Isolation of Acetic Acid Bacteria for Nano cellulose and Vinegar Production

Isolation of Acetic Acid Bacteria for Nano cellulose and Vinegar Production

Authors

  • KEOKENE.T
  • KEO-OUDONE.C
  • LORINHEUANG.C
  • Toshiharu Yakushi
  • Kallayanee Naloka
  • Gunjana Theeragool

Keywords:

Acetobacter, Komagataeibacter, nano-cellulose, Acetic acid bacteria, Vinegar Production

Abstract

A total of 33 acetic acid bacteria (AAB) isolates obtained from various kinds of fruits were collected from Laos. These isolates were divided into 2 groups, non-bacterial nanocellulose (BNC) and BNC-producing AAB. The 21 isolates of non-BNC-producing strains were preliminary determined for the ethanol-oxidizing ability in 5 ml of YPGD medium containing 4% ethanol incubated at 30 and 37ºC for 48 h. The five isolates (SNL223, SNL228, SNL229, SNL230 and SNL242) could produce higher amounts of acetic acid than the thermotolerant A. pasteurianus SKU1108 at 37ºC. Then isolate SNL230 further determined for the ethanol-oxidizing ability in 100 ml of coconut water medium containing (5% ethanol, 1% acetic acid incubated at 30ºC & 37 ºC, shaking at 200 rpm for 7 days and static condition compared with SKU1108.  The result showed that the higher yield of acetic acid produced by SNL230 was similar when incubated at 30 ºC & 37 ºC in two conditions (Shaking & static). Whereas the highest producing acetic acid could be observed in SKU1108 at 30ºC & 37 ºC than obtained in SNL230. Three isolates of acetic acid-producing strains were identified as Acetobacter species based on 16S rRNA gene.  The 12 isolates of BNC-producing strains were preliminary determined for BNC production in 5 ml of HS medium incubated at 30 and 37ºC for 7 days. The three isolates (SNL248.1, SNL252 and SNL253) exhibited a thick BNC pellicle when compared with thermotolerant K. xylinus MSKU 12 at 37ºC. The identification of the isolate SNL223, SNL229, and SNL242 by 16s rRNA sequence analysis revealed that they are Acetobacter pasteurianus, A. senegalensis /sicerae/ tropicalis and A. pasteurianus, respectively. In addition, based on 16S rRNA gene, the isolates SNL248.1, SNL252 and SNL253 were identified as Komagataeibacter intermedius whereas all of them species were identified as K. oboediens based on 16S-23S rRNA gene ITS sequences.

