The Study on Species Diversity of Acetic Acid

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International Journal of Advances in Science Engineering and Technology, ISSN(p): 2321 –8991, ISSN(e): 2321 –9009 Volume-6, Issue-2, Apr.-2018, http://iraj.in THE STUDY ON SPECIES DIVERSITY OF ACETIC ACID BACTERIA ISOLATED FROM TRADITIONAL NIPA SAP FERMENTED VINEGAR THAT PRODUCED IN THE EAST COAST SOUTHERN SUBREGION OF THAILAND

1TANAKWAN BUDSABUN, 2WATTANA PANPHUT, 3KITTHISAK KHLAEO CHANSUKH

1,2Department of Microbiology Faculty of Science and Technology, Suan Sunandha Rajabhat University, Bangkok 10300, Thailand, 3Department of Applied Thai Traditional Medicine College of Allied Health Science, Suan Sunandha Rajabhat University, Bangkok 10300, Thailand E-mail: [email protected], [email protected], [email protected]

Abstract - This study investigated the occurrence and identified the Acetic Acid Bacteria strain from samples, Traditional Nipa Sap Fermented Vinegar that Produced in the East Coast Southern Subregion of Thailand. The total of 46 isolates of Acetic Acid Bacteria strains were isolated from 17 samples of Traditional Nipa Sap Fermented Vinegar. The enrichment culture media at pH 3.5 were used to promote growth of acetic acid bacteria and the Glucose-Ethanol-Calcium- Carbonate agar was used to screen acid producing bacteria. The experiment was tested by culturing on the different carbon sources. The morphology, biochemical test and result of partial 16S rDNA sequencing analysis by Mega 7 program were used to identify all of acetic acid bacteria in this study into species. All of the 46 isolates were gram negative, rod shape, aerobic acid producing bacteria, oxidase negative and catalase positive. All of 46 strains of Acetic Acid Bacteria, 36 strains of them were routinely identified as Acetobacter pasteurianus, 2 strains of them were routinely identified as Acetobacter ghanensis and 8 strains of them were routinely identified as Komagataeibacter intermedius.

Index Terms - Isolation, Identification, Acetic Acid Bacteria, Thailand.

I. INTRODUCTION sorbitol, 1.5% peptone, 0.8% yeast extract), Manital medium (2% manital, 1.5% peptone, 0.8% yeast Acidic acid bacteria are classified in the alpha extract) and Methanol medium (2% methanol, 1.5% Proteobacteria, family Acetobacteraceae. Taxonomic peptone, 0.8% yeast extract) incubated at 3.5 pH and Study of acetic acid bacteria were continuing to this 30 °C for 3 – 5 days. When the microbial growth was day. The discovery of a new genus and species of observed, bacterial strains were streaked on Glucose acetic acid bacteria is still ongoing. Acetic acid ethanol CaCO3 agar plate (2% glucose, 0.3% ethanol, bacteria consist of 19 genera [1]. The traditional nipa 0.3% peptone, 0.3% yeast extract, 0.7% CaCO3 , 1.5% sap fermented vinegar is widely used in the east coast agar). Produce clear zone around the colony on southern subregion of Thailand. It is made from Glucose ethanol CaCO3 agar plate was used for fermented nectar from Nipa palm flowers. This study isolation of Acid producing bacteria [2]. aims to isolate and identify acetic acid bacteria from B. Characterization and Biochemical test traditional nipa sap fermented vinegar in order to Morphological and cultural characteristics of the obtain acetic acid bacteria for selecting special isolates were examined by Gram strain and Catalase characteristics for used in future. test, Oxidation - Fermentation test, Oxidation of acetate lactate test (0.3% peptone, 0.2% yeast extract, II. PROCEDURE 0.2% sodium acetate or sodium lactate, 0.002% bromthymol blue), growth on 30% Glucose medium A. Isolation of Acid is produced Bacteria (30% glucose, 0.5%yeast extract, 1.5% agar), Glucose The 8 and 9 samples of traditional nipa sap fermented without Acetic medium (1.5% glucose, 0.5% ethanol, vinegar were collected from Bang Bai Mai district 0.3% peptone, 0.3% yeast extract), Glucose with 0.3% Surat Thani province and Ban Bang Wam village Acetic acid medium (1.5% glucose, 0.5% ethanol, Bang Phra district Nakhon Sri Thammarat province, 0.3%peptone, 0.3% yeast extract, 0.3%acetic acid), Thailand. The samples were put into 5 enrichment Sorbitol medium (2% sorbitol, 0.3% peptone, 0.3% culture media ; Glucose ethanol – acetic acid medium yeast extract) and Sucose medium (2% sucrose, 0.3% (1.5% glucose, 0.5% ethanol, 1.5%peptone, 0.8% peptone, 0.3% yeast extract), growth on 1% and 7% yeast extract, 0.3%acetic acid ) , 30% glucose medium glutamate agar medium (1% glucose, 0.1% KH2HPO4, (30% glucose, 0.5% ethanol, 1.5%peptone, 0.8% yeast 0.02% MgSO4.7H2O, 0.01% KCl, 2% agar, 1% or 7% extract, 0.3% acetic acid), Sorbitol medium (2% Sodium glutamate) all of the methods as described by Yamada et al., 2000 [3]. ; Lisdiyanti et al. , 2000 [4]

