A novel osmotic pressure control fed-batch fermentation strategy for improvement of erythritol production by Yarrowia lipolytica from glycerol

Bioresource Technology 151 (2014) 120–127

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A novel osmotic pressure control fed-batch fermentation strategy for improvement of erythritol production by Yarrowia lipolytica from glycerol Li-Bo Yang, Xiao-Bei Zhan ⇑, Zhi-Yong Zheng, Jian-Rong Wu, Min-Jie Gao, Chi-Chung Lin ⇑ Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China

h i g h l i g h t s  Osmotic pressure affected the ratio of erythritol to mannitol production.  A novel osmotic pressure control strategy improved erythritol production.  Substrate-feeding kept the osmotic pressure at a relatively constant level.  Erythritol production reached 194.3 g/L.  High osmotic pressure and low pH protect the culture against contamination.

a r t i c l e

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Article history: Received 26 August 2013 Received in revised form 7 October 2013 Accepted 10 October 2013 Available online 18 October 2013 Keywords: Osmotic pressure control strategy Fed-batch Erythritol Yarrowia lipolytica High productivity

a b s t r a c t The effect of osmotic pressure on erythritol and mannitol production by an osmophilic yeast strain of Yarrowia lipolytica CICC 1675 using glycerol as the sole carbon source was investigated. Appropriately high osmotic pressure was found to enhance erythritol production and inhibit mannitol formation. A novel two-stage osmotic pressure control fed-batch strategy based on the kinetic analysis was developed for higher erythritol yield and productivity. During the first 96 h, the osmotic pressure was maintained at 4.25 osmol/kg by feeding glycerol to reduce the inhibition of cell growth. After 132 h, the osmotic pressure was controlled at 4.94 osmol/kg to maintain a high dpery/dt. Maximum erythritol yield of 194.3 g/L was obtained with 0.95 g/L/h productivity, which were 25.7% and 2.2%, respectively, improvement over the best results in one-stage fed-batch fermentation. This is the first report that a novel osmotic pressure control fed-batch strategy significantly enhanced erythritol production. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The global increase in petroleum prices leads to rapid growth of the biodiesel industry which generates large amount of waste glycerol. For the production of 100 kg of biodiesel by triglyceride transesterification, 10 kg of crude glycerol is expected to be formed as a by-product (Kolesarova et al., 2011). The glycerol-rich residual waste water generated during the process of biodiesel production has created its disposal problem. However, such crude glycerol can be utilized to produce many valuable fermentation products and thus resolved the disposal problem (Mattam et al., 2013). Biodiesel waste contains glycerol (up to 80%), oil residue, free fatty acids, sodium salts, and water, which may serve as a raw ⇑ Corresponding authors. Address: School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China (X.-B. Zhan, C.-C. Lin). Tel./fax: +86 510 85918299 (X.-B. Zhan). E-mail addresses: [email protected] (X.-B. Zhan), [email protected] (C.-C. Lin). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.10.031

material for various biotechnological processes. A lot of efforts were made for the microbiological conversion of glycerol into valuable products: 1,3-propanediol (Papanikolaou et al., 2008), microbial biomass and lipids (Papanikolaou and Aggelis, 2002; Cheirsilp and Louhasakul, 2013), erythritol (Rymowicz et al., 2008, 2009; Rywinska et al., 2009), mannitol (Andre et al., 2009), organic acids (Lee et al., 2001; Rymowicz et al., 2010). Erythritol, a four-carbon sugar alcohol, is a biological sweetener used extensively in the food and pharmaceutical industries. It has been shown to be safe even for daily up-take as a noncariogenic sweetener at reasonably high amount. Erythritol cannot be utilized by the bacteria. Consequently, unlike the most common sweetener: sucrose, erythritol does not promote dental caries (Goldberg, 1994). Erythritol is produced at the commercial industrial scale by fermentation processes using mainly sucrose, glucose or fructose syrup or enzymatically hydrolyzed wheat or corn starches as carbon sources (Ishizuka et al., 1989). New uses of inexpensive raw materials such as glycerol for microbial transformations into useful products are increasingly important

