P-ISSN 2586-9000 E-ISSN 2586-9027 Homepage : https://tci-thaijo.org/index.php/SciTechAsia Science & Technology Asia Vol. 22 No.3 July - September 2017 Page: [ 20 - 31 ] Original research article Screening and Preliminary Optimizations for Dihydroxyacetone Production from Glycerol by the Gluconobacter and Asaia Isolates Found in Thailand Issara Poljungreed1 , Siwarutt Boonyarattanakalin1,* 1Sirindhorn International Institute of Technology, School of Bio-Chemical Engineering and Technology, Thammasat University Rangsit Campus, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand Pattaraporn Yukphan2 2BIOTEC Culture Collection, Biodiversity and Biotechnological Resource Research Unit (BBR), National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand Received 17 March 2017; Received in revised form 23 May 2017 Accepted 31 May 2017; Available online 12 October 2017 ABSTRACT This study aims to investigate the feasibility of using a minimal glycerol medium with inorganic salt supplementation for a low-cost dihydroxyacetone (DHA) production by bacteria. Acetic acid bacterial isolates of Gluconobacter and Asaia, which are found in the Northern, North-Eastern, Middle, Western, and Southern regions of Thailand, were evaluated for their ability to produce DHA using glycerol as a carbon source. During the qualitative screening, 66 isolates, including Gluconobacter ( 61 isolates) and Asaia ( 5 isolates) , from a total of 486 isolates showed highly positive results by the Fehling test. The 5 isolates of Asaia gave low DHA production in the quantitative screening, whereas Gluconobacter isolates showed DHA production at low (0-5.70 g/L), medium (5.71-11.40 g/L), and high (11.41-16.89 g/L) levels. Preliminary culture medium optimizations for G. frateurii BCC 36199, a most promising microorganism for DHA production, were also carried out using a low-cost minimal glycerol medium supplemented with an inorganic salt. G. frateurii BCC 36199 produced 18.67 g/L of DHA with ysp of 95.44% (DHA moles/glycerol moles) at 30°C, 20 g/L of glycerol, and pH 4.5. The cultivation of G. frateurii BCC 36199 in the developed minimal glycerol medium is practical and can be further optimized in order to apply for industry. Keywords: Dihydroxyacetone; Glycerol; Gluconobacter; Asaia; Acetic acid bacteria *Corresponding author: [email protected] doi: 10.14456/scitechasia.2017.3 I. Poljungreed et al. | Science & Technology Asia | Vol.22 No.3 July – September 2017 1. Introduction 2. Materials and Methods Glycerol is currently generated in 2.1 Microorganisms excess amounts as a major by- product of The Gluconobacter (256 isolates) and biodiesel production processes [ 1] . Asaia (230 isolates) genera used in this study Consequently, the price of glycerol has were obtained from the BIOTEC Culture declined [2]. Glycerol has been studied as a Collection ( BCC) , BIOTEC, National carbon source for microbial fermentation to Science and Technology Department generate many useful chemicals [1]. Among Agency (NSTDA), Thailand. Before using, the chemicals obtained from glycerol the microorganisms, which were kept at - fermentation, dihydroxyacetone (DHA) is a 20°C, were activated by culturing them on a promising product because of its high price Glucose- Ethanol- Calcium carbonate Agar and versatility [3]. DHA is used in cosmetic (GECA) medium (20 g/L of glucose, 5 g/L products, pharmaceuticals, and various kinds of ethanol, 1.2 g/L of peptone, 3 g/L of yeast of chemicals syntheses [4]. extract, and 7 g/L of calcium carbonate) at Acetic acid bacteria are commonly 30°C for 24 h. used for DHA production using glycerol as a 2.2 Qualitative Screening for DHA carbon source. Microbial production of DHA Production from Glycerol from glycerol was first observed in the case The 486 isolates of Gluconobacter and of the bacteria sorbose bacillus [ 5] . Other Asaia were cultured in test tubes containing acetic acid bacteria such as Acetobacter 10 mL of an organic screening medium xylinum [6,7], Acetobacter suboxydans [8], (glycerol 60 g/L, peptone 10 g/L, and yeast and Gluconobacter melanogenus [ 9] have extract 5 g/ L) at 30°C for 24 h. After 24 also been reported for DHA production from hours, the fermented screening media were glycerol. In recent years, G. oxydans has tested for DHA generation by reacting with become the most commonly used the Fehling’ s reagent [ 18] . Yellow to red microorganism for converting glycerol to precipitate was inspected in the cultured DHA [4, 10-12]. media with the generated DHA. The non- Asaia, another genus of the acetic acid fermented organic screening medium added bacteria, also shows the ability to produce with the DHA standard was used as a positive DHA from glycerol [ 13,14] . Acetic acid control, and the negative control was the bacteria are normally found in fruits and unfermented organic screening medium. flowers [15]. These bacteria are also present in alcoholic beverages [16]. A