Bioresource Technology 142 (2013) 384389

Contents lists available at SciVerse ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Efcient production of dihydroxyacetone from biodiesel-derived crude glycerol by newly isolated Gluconobacter frateurii
Yu-Peng Liu a,1, Yang Sun a,1, Cong Tan b, Hua Li a, Xiao-Juan Zheng a, Kui-Qi Jin a, Gang Wang a,
a b

Institute of Bioengineering, School of Life Sciences, Henan University, Kaifeng 475004, PR China Changxing Pharmaceutical Co., Ltd., ChangXing 313100, PR China

h i g h l i g h t s
 Satisfactory dihydroxyacetone production by Gluconobacter frateurii on crude glycerol.  High production of dihydroxyacetone (up to 125.8 g l
1

) from crude glycerol.

 Optimization of dihydroxyacetone fermentation process by G. frateurii.

a r t i c l e

i n f o

a b s t r a c t
The efcient production of dihydroxyacetone (DHA) on biodiesel-derived glycerol based media was developed. A newly isolated strain, Gluconobacter frateurii CGMCC 5397, could convert crude glycerol to DHA with high yield and productivity. In shake-ask fermentation, the DHA concentration of 73.1 g l1 was attained at 48 h using an optimum medium containing biodiesel-derived crude glycerol. When fed-batch fermentation was carried out in a 7-l stirred bioreactor with crude glycerol, the DHA concentration, productivity, and yield were 125.8 g l1, 2.6 g l1 h1, and 90.5% at 48 h, respectively. This study suggests that the inexpensive biodiesel-derived crude glycerol could be utilized for efcient production of DHA by G. frateurii. 2013 Elsevier Ltd. All rights reserved.

Article history: Received 28 March 2013 Received in revised form 10 May 2013 Accepted 15 May 2013 Available online 23 May 2013 Keywords: Biodiesel-derived Crude glycerol Gluconobacter frateurii Dihydroxyacetone

1. Introduction Nowadays, fuel crisis has globally ounced the economy in every region, particularly the oil consuming countries due to the rapidly decreasing available global stocks. Due to this serious situation, biodiesel which comes from 100% renewable resources provides an alternative fuel option for future (Ayoub and Abdullah, 2012). Several sources for producing biodiesel have been studied such as rape seed, coal seed, palm oil, sunower oil, waste cooking oil, soybean oil, etc. (Zhang et al., 2003). Glycerol is produced as a byproduct at levels of approximately 10% (w/w) of the total biodiesel generated (Johnson and Taconi, 2007). Worldwide, crude glycerol derived from biodiesel conversion has increased from 200,000 tons in 2004 to 1.224 million tons in 2008 (Yang et al., 2012). Therefore, it is of great importance for scientists to nd new applications for crude glycerol. The crude glycerol is a relatively inexpensive raw material and has already been used for the production of a number of industrial important chemicals, such
Corresponding author. Tel.: +86 378 3887799.
1

E-mail address: wlwsw2012@yahoo.cn (G. Wang). These authors contributed equally to this work.

as ethanol (Oh et al., 2011), citric acid (Papanikolaou and Aggelis, 2003), docosahexaenoic acid (Chi et al., 2007), polyhydroxybuty et al., 2013), erythritol rate (Naranjo et al., 2013; Vrana poljaric (Rymowicz et al., 2009), 1,3-propanediol and 2,3-butanediol (Metsoviti et al., 2012). At the same time, the 1,3-dihydroxyacetone (DHA) is also a value-added product from crude glycerol. DHA is a very important chemical product, and used extensively in the cosmetic industry for making articial suntans (Brown, 2001; Levy, 1992; Nguyen and Kochevar, 2003). DHA has also been proposed to be involved in weight augmentation and fat loss, antioxidant activity, and increasing endurance capacity (Stanko et al., 1990), and it offers a safe and effective therapeutic option for recalcitrant vitiligo. Moreover, DHA serves as a variety of ne chemicals such as 1,2-propylene glycerol or lactic acid (Bicker et al., 2005; Hekmat et al., 2003). The production of DHA from glycerol can be done either via chemical or microbial route. It has been reported that the microbial route of DHA production was more efcient as compared to that of chemical route (Mishra et al., 2008). Commercial synthesis of DHA is done more economically via microbial route due to the expensive safety measurements required in case of chemical route. It has recently been reported that the enzyme catalyzed chemical