L.-B. Yang et al. / Bioresource Technology 151 (2014) 120–127

(Papanikolaou et al., 2008). The crucial reasons causing low yield of erythritol (conversion rate) and high level of by-products (mannitol or arabitol) during erythritol fermentation process have not been well understood. Several reports have found that high initial level of glucose favors erythritol biosynthesis by osmophilic microorganisms (Ishizuka et al., 1989; Park et al., 1998). Osmotic pressure has been known to exert profound influence on cell growth and its metabolism. When yeast cells are subjected to high osmotic pressure at the initial stage of the fermentation, sluggish fermentation is often observed. Ultimately the fermentation efficiency declines at the subsequent stage (Panchal and Stewart, 1980). High osmotic pressure, low water activity (aw) and by-products formation are key factors responsible for the inhibition of yeast growth and decrease fermentation performance under high substrate concentrations (Puligundla et al., 2011). The objectives of the present work are: (1) to investigate the effect of osmotic pressure on cell growth and polyols (erythritol and mannitol) production by Yarrowia lipolytica using glycerol as carbon source; (2) to develop a novel two-stage osmotic pressure control process in fed-batch fermentation for high yield, high concentration and high productivity of erythritol simultaneously. 2. Methods 2.1. Microorganism and media The strain Y. lipolytica CICC 1675 used in this study was originally obtained from the yeast culture collection of the China Center of Industrial Culture Collection. The yeast culture was maintained on yeast maltose agar (YM) slants at 4 °C. The growth medium for the inoculum preparation contained (g/L): glycerol, 50; yeast extract, 5; peptone, 5; MgSO47H2O, 0.5; KH2PO4, 0.2. Erythritol fermentation production was conducted in a medium consisting of (g/L): glycerol, 200; urea, 1.68; yeast extract, 1.0; MgSO47H2O, 1.0; KH2PO4, 0.2; CaCl22H2O, 0.5; ZnSO47H2O, 0.1; MnSO44H2O, 0.01; FeSO47H2O, 0.1, all media were sterilized at 115 °C for 15 min before the filter-sterilized CaCl22H2O and FeSO47H2O were added. 2.2. Culture methods For seed preparation, Y. lipolytica CICC 1675 from a fresh slant was inoculated into 50 mL growth medium in 250 mL flasks and cultivated in a rotary shaker at 250 rpm for 24 h. The shake-flask experiments were conducted for 120 h in 250 mL flasks containing 30 mL of the appropriate medium under the same conditions as described above. Samples were taken at the end of the fermentation. Batch and fed-batch fermentations were carried out in a 7 L stirred-tank reactor (BioFlo 115; New Brunswick Scientific Co., NJ, USA) with a working volume of 3 L. All fermentations were carried out at 30 °C and pH was maintained automatically at 3.0 by the addition of a 2.0 mol/L NaOH. The aeration rate was controlled at 1.0 vvm and the agitation speed was controlled at 800 rpm. During the fermentations, samples were taken at 6–12 h intervals. All the cultures were cultivated until the carbon source was consumed completely. The shake-flask experiments and all the batch cultures were performed in three replications, and the two-stage osmotic pressure control fed-batch fermentations were performed in five replications.

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distilled water. Optical density was measured by UV spectrophotometer (model UV-2000; UNICO, Dayton, NJ) at 600 nm; the path-length of a cuvette was 1 cm. Dry cell weight (DCW) was quantified with sample culture dried in a hot-air oven at 105 °C until a constant value was obtained:

DCW ¼ 1:12 ðOD600 Þ  0:19

ð1Þ

where DCW is dry cell weight, g/L; OD600 is optical density at 600 nm. 2.3.2. Substrate and product concentrations For the determination of the levels of glycerol, erythritol and mannitol, fermentation broth was centrifuged at 10,000g for 10 min. The supernatant was diluted 20 times and filtered through a microfiltration membrane with 0.22 lm pore size. Polyols in the supernatant were simultaneously determined by HPLC (Agilent 1200 series, Santa Clara, CA, USA) with a Aminex HPX-87H ionexclusion column (300 mm  7.8 mm; Bio-Rad Laboratories Inc., Hercules, CA, USA). The mobile phase was 5.0 mmol sulfuric acid in distilled, de-ionized water filtered with 0.22 lm microfiltration membrane. The column was eluted at 35 °C with the mobile phase at a flow rate of 0.6 mL/min, and sample injection volume was 5.0 lL. The mannitol, erythritol and glycerol were detected with a refractive index detector. All the values are the averages of three independent extraction processes. 2.3.3. Preparation of crude cell extract and determination of the activities of intracellular enzymes for polyols biosynthesis For the determination of intracellular enzymatic activity involved in erythritol and mannitol biosynthesis, cells were harvested by centrifugation (10,000g, 10 min, 4 °C) and washed twice with cold phosphate buffer (100 mmol/L, pH 6.0) and the washed cells were suspended in the buffer (100 mmol/L phosphate buffer, 10 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, and 1 mmol/L phenylmethanesulfonyl flouride (PMSF), pH 6.0) for 30 min. The cell suspension was homogenized by grinding with glass beads (0.5 mm, Sigma) in a bead beater (BiospecProducts Co., Bartlesville, OK, USA) for 5 cycles of 1 min each with 2 min of cooling. Cell lysates were centrifuged at 10,000g for 30 min at 4 °C to remove the cell debris, and the supernatant was used to determine the enzymatic activity. The activity of mannitol-1-phosphate dehydrogenase (M-1PDH) or erythrose reductase (ER) was determined at 30 °C from the reduction of NAD(P)H, by measuring the decrease in the absorbance at 340 nm. The assay mixture (1.0 mL) for the reduction consisted of 0.1 mL NAD(P)H (1.15 mol/L), 0.1 mL fructose-6phosphate or erythrose (0.1 mol/L), 0.1 mL cell extract, 0.1 mL H2O and 0.6 mL phosphate buffer (0.1 mol/L, pH 6.0). Before the addition of cell extract, the reaction mixture was allowed to stand for 1 min to eliminate the endogenous oxidation of NAD(P)H. One unit of enzyme activity was defined as the amount which will cause a decrease in optical density of 0.01 per minute during the first minute of the reaction under the above conditions. Specific activity is expressed in terms of units of enzyme per milligram of protein. Activities expressed are presented as average of triplicate assays. 2.3.4. Protein assays The protein content was determined by the Bradford method (Bradford, 1976) and using bovine serum albumin (Sigma–Aldrich Co. St. Louis, Missouri, USA) as the standard reference.

2.3. Analytical methods 2.3.1. Cell concentration For biomass detection, 500 lL of cultures were centrifuged at 10,000g for 10 min. The cell pellet was suspended with 10 mL

2.3.5. Osmotic pressure determination For the determination of osmotic pressure, each 50 lL of bulk sample was analyzed by freezing-point depression using an osmometer (Osmomat 030, Gonotec) (Wucherpfennig et al., 2011).

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2.3.6. Calculation of the kinetic parameters The specific cell growth rate (l, per hour), specific polyols formation rate (qp, per hour) were estimated from experimental or fitted data of cell growth (x, g/L), and polyols production (p, g/L) by Eqs. (2)–(4), respectively (Ji et al., 2009). The fitted data were obtained by interposing between experimental data of cell growth, residual glycerol concentration or polyols production yield at definite time (dt = 0.1 h) with the approximation method of cubic spline interpolation in Origin software (Version 8.1, OriginLab Corp., Northampton, MA, USA):

l¼

1 dx 1 Dx ¼ lim x dt x Dt!0 Dt

ð2Þ

dp Dp ¼ lim dt Dt!0 Dt qp ¼

ð3Þ

1 dp 1 Dp ¼ lim x dt x Dt!0 Dt

ð4Þ

2.3.7. Method for osmotic pressure control in fed-batch fermentation The relationship between osmotic pressure and glycerol concentration was established. In the fed-batch fermentation, feeding glycerol quantitatively to maintain the osmotic pressure at a preset value was based on Eq. (5):

Y osm ¼ 0:012C gly  0:017

ð5Þ

where Yosm is osmotic pressure (osmol/kg); Cgly is glycerol concentration (g/L). 3. Results and discussion 3.1. Effects of initial glycerol concentration on erythritol production To study the effect of initial glycerol concentration on cell growth and polyols production in Y. lipolytica suspension cells, a broad range of glycerol concentration (from 50 g/L to 300 g/L) was tested (Table 1), in which the carbon/nitrogen ratio (C/N) was maintained at a constant value. Glycerol was not only the carbon source but also the osmotic agent. With increasing glycerol concentration, the osmotic pressure of the media elevated accordingly ranging from 0.68 to 4.50 osmol/kg. The maximum biomass and total polyols concentration increased and reached 16.0 and 106.1 g/L, respectively. The total polyols yield (Yp/s) elevated with the initial glycerol concentration increasing, particularly from the range of 50–250 g/L, and reached 0.57 g/g with 250 g/L of initial glycerol. The resulting Yx/s were comparable below the 200 g/L glycerol concentration reaching 0.083 g/g, presumably because the biomass was limited by the constant C/N of the media.