The Study on Species Diversity of Acetic Acid Bacteria Isolated from Traditional NIPA Sap Fermented Vinegar that Produced in the East Coast Southern Subregion of Thailand

50 International Journal of Advances in Science Engineering and Technology, ISSN(p): 2321 –8991, ISSN(e): 2321 –9009 Volume-6, Issue-2, Apr.-2018, http://iraj.in and 2002 [5]. The 6 type strains; Acetobacter aceti were sent to Macrogen company (Seoul, South BCC 12455, Gluconobacter oxydans BCC 12337, Korea) for sequencing partial 16S rDNA regions. Swaminathania salitelerans BCC 17684, Kozakia The primer 800R (5' TACCAGGGTATCTAATCC balitensis BCC 12275, Gluconacetobacter 3'), universal 16S rDNA sequencing primers was liquefaciens BCC 12274, Asaia bogorensis BCC used for sequencing. Multiple sequence alignment 12264 were used as the reference strains for this was performed with the program BioEdit (version paper. 7.2.5). Sequence gaps and ambiguous bases were C. Species identifications by partial sequencing of excluded. Sequence comparisons were made for 16S-rDNA bases distance matrices were calculated by Bacterial 16S rDNA was amplified by PCR two-parameter method of Kimura. Phylogenetic trees technique with primer 20F (5'- based on partial 16S rDNA gene sequence derived GAGTTTGATCCTGGCTCAG - 3') and 1500R (5'- from the neighbor-joining method was constructed GTTACCTTGTTACGACTT - 3') (Yukphan et al., by use of the program MEGA (version 7). 2004b). PCR amplification was carried out on a Robustness of individual branches were estimated by GeneAmp PCR System 2400 (AB Applied bootstrapping with 1,000 replications [6], [7], [8], Biosystem, USA). The purified PCR product samples [9].

III. RESULT AND DISCUSSION

Fig. 1. Phylogenetic relationships of Acetobacter type strains and 16 Acetobacter strains that isolated from Ban Bang Wam village Bang Phra district Nakhon Sri Thammarat province. The phylogenetic tree based on partial 16S rDNA sequence was constructed by the neighbour-joining method. Numerals at the nodes indicate bootstrap percentages derived from 1,000 replications.

The Study on Species Diversity of Acetic Acid Bacteria Isolated from Traditional NIPA Sap Fermented Vinegar that Produced in the East Coast Southern Subregion of Thailand

51 International Journal of Advances in Science Engineering and Technology, ISSN(p): 2321 –8991, ISSN(e): 2321 –9009 Volume-6, Issue-2, Apr.-2018, http://iraj.in

Fig. 2. Phylogenetic relationships of Acetobacter type strains and 18 Acetobacter strains that isolated from Bang Bai Mai district Surat Thani province. The phylogenetic tree based on partial 16S rDNA sequence was constructed by the neighbour-joining method. Numerals at the nodes indicate bootstrap percentages derived from 1,000 replications.