0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.05.055

Y.-P. Liu et al. / Bioresource Technology 142 (2013) 384389

385

bioconversions are more acceptable by the pharmaceutical and chemical industries as a practical alternative to chemical synthesis methods due to the intractable synthetic problems involved with the chemical synthesis methods and also because of stringent environmental constraints. Since Bertrand rst observed production of DHA from glycerol through bacterial route in the year 1898, various microorganisms used for DHA production have been reported (Mishra et al., 2008). Gluconobacter strains produce DHA via incomplete oxidation of glycerol with the activity of membrane-bound glycerol dehydrogenase (Deppenmeier et al., 2002), and have been the most extensively used microorganisms in the present research for DHA production (Hu et al., 2010; Lu et al., 2012; Tkac et al., 2001; Wethmar and Deckwer, 1999). In this article, we systematically reported, for the rst time, the efcient production of DHA by newly isolated Gluconobacter frateurii from biodiesel-derived crude glycerol. The effects of initial glycerol concentration and complex nitrogen sources on cell growth and DHA production from biodiesel-derived glycerol were examined. Moreover, the crude glycerol-fed batch fermentation was also developed to enhance the production of DHA. 2. Methods 2.1. Crude glycerol Crude glycerol used in this work was obtained from palm oilbased biodiesel plant in Malaysia operated by Vance Bioenergy [composition: 80.5% (w/w) glycerol, 10.1% (w/w) water, 5.2% (w/ w) sodium salts, 0.4% (w/w) potassium salts, 0.3% (w/w) other salts, 0.5% (w/w) methanol, 2% (w/w) other organics (esters, free fatty acids, soaps, etc.)]. Crude glycerol was used as carbon source in culture medium without purication. The purity of crude glycerol (80.5%, w/w) used in each experiment has been taken into consideration and the appropriate calculations were made, thus, in any case the initial concentrations of glycerol quoted refer to pure glycerol. 2.2. Isolation of crude glycerol-utilizing microorganism Screening plates and fermentation screening medium were all prepared by crude glycerol in this study. Samples were collected at various locations in Kaifeng (PR China), that contained sewage, sludge, rotted fruits, soil, honey, etc. All samples were stored at 4 C before isolation, ten grams of each sample was suspended in 90 ml of sterile distilled water and shaking for an hour at 30 C with 100 rpm, respectively. Aliquots of the cultures (0.2 ml) were spread on screening agar plates (100 g l1 glycerol, 15 g l1 yeast extract, 3 g l1 KH2PO4, 20 g l1 agar, pH 6.0) and incubated at 30 C for 3 days. Strains utilizing crude glycerol were selected from agar plates and pure cultures were obtained by slant culture. An overnight culture of isolated strains (2% inocula) was inoculated into 250 ml shake-asks with 30 ml of fermentation screening medium (80 g l1 glycerol, 15 g l1 yeast extract, 3 g l1 KH2PO4, pH 6.0) and the asks were incubated at 220 rpm shaking under 30 C for 48 h. The cultures were collected and subjected for further assay by measuring the concentration of DHA. 2.3. Microbial identication The 16S rRNA gene was amplied from genomic DNA by PCR using the bacterial primers. The sequences of the primers used for amplication were 50 -AGAGTTTGATCATGGCTCAG-30 (forward) and 50 -AAGGAGGTGATCCAGCCGCA-30 (reverse), and the PCR product was puried and the sequence was determined by TaKaRa Bio-

