 100500 0.40 500 250 100 0.10 250 100 0.20 100 0.00 0.00

Fig. 7 Restriction analysis of 16S-23S rRNA gene ITS 02468 Fig. 7 PCR products of isolates of Group 7 by digestion with Initial ethanol (%) Fig. 7 (a) HpaII and (b) AvaII. 1, A. orleanensis NBRC 13752T; 2.50 0.45 (b) 2.04 2, isolate PHD-85 (Group 7); 3, A. cerevisiae LMG 1625T; 0.40 T T 4, A. syzygii NBRC 16604 , 5, A. ghanensis LMG 23848 ; 2.00 0.35 T 1.67 6, A. cibinongensis NBRC 16605 ; 7, A. estunensis NBRC 0.30 1.49 T T 1.50 13751 ; 8, A. peroxydans NBRC 13755 ; 9, A. senegalen- 0.25 sis LMG 23690T; 10, A. tropicalis NBRC 16470T; 11, 1.18 0.20 T 1.00 0.80 A. indonesiensis NBRC 16471 ; 12, A. lovaniensis NBRC OD600 (nm) Acetic acid Acetic (%) 0.15 13753T; 13, A. malorum LMG 1746T; 14, A. nitrogenifigens LMG 23498T; 15, A. orientalis NBRC 16606T; 16, A. pas- 0.50 0.10 teurianus TISTR 1056T; 17, A. aceti IFO 14818T; M, 50 bp 0.05 DNA markers. 0.00 0.00 0 1 1.5 2 2.5 Initial acetic acid (%) from one another by TaqI and AluI digestions, while Fig. 8 (a) Effect of initial ethanol concentrations on acetic the type strains of A. cerevisiae, A. ghanensis, and acid production of A. pasteurianus isolate PHD-23 at 0, 2, A. malorum were differentiated from the type strains 4, 6, 8% ethanol and (b) effect of initial acetic acid concen- of other Acetobacter species by HpaII digestion. In tration on acetic acid production of A. pasteurianus isolate addition, the type strains of A. syzygii and A. lovanien- PHD-23 at 0, 1, 1.5, 2, 2.5% acetic acid. Symbols: acetic sis were distinguished from the type strains of other acid (column), growth (line), the values are as percentages Acetobacter species by AvaII and HpaII digestion. and growth is expressed as Optical Density (nm). Tests The 16S-23S rRNA gene ITS restriction analysis were in triplicate to obtain valid statistical evaluation of the described above is therefore one of the most rapid results, expressed as mean ± s.e.m. methods for molecular identification, when used to- gether with phenotypic and chemotaxonomic characterizations as well as 16S rRNA gene sequence (v/v). The strain produced 1.5% acetic acid when analysis. 4.0% ethanol was used as a carbon source. The effect of initial acetic acid concentration (0–2% v/v) on Acetic acid production by Acetobacter strains acetic acid production in isolate PHD-23 showed that isolated in Thailand the isolate oxidized ethanol and accumulated acetic The 47 isolates assigned to the genus Acetobac- acid until the initial concentrations of acetic acid that ter showed that ADH activity ranged from 2.05– was less than 2% (Fig. 8b). The data obtained sug- 7.01 units/mg protein at 30 °C (Table 1). Isolate gested that isolate PHD-23 could produce the highest PHD-23 identified as A. pasteurianus showed the acetic acid concentration when there was no addition highest ADH activity, and was therefore selected for of acetic acid. The growth of the isolate decreased optimization of acetic acid production. A. pasteuri- when the initial acetic acid concentration was more anus isolate PHD-23 produced 0.35, 1.02, 1.50, 0.75, than 1.0% (v/v). At 2.5% acetic acid (v/v), the and 0.56% acetic acid (v/v) without lag time when bacterial growth was not observed (Fig. 8b). ethanol concentrations were changed, respectively, to Saeki et al 27 reported that a thermotolerant acetic 0, 2.0, 4.0, 6.0, and 8.0% (v/v) (Fig. 8a). The growth acid bacterium, isolate SKU1180 isolated from Thai- increased maximally at ethanol concentration of 4.0% land, could grow at 37–40 °C and produced 5.06–

www.scienceasia.org 52 ScienceAsia 38 (2012)