A total of 46 strains of Acitic acid bacteria were isolated from 17 samples of traditional nipa sap fermented vinegar that 8 sample were collected from Bang Bai Mai district Surat Thani province and 9 samples were collected from Ban Bang Wam village Bang Phra district Nakhon Sri Thammarat province, Thailand by an enrichment culture method. All of 46 strains were classified to acetic acid bacteria because all of them were gram-negative rods, aerobic respiration, oxidase-negative, catalase-positive and produced clear zone on Glucose ethanol CaCO3 agar plate. The colonies of the isolates were white or cream, circular, raised or convex with entire margin on Glucose ethanol CaCO3 agar plates. All of 46 isolates of acetic acid bacteria, there were 38 isolates that identified to genus Acetobacter because all of their acetate or lactate oxidation is very strong and they grew on Glucose with 0.3% acetic acid medium and glutamate agar but did not grow on 30% glucose medium. All of the 38 acetic acid bacteria isolate grew at pH

The Study on Species Diversity of Acetic Acid Bacteria Isolated from Traditional NIPA Sap Fermented Vinegar that Produced in the East Coast Southern Subregion of Thailand

52 International Journal of Advances in Science Engineering and Technology, ISSN(p): 2321 –8991, ISSN(e): 2321 –9009 Volume-6, Issue-2, Apr.-2018, http://iraj.in 3.5-6. In a phylogenetic tree based on partial 16S rDNA sequence constructed by the neighbour-joining method. All of the 38 isolates formed a cluster together with genus Acetobacter pasteurianus , 16 strains were isolate from Ban Bang Wam village Bang Phra district Nakhon Sri Thammarat province (Fig. 1). and 18 strains were isolate from Bang Bai Mai district Surat Thani province (Fig.2). The other 8 isolates from 46 isolates were identified to genus Komagataeibacter because all of their acetate or lactate oxidation is strong and they grew on Glucose with 0.3% acetic acid medium, 30% glucose medium and glutamate agar. All of them grew at pH 3.5-6. In a phylogenetic tree based on partial 16S rDNA sequence constructed by the neighbour-joining method. All of the 8 isolates formed a cluster together with genus Komagataeibacter intermedius (Fig.3). The 8 Strains of Komagataeibacter intermedius in this study, strain SI002, SI003, SI009, SI013, SI019 and SI022 were isolated from Ban Bang Wam village Bang Phra district Nakhon Sri Thammarat province and strain M03 and M19 were isolated from Bang Bai Mai district Surat Thani province.

Fig. 3. Phylogenetic relationships of Komagataeibacter type strains and 8 Komagataeibacter strains that isolated from Bang Bai Mai district Surat Thani province (SI) and Bang Bai Mai district Surat Thani province (M).. The phylogenetic tree based on partial 16S rDNA sequence was constructed by the neighbour-joining method. Numerals at the nodes indicate bootstrap percentages derived from 1,000 replications.

The Study on Species Diversity of Acetic Acid Bacteria Isolated from Traditional NIPA Sap Fermented Vinegar that Produced in the East Coast Southern Subregion of Thailand