technology (Dalian) Co., Ltd. The sequence was aligned with reference sequences obtained from databases using ClustalW program. Pairwise evolutionary distances of them were calculated using Kimuras two-parameter model. A phylogenetic tree from distance matrices was constructed by the neighbor-joining method. 2.4. Physiological and biochemical characterization of strain HD924 To investigate the physiological and biochemical characteristics, standard techniques were performed, including Gram staining, the oxidase reaction, catalase, production of water-soluble brown pigment, production of DHA and 5-keto-D-gluconic acid, acid production from carbohydrates, the G + C (mol %) of DNA and etc. 2.5. Fermentation in shake-asks Cells were grown in 250 ml shake-asks containing 30 ml medium prepared by crude glycerol. The seed medium containing per liter: 15 g glycerol, 15 g yeast extract, 3 g KH2PO4. The fermentation medium containing per liter: 100 g glycerol, 15 g yeast extract (or 24 g corn steep liquor), 3 g CaCO3. The pH of the medium was adjusted to 6.0 with 2 mol l1 NaOH, and the medium was heat sterilized (20 min at 121 C). The seed medium was inoculated with 2 ml stock culture from 80 C ultra-low temperature freezer and incubated at 30 C for 16 h. For the fermentation experiments, the medium was inoculated with 5% of the seed culture and incubated at 220 rpm shaking under 30 C for 48 h. 2.6. Fermentation in stirred bioreactors Batch fermentation was carried out at 30 C with 4 l medium in 7-l stirred bioreactors (BioFlo 4000, New Brunswick Scientic, Edison, NJ, USA). The fermentation medium was prepared by crude glycerol (or pure glycerol) and pH was controlled at 5.5 with 2 mol l1 NaOH during the fermentation process. Air rate and agitation speed were controlled at 1.5 vvm and 350 rpm. Fed-batch fermentation was carried out under the same conditions as batch fermentation except the initial glycerol concentration was 20 g l1. When the concentration of glycerol was lower than 5 g l1, a sterilized crude glycerol was fed into the stirred bioreactor using a peristaltic pump to maintain the glycerol concentration within 515 g l1 during the fermentation process. 2.7. Analytical methods After the desired incubation period, the culture was diluted with 0.2 mol l1 HCl, and the cell concentration (OD560) was determined by measuring the absorbance at 560 nm using a spectrophotometer (U-752). DHA and glycerol were analyzed by high-performance liquid chromatography (HPLC). Fermentation samples were centrifuged (8000g, 10 min) at 4 C, and the concentrations of DHA and glycerol in supernatants were quantied using a HPLC system (Waters, Milford, MA) equipped with a refractive index detector, an UV detector, and an Aminex HPX-87H column (300 7.8 mm, 9 lm; BioRad Chemical Division, Richmond, Calif.). The mobile phase was 8 mmol l1 H2SO4 solution at a ow rate of 0.5 ml/min, and the column was operated at 55 C. DHA yield was dened as the amount of DHA produced from each gram of glycerol consumed (expressed in percentage), and was normalized in accordance with the dilution factor of base or crude glycerol solution. The glycerol utilization was calculated as glycerol consumed/initial glycerol and expressed in percentage.

386

Y.-P. Liu et al. / Bioresource Technology 142 (2013) 384389

3. Results and discussion 3.1. Screening of microorganisms producing DHA from crude glycerol Among about 200 samples tested, 42 microbial strains were isolated based on their abilities to grow on crude glycerol as a sole carbon source. Through production of DHA analysis for these candidates by HPLC, strain HD924 isolated from rotted fruits of Kaifeng (PR China) was nally selected for its highest DHA production (Table 1). The 16S rRNA gene sequence (1364 bases, GenBank accession number JQ044419) was compared with the sequence data in GenBank database by using the BLAST algorithm. According to the BLAST analysis on sequence similarity, the phylogenetic analysis was carried out by the neighbor-joining method (Fig. 1). It was shown that the closest relatives of the strain HD924 were G. frateurii NBRC 3254 (99.9%), Gluconobacter oxydanys NBRC3289 (99.9%), Gluconobacter thailandicus F149-1 (99.8%), Gluconobacter cerinus NBRC3262 (99.8%), and Gluconobacter japonicus NBRC3272 (99.8%). Furthermore, physiological and biochemical characterizations of strain HD924 were investigated and compared with those of the other related Gluconobacter species (date not shown). According to its phenotypic characteristics, strain HD924 is apparently closer to G. frateurii than to other Gluconobacter species (Malimas et al., 2008). On the basis of sequence similarity as well as physiological, and biochemical analysis, it was concluded that newly isolated bacterial strain HD924 belongs to G. frateurii. This strain was deposited in China General Microbiological Culture Collection Center with the accession number of CGMCC5397. We propose the name G. frateurii CGMCC5397 for strain HD924.