4.42 g/l/h of acetic acid using higher concentrations 3. Lisdiyanti P, Kawasaki H, Seki T, Yamada Y, Uchimura of ethanol up to 9%, without any appreciable lag time. T, Komagata K (2000) Systematic study of the genus Lu et al 28 reported that a thermotolerant acetic Acetobacter with descriptions of Acetobacter indone- acid bacterium, Acetobacter isolate I14-2 isolated siensis sp. nov., Acetobacter tropicalis sp. nov., Ace- from a spoiled banana in Taiwan, produced 41 g/l tobacter orleanensis (Henneberg 1906) comb. nov., acetic acid at 37 °C during six-day cultivation in a Acetobacter lovaniensis (Frateur 1950) comb. nov., and Acetobacter estunensis J Gen medium containing 2 g/l acetic acid and 5% (v/v) (Carr 1958) comb. nov. Appl Microbiol 46, 147–65. ethanol. The isolate retained 68% acetic acid-pro- 4. Lisdiyanti P, Kawasaki H, Seki T, Yamada Y, Uchimura ducing activity when compared with cultivation at T, Komagata K (2001) Identification of Acetobacter 30 °C. strains isolated from Indonesian sources, and proposals 29 Ndoye et al reported two isolates, A. tropicalis of Acetobacter syzygii sp. nov., Acetobacter cibinon- CWBI-B418 and A. pasteurianus CWBIB419, which gensis sp. nov., and Acetobacter orientalis sp. nov. were isolated, respectively, from mango and cereal. J Gen Appl Microbiol 47, 119–31. The two isolates showed their growth without any 5. Skerman VBD, McGowan V, Sneath PHA (1980) Ap- appreciable lag phase and a high level of acetic acid proved lists of bacterial names. Int J Syst Bacteriol 30, production at 35 and 38 °C, respectively. At 30 °C, 225–420. isolates CWBI-B418 and CWBI-B419 produced 1.5% 6. Yamada Y, Aida K, Uemura T (1968) Distribution of and 2% acetic acid w/v, respectively. Isolate CWBI- ubiquinone 10 and 9 in acetic acid bacteria and its B419 produced 2% acetic acid (w/v) at 35 and 38 °C, relation to the classification of genera Gluconobacter Acetobacter and isolate CWBI-B418 produced 2% and 1.8% acetic and , especially of so-called intermediate strains. Agr Biol Chem 32, 786–8. acid (w/v) at 35 and 38 °C, respectively. 7. Matsushita K, Tomaya H, Adachi O (1994) Respiratory In comparison with the acetic acid production chain and bioenergetics of acetic acid bacteria. In: mentioned above, a new isolate, A. pasteurianus iso- Rose AH, Tempest DW (eds) Advances in Microbial late PHD-23 reported in this study oxidized higher Physiology, Academic Press, London, pp 247–301. concentrations of ethanol up to 8% without any ap- 8. Kommanee J, Akaracharanya A, Tanasupawat S, Mal- preciable lag time, which was similar to the thermo- imas T, Yukphan P, Nakagawa Y, Yamada Y (2008) tolerant acetic acid bacteria reported by Saeki et al 27. Identification of Acetobacter strains isolated in Thai- In the acetic acid production at 30 °C, isolate PHD-23 land based on 16S-23S rRNA gene ITS restriction and produced acetic acid similar to isolates CWBI-B418 16S rRNA gene sequence analyses. Ann Microbiol 58, and CWBI-B419 29. 319–24. Considering the data obtained above, A. pasteuri- 9. Seearunruangchai A, Tanasupawat S, Keeratipibut S, anus isolate PHD-23 has advantages of (1) resistance Thawai C, Itoh T, Yamada Y (2004) Identification of to ethanol, (2) high acetic acid productivity, and acetic acid bacteria isolated from fruits and related materials collected in Thailand. J Gen Appl Microbiol (3) easy preservation by lyophilization, so that the 50, 47–53. isolate is suitable for vinegar making. 