53 International Journal of Advances in Science Engineering and Technology, ISSN(p): 2321 –8991, ISSN(e): 2321 –9009 Volume-6, Issue-2, Apr.-2018, http://iraj.in CONCLUSIONS [2] Y. Yamada, R. Hosono, P. Lisdiyanti, Y. Widyastuti, S. Saono, T. Uchimura, and K. Komagata, “ Identification of acetic acid bacteria from Indonesian sources, especially of isolates classified From the results, it is noteworthy that there are only in the genus Gluconobacter, ” J. Gen. Appl. Microbiol, vol. 45, two genera that can be isolated from the the two pp. 23-28, Feb 1999. [3] Y. Yamada, K. Katsura, J. Kawasaki, Y. Widyastuti, S. Saono, sources of traditional nipa sap fermented vinegar, T. Seki, T. Uchimura, and K. Komagata, “ Asaia bogorensis Genus Acetobacter and Genus Komagataeibacter. gen. Nov., sp. Nov., an unusual acetic acid bacterium in the Both of these genera are closely relate, Genus Alpha-Proteobacteria,” Int. J. Syst. Evol. Microbiol, vol. 52, pp. Komagataeibacter was separated from the genus. 1-5, Mar 2000. [4] P.Lisdiyanti, H. Kavasaki, T. Seki, Y. Yamada, T. Uchimura, Gluconacetobacter, which was separated from the and K Komagata, “.Systematic study of the genus Acetobacter genus Acetobacter. In 1998 Boesch Cornelia et al. with description of Acetobacter indonesiensis sp. Nov., proposed a new species of acetic acid bacteria, Acetobacter tropicalis sp. Nov., Acetobacter orieanensis (Henneberg 1906) comb. Nov. , Acetobacter lovanieensis ( Acetobacter intermedius, which was later was moved Frateur 1950 ) comb. Nov., and Acetobacter estunensis (Carr from the genus Acetobacter to Komagataeibacter and 1958 ) comb. Nov, ” J. Gen. Appl. Microbiol, vol.46, use the name Komagataeibacter intermedius. The pp.147-165, Jun 2000. [5] P.Lisdiyanti, H. Kavasaki, Y. Widyastuti, S., Saono, T. Seki, , special properties of K. intermedius is cellulose Y. Yamada, T, Uchimura, and K.Komagata, “ Kozakia baliensis production. This is an interesting in selection of [6] gen. Nov., sp. Nov., a novel acetic acid bacteria in the cellulose-producing species for use in commercial Alpha-Proteobacteria,” Int. J. Syst. Evol. Microbiol, vol 52, pp1-5, May.2002. cellulose production in future. [7] P.Yukphan, T. Malimas, M. Takahashi, W. Potacharoen, T. Busabun, , S. Tanasupawat, Y. Nakagawa, M. Tanticharoen, ACKNOWLEDGEMENTS and Y. Yamada, “Re-identification of Gluconobacter strains based on restriction analysis of 16S-23S rDNA internal transcribed spacer regions,” J. Gen. Appl. Microbiol, vol 50, This study was supported by Industrial Microbiology pp.189-195, Aug 2004a. Department, Faculty of Science and Technology, Suan [8] T. Malimas, P. Yukphan, M. Tahashi, M. Kaneyasu, W. Potacharoen, S.Tanasupawat, Y. Nakagawa, M Tanticharoen, sunandha Rajabhat University. Support was also and Y. Yamada, Gluconobacter kondonii sp. nov., an Acetic acid provided by Institute for Research and Development, bacterium in the α-Proteobacteria, J. Gen. Appl. Microbiol, vol. Suan sunandha Rajabhat University. 53, pp.301-307, Oct 2007. [9] T.Malimas, P.Yukphan, M. Tahashi, M. Kaneyasu, W. Potacharoen, S. ,Tanasupawat, Y. Nakagawa, M. Tanticharoen, REFERENCES and Y. Yamada, “Gluconobacter roseus(ex Asai) sp. nov., nom. rev., a pink-colored acetic acid bacterium in the [1] J. Treck, F. Barja, “Updates on quick identification of acetic acid Alphaproteobacteria,” J. Gen. Appl. Microbiol, vol. 54, pp. bacteria with a focus on the.16S-23S rDNA gene internal 119-215, Apr 2008. transcribed spacer and the analysis of cell proteins by [10] P. Yukphan, W. Potacharoen, S. Tanasupawat, M. MALDITOF mass spectrometry, ” Int J Food Microbiology, vol. Tanticharoen, and Y. Yamada, “ Asaia krungthepensis sp. nov., 196, pp.137-44, Mar 2015. an acetic acid bacterium in the α-Proteobacteria,” IJSEM, vol. 54, pp. 313-316, Mar 2004b.

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The Study on Species Diversity of Acetic Acid Bacteria Isolated from Traditional NIPA Sap Fermented Vinegar that Produced in the East Coast Southern Subregion of Thailand