3.2. Effect of different nitrogen sources To optimize the nitrogen sources, the effect of different inorganic and organic nitrogen sources (in same percent equivalent) were compared with yeast extract (15.0 g l1) in shake-asks, including carbamide (2.9 g l1), (NH4)2SO4 (6.3 g l1), KNO3 (9.6 g l1), peptone (12 g l1), corn steep liquor (CSL, 24 g l1) and beef extract (15 g l1). As shown in Fig. 2, poor cell growth was observed with all the inorganic nitrogen sources tested, and corn steep liquor (CSL) was found to be the best nitrogen source for DHA production by G. frateurii CGMCC5397. Hu et al. (2010) reported that yeast extract, peptone, and corn steep liquor were all favorable nitrogen sources for DHA production by G. oxydans ZJB09112, and there was no remarkable difference in DHA production using these three ones. Different from the above report, the DHA production using CSL (72.6 2.2 g l1) was higher than other nitrogen sources tested, and peptone was not a favorable nitrogen source in this study. When yeast extract was used as a complex nutrient, 71.8 1.9 g l1 DHA was obtained, which was little difference compared with that of CSL. Since yeast extract and peptone are expensive, it is desirable to nd a least expensive complex nitrogen source to produce DHA. Therefore, CSL was selected as the nitrogen source for its effectiveness and low cost in this study. However, the inorganic compounds tested in this study were not favorable nitrogen sources for DHA production by G. frateurii CGMCC5397. 3.3. Effect of initial glycerol concentration A few reports have showed that the initial high glycerol concentration exerts an inhibitory effect on DHA production (Bauer

Table 1 Part of the isolated strains producing DHA from crude glycerol.a Strains HD924 (this work) HD385 (this work) HD532 (this work) HD389 (this work) HD921 (this work) HD426 (this work) Gluconobacter oxydans 1.00637c
a b c

Cell concentration (OD560) 6.8 0.2 4.5 0.2 6.1 0.4 7.6 0.3 6.5 0.5 4.0 0.3 6.0 0.3
b

DHA concentration (g l1) 66.7 2.1 51.2 0.9 24.5 2.3 31.5 1.7 56.3 3.7 17.2 3.1 50.4 2.2

Cells were grown in 250 ml shake-asks for 48 h with the crude glycerol fermentation screening medium (80 g l1 glycerol, 15 g l1 yeast extract, 3 g l1 KH2PO4, pH 6.0). Each value is an average of three parallel replicates and is represented as mean standard deviation. Gluconobacter oxydans 1.00637 was obtained from the China General Microbiological Culture Collection Center (Beijing, PR China).

Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences similarity of strain HD924 and the reference strains.

Y.-P. Liu et al. / Bioresource Technology 142 (2013) 384389

387

3.4. Biodiesel-derived crude glycerol vs. pure glycerol fermentation in stirred bioreactors Impurities in crude glycerol can greatly inuence the conversion of glycerol into other products. In some biological conversion processes, pollutants in crude glycerol, inhibit cell growth and result in lower production rates and product yields (compared with pure or commercial glycerol under the same culture conditions). Based on the above results obtained in shake-asks, batch fermentations of biodiesel-derived crude glycerol and pure glycerol were carried out in 7-l stirred bioreactors. In the fermentation of biodiesel-derived crude glycerol (Fig. 4a), the cell concentration reached the maximum value of OD560 (7.5 0.4) at 16 h and did not grow afterwards. At the end of fermentation (48 h), the concentration of DHA was 80.2 3.6 g l1, and the glycerol left only 10.7 1.9 g l1. The DHA production increased 9.7%, compared with that of shake-asks fermentation in Fig. 3 (73.1 1.4 g l1). Claret et al. (1994) reported that the key enzyme responsible for DHA production was membrane-bound glycerol dehydrogenase, which employs oxygen as the nal acceptor of reduced equivalents without NADH mediation. Flickinger and Perlman (1977) have proved that the amount of glycerol converted to DHA could be increased by improving the dissolved oxygen of fermentation media. In this study, the more oxygen supplied in 7-l stirred bioreactors, which led to a higher glycerol utilization compared with that in shake-asks fermentation.