10. Sievers M, Sellmer S, Teuber M (1992) Acetobacter europaeus sp. nov., a main component of industrial Acknowledgements: This study was supported by the vinegar fermenters in Central Europe. Syst Appl Micro- Faculty of Pharmaceutical Sciences Research Fund, 2008, biol 15, 386–92. Chulalongkorn University, Thailand and Graduate Institute 11. Yamada Y, Okada Y, Kondo K (1976) Isolation and of Science and Technology, TGIST, 2007, National Science characterization of “polarly flagellated intermediate and Technology Development Agency, Thailand. strains” in acetic acid bacteria. J Gen Appl Microbiol 22, 237–45. REFERENCES 12. Hucker GJ, Conn HJ (1923) Method of Gram staining. 1. Cleenwerck I, Camu N, Engelbeen K, de Winter T, NY State Agric Exp Stn Tech Bull 93, 3–37. Vandemeulebroecke K, de Vos P, de Vuyst L (2007) 13. Asai T, Iizuka H, Komagata K (1964) The flagellation Acetobacter ghanensis sp. nov., a novel acetic acid and taxonomy of genera Gluconobacter and Aceto- bacterium isolated from traditional heap fermentations bacter with reference to the existence of intermediate of Ghanaian cocoa bean. Int J Syst Evol Microbiol 57, strains. J Gen Appl Microbiol 10, 95–126. 1647–52. 14. Gossele´ J, Swings J, De Ley J (1980) A rapid, sim- 2. Cleenwerck I, Gonzalez A, Camu N, Engelbeen K, ple and simultaneous detection of 2-keto, 5-keto- and de Vos P, de Vuyst L (2008) Acetobacter fabarum sp. 2,5-diketogluconic acid by thin layer chromatography nov., an acetic acid bacterium from a Ghanaian cocoa in culture media of acetic acid bacteria. Zentralbl Bak- bean heap fermentation. Int J Syst Evol Microbiol 58, teriol Parasitenk Infektionskrankh Hyg I Abt Orig C 1, 2180–5. 178–81. www.scienceasia.org ScienceAsia 38 (2012) 53

15. Tamaoka J, Katayama-Fujimura Y, Kuraishi H (1983) Analysis of bacterial menaquinone mixtures by high performance liquid chromatography. J Appl Bacteriol 54, 31–6. 16. Saito H, Miura K (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72, 619–29. 17. Tamaoka J, Komagata K (1984) Determination of DNA base composition by reversed-phase high performance liquid chromatography. FEMS Microbiol Lett 25, 125–8. 18. Brosius J, Dull TJ, Sleeter DD, Noller HF (1981) Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol 148, 107–27. 19. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL X windows inter- face: flexible strategies for multiple sequence align- ment aided by quality analysis tools. Nucleic Acids Res 25, 4876–82. 20. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through com- parative studies of nucleotide sequences. J Mol Evol 16, 111–20. 21. Saitou N, Nei M (1987) The neighboring-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–25. 22. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) soft- ware version 4.0. Mol Biol Evol 24, 1596–9. 23. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–91. 24. Trcekˇ J, Teuber M (2002) Genetic and restriction analysis of the 16S–23S rDNA internal transcribed spacer regions of the acetic acid bacteria. FEMS Microbiol Lett 208, 69–75. 25. Adachi O, Miyagawa E, Shinagawa E, Matsushita K, Ameyama M (1978) Purification and properties of par- ticulate alcohol dehydrogenase from Acetobacter aceti. Agr Biol Chem 42, 2331–40. 26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193, 265–75. 27. Saeki A, Theeragool G, Matsushita K, Toyama H, Lo- tong N, Adachi O (1997) Development of thermotolerant acetic acid bacteria useful for vinegar fermentation at higher temperatures. Biosci Biotechnol Biochem 61, 138–45. 28. Lu SF, Lee FL, Chen HK (1999) A thermotolerant and high acetic acid-producing bacterium Acetobacter sp. I14-2. J Appl Microbiol 86, 55–62. 29. Ndoye B, Lebecque S, Dubois-Dauphin R, Tounkara L, Guiro AT, Kere C, Diawara B, Thonart P (2006) Thermoresistant properties of acetic acids bacteria isolated from tropical products of Sub-Saharan Africa and destined to industrial vinegar. Enzym Microb Tech 39, 916–23.