การผลิตเอทานอลและมวลชีวภาพสำหรับ กระบวนการไบโอทรานส์ฟอร์เมชั่น จากจุลินทรีย์ 15 สายพันธุ์ ในสภาวะตั้งนิ่ง Production of Ethanol and Biomass for Biotransformation Process from 15 Microbial Strains in the Stand Still Condition ฐิติพร กันจันวงศ์ (Titiporn Kanchanwong), ณัฐพงษ์ กาละปัน (Nattapong Kalapan), นพพล เล็กสวัสดิ์ (Noppol Leksawasdi) บทคัดย่อ ทำการทดลองเพาะเลี้ยงกล้าเชื้อจุลินทรีย์ 15 สายพันธุ์ในระบบตั้งนิ่งที่ระดับ 10 ml เป็นเวลา 24 h ในอาหารเลี้ยงเชื้อที่มีกลูโคสเป็นแหล่งอาหารคาร์บอนเท่านั้น พบว่า Zymomonas mobilis TISTR No. 405 ผลิตเอทานอลได้สูงสุดที่ 9.47 ± 0.47 g/l ส่วนการทดลองในระบบตั้งนิ่งปริมาตร 100 ml ที่มีกลูโคส 63.5 – 64.4 g/l เป็นแหล่งอาหารคาร์บอน เป็นเวลา 48 h พบว่า Saccharomyces cerevisiae TISTR No ผลิต เอทานอล (g/l) ได้ระดับสูงสุดที่ 23.5 ± 2.6 g/l และค่า Yp/s = 0.37 ± 0.04 g เอทานอลต่อ g กลูโคส ตามด้วย Z. mobilis TISTR No. 405 (23.5 ± 4.0, 0.36 ± 0.06) 550 (21.9 ± 3.1, 0.34 ± 0.05) และ S. cerevisiae TISTR No (21.0 ± 0.8, 0.33 ± 0.01) การ ทดลองไบโอทรานส์ฟอร์เมชั่นด้วยมวลชีวภาพของจุลินทรีย์เหล่านี้ในระบบตั้ง นิ่งที่ทำการแทนที่สารละลายบัฟเฟอร์ด้วยตัวทำละลาย 4 ชนิด ( น้ำ ออกทานอล ไดโพรพิลีนไกลคอล และตัวทำละลายผสมระหว่างออกทานอลและไดโพรพิลีน ไกลคอลในอัตราส่วน 1 : 1) ไม่พบการผลิต R-phenylacetyl-carbinol (PAC) ในตัวทำละลายทั้งสี่ชนิด Abstract The cultivation of seed cultures for 15 microbial strains was investigated in the stand still system at 10 ml level for 24 h in the cultivation medium with glucose as a sole carbon source indicated that Zymomonas mobilis TISTR No. 405 produced the highest ethanol concentration of 9.47 ± 0.47 g/l. The cultivation experiment in a 100 ml stand still system with g/l glucose as carbon source for 48 h showed that Saccharomyces cerevisiae TISTR No produced ethanol at the highest level of 23.5 ± 2.6 g/l with Yp/s = 0.37 ± 0.04 g ethanol/g glucose. This was followed by Z. mobilis TISTR No. 405 (23.5 ± 4.0, 0.36 ± 0.06), 550 (21.9 ± 3.1, 0.34 ± 0.05) and S. cerevisiae TISTR No (21.0 ± 0.8, 0.33 ± 0.01). The subsequent biotransformation experiment was conducted by utlizing the wet biomass of these microbial strains in the stand still condition at which the 10 ml buffer solution was replaced by 4 solvents (distilled water, octanol, dipropylene glycol as well as a mixture of octanol and dipropylene glycol in 1:1 ratio) of equal volume. However, the production of R- phenylacetylcarbinol (PAC) was not detected in any of the examined solvents. Results, Discussion & Conclusion ภาควิชาวิศวกรรมอาหาร คณะอุตสาหกรรมเกษตร มหาวิทยาลัยเชียงใหม่ (Department of Food Engineering, Faculty of Agro-Industry, Chiang Mai University) Introduction Materials & Methods [Please contact authors for detailed references] Longan has been considered as one of the crucial economic crop of Thailand as evident from the export values in 2007 as followed (millions $US); pineapple (361), durian (87), young corn (58), and fresh longan (57.8) (OAE 2007). In 2005, thirty percents of all longan products were for domestic consumption while forty percents were in some sorts of processed forms. The remaining seventy percents of longan products were exported overseas (DOA 2005). However, the substandard and overproduced dried longan during of more than 30,000 MT in various subdistrict stocks, that scattered throughout eight provincial areas in Northern part of Thailand (NaewNa, 2005), has yet remained one of the challenging problems for Thai government to find the best solution (TISC 2005). The objectives of current study were to (1) investigate the ability of 15 microbial strains for ethanol production in a stand still condition as well as (2) evaluate the possibility of performing biotransformation under non-aqueous condition using the biomass obtained previously. [Please contact authors for detailed references] Five species of microbes and the respective strains were ordered from Thailand Institute of Scientific and Technological Research. Each culture was stood aside at 25.6 o C (average temperature of Chiang Mai). The freeze dried stock of each culture was proliferated in the corresponding medium as recommended by TISTR (YM-Yeast Media for C. utilis and S. cerevisiae, NB-Nutrient Broth for E. coli and Klebsiella sp., ZM-Zymomonas Media for Z. mobilis) and stored as glycerol stock at -20 o C in 1 ml aliquot. In order to investigate the effect of 24 h incubation period on the growth of each microbe, the glycerol stock aliquot was transferred to the sterilized inoculum medium of 10 ml.. Further investigation was carried out in the larger scale of 100 ml with the initial glucose concentration of 63.5 – 64.4 g/l and corresponding 1.5 folds of nitrogen source concentration used previously in the inoculum medium. The biotransformation was performed at 4 and 20 o C in the stand still condition for 24 h with 0.53 g dry biomass equivalent/l. The biotransformation system contained 4 species of solvents, namely, water, octanol, dipropylene glycol (DPG), and DPG mixed with octanol in 1:1 volume ratio. The yield of ethanol produced over glucose consumed in 10 ml was the highest with C. utilis TISTR No (0.51 g ethanol/g glucose) while the highest ethanol producer (10.6 ± 0.5 g/l), Z. mobilis TISTR No. 405, achieved the ethanol yield of 0.48 g/g. In term of ethanol production in a stand still condition of 100 ml scale, the high ethanol producers included S. cerevisiae TISTR No (23.5 ± 2.6 g/l ethanol produced at 48 h minus 0 h), Z. mobilis TISTR No. 405 (23.5 ± 4.0 g/l), TISTR No. 550 (21.9 ± 3.1 g/l), and S. cerevisiae TISTR No (21.0 ± 0.8 g/l) with the corresponding ethanol yields of 0.37, 0.36, 0.34, and 0.33 g ethanol/g glucose, respectively. The biotransformation using harvested cells from the previous 100 ml stand still experiment in a single phase solvent system indicated that after 8 h reaction period benzaldehyde was the most soluble in dipropylene glycol with measured concentration range of 112 – 134 mM in comparison to the initial benzaldehyde concentration of 150 mM. In the case of pyruvate, the measured range of pyruvate and initial pyruvate concentrations were 166 – 223 and 180 mM, respectively. None of PAC was detected in these reaction systems with production of acetaldehyde at the concentration level of lesser than 0.6 mM. Acknowledgements The research team would like to express our gratitude to NRCT and TRF who provided us with the funding support (NRCT & TRF Fundings, 2006).