Fig. 2. Effects of different nitrogen sources on the production of DHA from crude glycerol. Cells were grown in 250 ml shake-asks for 48 h with an initial glycerol concentration of 100 g l1, and were supplied with different nitrogen sources. Symbols are the DHA ( ) and OD560 ( ). The error bars in the gure indicate the standard deviations from three independent samples.

Fig. 3. Effects of initial glycerol concentration on the production of DHA from crude glycerol. Cells were grown in 250 ml shake-asks for 48 h with different initial glycerol concentrations from 20 to 200 g l1, and the corn steep liquor concentration was 24 g l1. Symbols are the DHA ( ) and OD560 ( ). The error bars in the gure indicate the standard deviations from three independent samples.

et al., 2005; Claret et al., 1992, 1994). Yamada et al. (1979) also indicated that the rate of DHA production was strongly affected by an increase in the initial content of glycerol. Therefore, DHA productions with crude glycerol in different dilution rates were investigated. In this study, the biomass and DHA production were also affected with the high initial glycerol concentration (Fig. 3). The results showed that G. frateurii CGMCC5397 could grow in the medium containing 200 g l1 of glycerol, while the cell (OD560 = 0.9 0.1) and DHA concentration (10.9 1.7 g l1) were all low at 48 h. The production of DHA increased signicantly with the increase of initial glycerol concentrations from 20 g l1 to 100 g l1. However, the DHA concentration and biomass decreased when the initial glycerol concentration was over 100 g l1. The maximum values of the cell (OD560 = 6.9 0.2) and DHA concentration (73.1 1.4 g l1) were obtained in the culture with an initial glycerol concentration of 100 g l1 and the yield was 89.5 1.7%.

Fig. 4. Time courses of cell growth and production of DHA in 7-l stirred bioreactors with crude glycerol (a) and pure glycerol (b). Cells were grown with 100 g l1 initial glycerol and 24 g l1 corn steep liquor. Symbols are the DHA (4), glycerol (s) and OD560 (j). The error bars in the gure indicate the standard deviations from three independent samples.

388

Y.-P. Liu et al. / Bioresource Technology 142 (2013) 384389

For pure glycerol fermentation (Fig. 4b), the cell concentration reached the maximum value of OD560 (8.3 0.3) at 16 h, which was 11% increased compared with that in crude glycerol fermentation. At 48 h, the DHA concentration was increased to 85.9 2.9 g l1, 5.7 g l1 higher than that in (Fig. 4a). Furthermore, the concentration of glycerol was only 5.9 0.8 g l1 left, which led to the glycerol utilization from 89.3% to 94.1%. However, the DHA yield was constant (about 90%) between the two fermentation methods. The results indicated that cell growth and DHA production had no remarkable inuence between the biodiesel-derived crude glycerol and pure glycerol fermentation, except that the DHA production of crude glycerol was a little lower. This indicated that it may have some poisonous contents which affect the DHA production left in the biodiesel-derived crude glycerol.

3.5. Effect of chloride salts on the production of DHA The chemical composition of crude glycerol mainly varies with the type of catalyst used to produce biodiesel. Accordingly, methanol, soap, catalysts, salts, non-glycerol organic matter, and water impurities usually are contained in the crude glycerol (Yang et al., 2012). Base catalysts such as sodium hydroxide (NaOH) and potassium hydroxide (KOH) are the most commonly used catalysts in industrial biodiesel production using palm oil (Lam and Lee, 2011). The transesterication of triglycerides with sodium hydroxide generates a methyl-ester phase and a glycerol phase. The glycerol phase is typically neutralized with acid and the cationic component of the catalyst is incorporated as a salt. For example, sodium chloride is formed in the neutralization with hydrochloric acid of the glycerol phase containing sodium ion, and the concentrated crude glycerol (used in this work) was obtained after the excess of methanol was distilled off. In this study, the main impurities in the crude glycerol were sodium chloride, potassium chloride, methanol and other organics. Because the content of methanol (0.5%, w/w) in crude glycerol were very low, and part of toxic methanol presented in the crude glycerol had been evaporated during sterilization, the methanol left in fermentation media had no inuence on the production of DHA (data not shown). In addition, most of the other organics [2% (w/w), includes esters, free fatty acids, soaps, etc.] left in crude glycerol fermentation media were nontoxic to cell growth of G. frateurii CGMCC5397. The effects of chloride salts (sodium chloride,