diversity of 2.3 Quantitative Screening for High acetic acid bacteria in the genera of DHA Production Microorganisms Gluconobacter and Asaia has been found in The acetic acid bacteria which showed Thailand [17]. These two bacterial genera, highly positive results from the qualitative which have been reported to consume screening step were selected to be studied in glycerol, could be promising this section. Microbial inoculums were microorganisms that have the ability to prepared by culturing the bacteria in a 150- produce DHA from glycerol. Therefore, this mL Erlenmeyer flask containing 30 mL of study aimed to evaluate the feasibility of the inoculum medium (30 g/L of glycerol, 10 DHA production from glycerol using the g/L of peptone, and 5 g/L of yeast extract) at acetic acid bacteria genera of Gluconobacter 30°C for 24 h with a shaking speed of 150 and Asaia to determine whether these rpm. The inoculum (1 mL), with absorbance bacteria are promising DHA- producing at 560 nm ( OD560) around 1. 3, was then microorganisms. transferred into a 150-mL Erlenmeyer flask containing a minimal medium ( 50 mL) composed of only glycerol ( 60 g/ L) and 21 S. Boonyarattanakalin et al. | Science & Technology Asia | Vol.22 No.3 July – September 2017 diammonium phosphate [(NH4) 2HPO4, 2.8 fitting using DOE PRO XL 2007 ( Sigma g/L]. (NH4)2HPO4 was used as an inorganic zone) software. Two levels of three nitrogen source for the microorganisms in numerical factors (Table 1) were contributed place of the usual organic nitrogen sources in the statistical calculations. ( yeast extract and peptone) in order to 2.5 Determination of Glycerol and observe the capability of the screened DHA in Glycerol Fermentation bacteria for DHA production in a low cost Fermentation samples were centrifuged culture medium. The inoculated inorganic (5,000 rpm, 10 min) and then analyzed for the screening medium was cultured at 30°C for amounts of DHA and glycerol by HPLC. The 24 h with a shaking speed of 150 rpm, and HPLC system was equipped with a CarboSep the cultured medium was then monitored for CHO 682 (7.8 mm × 300 mm, Transgenomic) DHA and glycerol concentrations by high column as the stationary phase operated at performance liquid chromatography 80°C. De-ionized (DI) water was used as the ( HPLC) . The DHA production of the mobile phase with a flow rate of 0.5 mL/min. screened bacteria was presented as DHA Sucrose was used as an internal standard. concentration (p, g/L) and DHA yield (y , % sp Culture samples were filtered (VertiCleanTM of DHA moles/glycerol moles). The y was sp PTFE syringe Filters, 13mm, 0.2µm) before calculated by Eq. (2.1) : the analysis. A Refractive Index Detector (RID) was used as a detector for the HPLC 풚 = (푫푯푨 풎풐풍풆풔⁄풊풏풊풕풊풂풍 품풍풚풄풆풓풐풍 풎풐풍풆풔) 풔풑 system. × ퟏퟎퟎ (Eq. 2.1) 2.4 Preliminary Optimizations for Table 1. Culture parameters for preliminary DHA Production from Glycerol optimization. Gluconobacter frateurii BCC 36199, level which showed the highest DHA production parameter code from the screening, was cultivated under 8 -1 1 different culture conditions based on the full Glycerol (g/L) A 20 60 factorial design of experiment in order to pH B 4.5 7.0 primarily optimize for DHA production by Temperature (°C) C 25 30 G. frateurii BCC 36199. The culture parameters that were studied in the preliminary optimization were glycerol 3. Results and Discussion concentration, pH, and temperature ( Table 3.1 Qualitative Screening for 1) . The media were supplemented with Glycerol Utilizing Microorganisms with (NH4)2HPO4 as an inorganic nitrogen source. the Ability to Generate DHA The cultivation was done at 30°C for 72 h Qualitative screening was used as a first with a shaking speed of 250 rpm in 150-mL step to select DHA- producing Erlenmeyer flask containing 50 mL of the microorganisms using the Fehling’ s reagent optimizing media. Fermentation samples test. The unfermented glycerol medium were collected daily and analyzed for DHA without DHA products, which showed a production. purple color with the Fehling’s test reagent, Regression models to describe the was used as the negative control. The relationship between the significant unfermented glycerol medium with the added cultivation parameters and the DHA DHA (30 g/L), which showed a yellow color production are derived based on a full with the Fehling’s test reagent, was used as the factorial design [19]. Statistical analysis of positive control. Cultured samples that the responses was performed by least squares showed negative, low positive, and highly 22 I. Poljungreed et al. | Science & Technology Asia | Vol.22 No.3 July – September 2017 positive results for DHA production gave sp., As. bogorensis, As. siamensis, As. purple, yellow- green, and yellow colors, krungthepensis, and G. cerinus produced respectively, with the Fehling’s test reagent. DHA at low levels. Isolates of Gluconobacter Table 2 shows the bacterial isolates which sp.