ຜົນການແຍກເຊື້ອອາເຊຕິກອາຊິດແບັກທີເຣຍໄດ້ທັງໝົດ 33 isolates ເຊຶ່ງແຍກມາຈາກໝາກໄມ້ຫຼາຍຊະນິດຂອງລາວເຮົາ. ເຊື້ອທີ່ໄດ້ແມ່ນແບ່ງອອກເປັນ 2 ກຸ່ມ ຄື: ກຸ່ມທີ່ 1 ບໍ່ສາມາດຜະລິດ cellulose ໄດ້ non-bacterial  nanocellulose (BNC) ມີ 21 isolates ເມື່ອນໍາເຊື້ອກຸ່ມນີ້ມາທົດສອບຄວາມສາມາດໃນການໝັກອາຊິດອາເຊຕິກ ໂດຍການ oxidize ເຫຼົ້າໄປເປັນອາຊິດ  (ethanol-oxidizing ability) ໂດຍທົດສອບໃນອາຫານ YPGD medium  ບໍລິມາດ 5 ml ທີ່ໃສ່ເຫຼົ້າ 4% ethanol ແລ້ວນໍາໄປບົ່ມທີ່ອຸນຫະພູມ 30ºC ແລະ 37ºC, 200 rpm ເປັນເວລາ 48 h. ພົບວ່າ ມີ 5 isolates ຄື: SNL223, SNL228, SNL229, SNL230 ແລະ SNL242 ສາມາດຜະລິດອາເຊຕິກອາຊິດໃນປະລິມານທີ່ສູງກວ່າໂຕນໍາມາປຽບທຽບ A. pasteurianus SKU1108 ທີ່ອຸນຫະພູມ 37ºC. ຈາກນັ້ນເລືອກເອົາ isolates SNL230 ມາທົດສອບການໝັກໃນນໍ້າ ໝາກພ້າວບໍລິມາດ 100 ml ທີ່ເຕີມ 5% ethanol, 1% acetic acid  ແລ້ວນໍາໄປບົ່ມທີ່ອຸນຫະພູມ 30ºC & 37 ºC, ໃນສະພາວະສັ່ນ 200 rpm  ແລະ ໃນສະພາວະບໍ່ສັ່ນເປັນເວລາ 7 ວັນ ໂດຍທຽບກັບເຊື້ອ SKU1108. ພົບວ່າ  isolate SNL230 ສາມາດຜະລິດອາເຊຕິກອາຊິດໄດ້ທັງສອງສະພາວະ ແລະ ທັງສອງອຸນຫະພູມ, ໃນສະພາວະສັ່ນຂອງມື້ທີ 6 ພົບວ່າເຊື້ອ SNL230 ແລະ SKU1108 ສາມາດຜະລິດອາເຊຕິກອາຊິດໄດ້ເຖິງ 5% ໃນສອງອຸນຫະພູມ 30ºC & 37 ºC. ສ່ວນສະພາວະບໍ່ສັ່ນ ພົບວ່າ ເຊື້ອ SKU1108  ສາມາດຜະລິດອາເຊຕິກອາຊິດໄດ້ເຖິງ 6% ໃນມື້ທີ 6 ທີ່ອຸນຫະພູມ 37 ºC ສ່ວນ isolate SNL230 ສາມາດຜະລິດອາເຊຕິກອາຊິດສູງສຸດໄດ້ພຽງແຕ່ 5% ໃນມື້ທີ 7 ທີ່ອຸນຫະພູມ 37 ºC. ຜົນການຈັດຈໍາແນກທາງດ້ານໂມເລກຸນລາ 16s rRNA sequence analysis ພົບວ່າ: ທັງ 3 ໄອໂຊເລສເປັນຊະນິດ Acetobacter. ສ່ວນກຸ່ມທີ່ 2 ສາມາດຜະລິດວຸ້ນໄດ້ BNC-producing Acetic Acid Bacteria ມີ 12 isolates. ເມື່ອນໍາເຊື້ອກຸ່ມນີ້ມາທົດສອບຄວາມສາມາດໃນການສ້າງ cellulose ໂດຍທົດສອບໃນອາຫານ HS medium  ບໍລິມາດ 5 ml ແລ້ວນໍາໄປບົ່ມທີ່ອຸນຫະພູມ 30ºC ແລະ 37ºC ເປັນເວລາ 7 ວັນ ພົບວ່າ ມີ 3 isolates (SNL248.1, SNL252 and SNL253) ທີ່ຜະລິດ cellulose ໄດ້ດີເມື່ອທຽບກັບ K. xylinus MSKU 12 ທີ່ອຸນຫະພູມ 37ºC. ຜົນການຈັດຈໍາແນກທາງດ້ານໂມເລກຸນລາ 16s rRNA sequence analysis ພົບວ່າ: isolate SNL223, SNL229 ແລະ SNL242 ເປັນຊະນິດ Acetobacter pasteurianus, A. senegalensis /sicerae/ tropicalis and A. pasteurianus. ສ່ວນ isolates SNL248.1, SNL252 ແລະ SNL253 ໂດຍອີງໃສ່ຜົນການຈັດຈໍາແນກທາງດ້ານໂມເລກຸນລາ 16s rRNA sequence analysis ພົບວ່າເປັນຊະນິດ Komagataeibacter intermedius ແຕ່ເມື່ອອີງໃສ່ຜົນການກວດສອບ 16S-23S rRNA gene ITS sequences ພົບວ່າເປັນຊະນິດ Komagataeibacter oboediens.

 

Published

2024-05-21

How to Cite

KEOKENE, T., KEO-OUDONE, C., LORINHEUANG, C., Yakushi, T., Naloka, K., & Theeragool, G. (2024). Isolation of Acetic Acid Bacteria for Nano cellulose and Vinegar Production. Scientific Journal of National University of Laos, 22. Retrieved from https://lajd.org/index.php/sjnuol/article/view/143

Issue

Section

ວິທະຍາສາດທໍາມະຊາດ

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