potassium chloride) on cell growth and DHA production were investigated by adding 00.2 mol l1 chloride salts in pure glycerol fermentation media (Fig. 5). From Fig. 5a, it can be seen that high initial concentration of sodium chloride (>0.1 mol l1) affect cell growth and DHA production of G. frateurii CGMCC5397, while potassium chloride has minor effect on them (Fig. 5b). When crude glycerol was used in batch fermentation (Fig. 4a), the initial sodium ion concentration of fermentation medium reached to 0.11 mol l1, which was harmful to the cell growth and DHA production of G. frateurii CGMCC5397 compared with those of pure glycerol fermentation (Fig. 4b). Sodium ion is involved in the formation of transmembrane pH gradient, cell motility and intracellular pH regulation (Lee et al., 1999). In this present study, chloride salts were used to investigated the effect of different sodium ion concentrations, and both cell growth and DHA production were slightly affected by the concentration of anions (chloride, sulfate or nitrate ions) from 0 to 0.2 mol l1 (data not shown). These results suggest that high concentration sodium ion in biodiesel-derived crude glycerol (this work) may affect the DHA production.

3.6. Fed-batch fermentation of biodiesel-derived crude glycerol in stirred bioreactor The above results (Fig. 4) have shown that substrate inhibition was a clear limitation to the DHA production in batch fermentation. Claret et al. (1992) reported that a longer lag period for beginning of growth was observed with a high initial glycerol concentration for G. oxydans. The purpose of glycerol feeding was to maintain the cell growth without substrate inhibition, and a few feeding strategies on DHA production with G. oxydans have been reported (Hu et al., 2010, 2011). Fed batch fermentation with biodiesel-derived crude glycerol was investigated in this study to enhance the production of DHA (Fig. 6). When the glycerol concentration was maintained at a lower level (515 g l1) during the fermentation process, cells grew faster and the steady phase prolonged due to the elimination of the substrate inhibition. The value OD560 was 8.7 at 8 h, and the DHA productivity (2.6 g l1 h1) was higher than that in the batch fermentation (1.7 g l1 h1) because of the elimination of substrate inhibition. At the end of fermentation (48 h), the DHA concentration and yield were 125.8 g l1 and 90.5%, respectively.

Fig. 5. Effects of chloride salts on the production of DHA with pure glycerol fermentation. Cells were grown in 250 ml shake-asks for 48 h with different initial concentrations of NaCl (a) and KCl (b) added to the pure glycerol fermentation medium (100 g l1 glycerol, 24 g l1 corn steep liquor, 3 g l1 CaCO3, pH 6.0). Symbols are the DHA ( ) and OD560 ( ). The error bars in the gure indicate the standard deviations from three independent samples.

Y.-P. Liu et al. / Bioresource Technology 142 (2013) 384389

389

Fig. 6. Time courses of cell growth and production of DHA in fed batch fermentation with crude glycerol. Cells were grown in a 7-l stirred bioreactor with 20 g l1 initial glycerol and 24 g l1 corn steep liquor. After 8 h, the glycerol concentration was maintained at 515 g l1 during the fermentation process. Symbols are the DHA (4), glycerol (s) and OD560 (j). The experiment was repeated twice and the data points are the mean of two replicates.

4. Conclusions A DHA producing strain HD924 was newly isolated from the rotted fruits, and was identied as G. frateurii. After optimization, the DHA concentration of 73.1 g l1 was obtained in shake-ask culture. During the fed batch fermentation in a 7-l stirred bioreactor, the DHA concentration (125.8 g l1) and productivity (2.6 g l1 h1) were higher than those in the batch fermentation. In conclusion, biodiesel-derived crude glycerol could be used as an economical and feasible carbon source for the DHA production by Gluconobacter strains. The present study should be potentially useful for the efcient DHA production on industrial scale. Acknowledgements This research was supported by grants from the National Natural Science Foundation of China (No. 30971952), the Key Project of Science and Technology of Henan Province (No. 132102210387), and Changxing Pharmaceutical Co., Ltd. References
Ayoub, M., Abdullah, A.Z., 2012. Critical review on the current scenario and signicance of crude glycerol resulting from biodiesel industry towards more sustainable renewable energy industry. Renewable Sustainable Energy Rev. 16, 26712686. Bauer, R., Katsikis, N., Varga, S., Hekmat, D., 2005. Study of the inhibitory effect of the product dihydroxyacetone on Gluconobacter oxydans in a semi-continuous two-stage repeated-fed-batch process. Bioproc. Biosyst. Eng. 28, 3743. Bicker, M., Endres, S., Ott, L., Vogel, H., 2005. Catalytical conversion of carbohydrates in subcritical water: a new chemical process for lactic acid production. J. Mol. Catal. A Chem. 239, 151157. Brown, D.A., 2001. Skin pigmentation enhancers. J. Photochem. Photobiol. B 63, 148161. Chi, Z., Pyle, D., Wen, Z., Frear, C., Chen, S., 2007. A laboratory study of producing docosahexaenoic acid from biodiesel-waste glycerol by microalgal fermentation. Process Biochem. 42, 15371545. Claret, C., Bories, A., Soucaille, P., 1992. Glycerol inhibition of growth and dihydroxyacetone production by Gluconobacter oxydans. Curr. Microbiol. 25, 149155. Claret, C., Salmon, J.M., Romieu, C., Bories, A., 1994. Physiology of Gluconobacter oxydans during dihydroxyacetone production from glycerol. Appl. Microbiol. Biotechnol. 41, 359365.

Deppenmeier, U., Hoffmeister, M., Prust, C., 2002. Biochemistry and biotechnological applications of Gluconobacter strains. Appl. Microbiol. Biotechnol. 60, 233242. Flickinger, M.C., Perlman, D., 1977. Application of oxygenenriched aeration in the conversion of glycerol to dihydroxyacetone by Gluconobacter melanogenus IFO 3293. Appl. Environ. Microbiol. 33, 706712. Hekmat, D., Bauer, R., Fricke, J., 2003. Optimization of the microbial synthesis of dihydroxyacetone from glycerol with Gluconobacter oxydans. Bioproc. Biosyst. Eng. 26, 109116. Hu, Z.C., Liu, Z.Q., Zheng, Y.G., Shen, Y.C., 2010. Production of 1,3-dihydroxyacetone from glycerol by Gluconobacter oxydans ZJB09112. J. Microbiol. Biotechnol. 20, 340345. Hu, Z.-C., Zheng, Y.-G., Shen, Y.-C., 2011. Use of glycerol for producing 1,3dihydroxyacetone by Gluconobacter oxydans in an airlift bioreactor. Bioresour. Technol. 102, 71777182. Johnson, D.T., Taconi, K.A., 2007. The glycerin glut: options for the value-added conversion of crude glycerol resulting from biodiesel production. Environ. Prog. 26, 338348. Lam, M.K., Lee, K.T., 2011. Production of biodiesel using palm oil. In: Pandey, A., Larroche, C., Ricke, S.C., Dussap, C.-G., Gnansounou, E. (Eds.), Biofuels: Alternative Feedstocks and Conversion Processes, 1st Ed. Academic Press, San Diego, pp. 356359. Lee, P.C., Lee, W.G., Lee, S.Y., Chang, H.N., 1999. Effects of medium components on the growth of Anaerobiospirillum succiniciproducens and succinic acid production. Process Biochem. 35, 4955. Levy, S.B., 1992. Dihydroxyacetone-containing sunless or self-tanning lotions. J. Am. Acad. Dermatol. 27, 989993. Lu, L., Wei, L., Zhu, K., Wei, D., Hua, Q., 2012. Combining metabolic engineering and adaptive evolution to enhance the production of dihydroxyacetone from glycerol by Gluconobacter oxydans in a low-cost way. Bioresour. Technol. 117, 317324. Malimas, T., Yukphan, P., Takahashi, M., Muramatsu, Y., Kaneyasu, M., Potacharoen, W., Tanasupawat, S., Nakagawa, Y., Tanticharoen, M., Yamada, Y., 2008. Gluconobacter sphaericus (Ameyama 1975) comb. nov., a brown pigmentproducing acetic acid bacterium in the Alphaproteobacteria. J. Gen. Appl. Microbiol. 54, 211220. Metsoviti, M., Paraskevaidi, K., Koutinas, A., Zeng, A.P., Papanikolaou, S., 2012. Production of 1,3-propanediol, 2,3-butanediol and ethanol by a newly isolated Klebsiella oxytoca strain growing on biodiesel-derived glycerol based media. Process Biochem. 47, 18721882. Mishra, R., Jain, S.R., Kumar, A., 2008. RETRACTED: microbial production of dihydroxyacetone. Biotechnol. Adv. 26, 293303. Naranjo, J.M., Posada, J.A., Higuita, J.C., Cardona, C.A., 2013. Valorization of glycerol through the production of biopolymers: the PHB case using Bacillus megaterium. Bioresour. Technol. 133, 3844. Nguyen, B.C., Kochevar, I.E., 2003. Factors inuencing sunless tanning with dihydroxyacetone. Br. J. Dermatol. 149, 332340. Oh, B.R., Seo, J.W., Heo, S.Y., Hong, W.K., Luo, L.H., Joe, M.H., Park, D.H., Kim, C.H., 2011. Efcient production of ethanol from crude glycerol by a Klebsiella pneumoniae mutant strain. Bioresour. Technol. 102, 39183922. Papanikolaou, S., Aggelis, G., 2003. Modelling aspects of the biotechnological valorization of raw glycerol: production of citric acid by Yarrowia lipolytica and 1,3-propanediol by Clostridium butyricum. J. Chem. Technol. Biotechnol. 78, 542547. ska, A., Marcinkiewicz, M., 2009. High-yield production of Rymowicz, W., Rywin erythritol from raw glycerol in fed-batch cultures of Yarrowia lipolytica. Biotechnol. Lett. 31, 377380. Stanko, R.T., Robertson, R.J., Spina, R.J., Reilly Jr., J.J., Greenawalt, K.D., Goss, F.L., 1990. Enhancement of arm exercise endurance capacity with dihydroxyacetone and pyruvate. J. Appl. Physiol. 68, 119124. Tkac, J., Navratil, M., Sturdik, E., Gemeiner, P., 2001. Monitoring of dihydroxyacetone production during oxidation of glycerol by immobilized Gluconobacter oxydans cells with an enzyme biosensor. Enzyme Microb. Technol. 28, 383388. , I., Lopar, M., Koller, M., Muhr, A., Salerno, A., Reiterer, A., Malli, K., Vrana poljaric Angerer, H., Strohmeier, K., Schober, S., Mittelbach, M., Horvat, P., 2013. Mathematical modeling of poly[(R)-3-hydroxyalkanoate] synthesis by Cupriavidus necator DSM 545 on substrates stemming from biodiesel production. Bioresour. Technol. 133, 482494. Wethmar, M., Deckwer, W.D., 1999. Semisynthetic culture medium for growth and dihydroxyacetone production by Gluconobacter oxydans. Biotechnol. Technol. 13, 283287. Yamada, S., Nabe, K., Izuo, N., Wada, M., Chibata, I., 1979. Fermentative production of dihydroxyacetone by Acetobacter suboxydans ATCC621. J. Ferment. Technol. 57, 215220. Yang, F., Hanna, M., Sun, R., 2012. Value-added uses for crude glycerol-a byproduct of biodiesel production. Biotechnol. Biofuel. 5, 110. Zhang, Y., Dub, M.A., McLean, D.D., Kates, M., 2003. Biodiesel production from waste cooking oil: 1. Process design and technological assessment. Bioresour. Technol. 89, 116.