However, the cell mass and total polyols concentration decreased as initial glycerol concentration was higher than 200 g/L suggesting that an optimal initial glycerol concentration or osmotic pressure was required for polyols biosynthesis by the osmophilic yeast. Y. lipolytica grows well by assimilating glycerol as a sole carbon source, which has been verified (Fontanille et al., 2012). The production of various products such as erythritol, mannitol, citric acid, and a-ketoglutaric acid by Y. lipolytica with crude or raw glycerol from the biodiesel industry have been reported (Rymowicz et al., 2010; Tomaszewska et al., 2012; Yu et al., 2012). Y. lipolytica can tolerate the high concentration of glycerol and salt at low pH (Praphailong and Fleet, 1997). It is conceivable that the production of polyols resulted from the exposure of yeast to higher osmotic pressure environment generated by the high concentrations of glycerol and NaCl. In this study, Y. lipolytica CICC 1675 can produce high level of erythritol as the predominant polyol and with higher concentrations (>50% w/w in shake flasks) as well as mannitol in response to a high external osmotic environment compensating for the difference between the extracellular and intracellular water potential. 3.2. Effects of constant osmotic pressure on polyols production To separate the effect of osmotic pressure on polyol production from various substrate concentrations, a set of experiments were conducted using NaCl as the osmotic agent to adjust the initial osmotic pressure of the fermentation system to the same level. The C/N ratio was maintained at a constant level. Comparing the Yx/s and Yp/s with various initial osmotic pressure is more meaningful for the evaluation of the cell growth and polyols production. As shown in Table 2, the Yp/s was 0.54 g/g. The yield of Yx/s was maintained at about 0.060 g/g with appropriate concentrations of NaCl ranged from 25 to 75 g/L, whereas 0.084 g/g was reached without NaCl addition. The substrate concentration and salinity are both related to osmotic pressure which plays an important role in polyols fermentation (Dragosits et al., 2010). While the quantitative and stoichiometric analyses suggest that initial osmotic pressure rather than the substrate concentration is the more important factor to enhance polyol production, the ratio of erythritol to mannitol increases with the higher concentrations of NaCl in spite of the constant osmotic pressure environment. 3.3. Determination of the optimal osmotic pressure in polyols production The above experimental results indicate that certain concentration of initial glycerol was not utilized when the growth of Y. lipolytica CICC 1675 was suppressed under high substrate concentration.

Table 1 Effects of initial glycerol concentration on polyols fermentation. Parameters

Initial glycerol conc. (g/L) Residual glycerol (g/L) Mannitol (g/L) Erythritol (g/L) Total polyols (g/L) x (g/L) Yp/s (g/g) Yx/s (g/g) rE/Ma (g/g)

Initial osmotic pressure (osmol/kg) 0.68 ± 0.03

1.37 ± 0.03

2.23 ± 0.06

3.21 ± 0.05

3.89 ± 0.05

4.50 ± 0.07

50 0.83 ± 0.20 14.9 ± 1.4 2.15 ± 0.5 17.0 ± 1.8 4.22 ± 0.13 0.35 ± 0.02 0.085 ± 0.003 0.14 ± 0.02

100 0.17 ± 0.13 30.5 ± 3.5 16.8 ± 0.7 47.4 ± 2.8 8.23 ± 0.22 0.47 ± 0.03 0.082 ± 0.004 0.55 ± 0.03

150 0.10 ± 0.16 36.8 ± 3.1 41.3 ± 1.3 78.1 ± 3.6 12.4 ± 0.3 0.52 ± 0.01 0.083 ± 0.005 1.12 ± 0.03

200 10.3 ± 1.4 50.4 ± 2.6 55.7 ± 3.7 106.1 ± 4.4 16.0 ± 0.4 0.56 ± 0.02 0.084 ± 0.002 1.11 ± 0.01

250 81.4 ± 3.0 39.1 ± 4.4 56.5 ± 2.3 95.6 ± 6.1 12.5 ± 0.2 0.57 ± 0.03 0.074 ± 0.004 1.45 ± 0.04

300 131.1 ± 4.5 28.8 ± 3.3 47.6 ± 2.4 76.4 ± 5.5 8.5 ± 0.2 0.45 ± 0.01 0.050 ± 0.005 1.65 ± 0.02

Data are given as means ± standard deviations, n = 3. a rE/M: the ratio of final production of erythritol to mannitol.

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L.-B. Yang et al. / Bioresource Technology 151 (2014) 120–127 Table 2 Effects of the initial constant osmotic pressure on polyols production. Parameters

Initial osmotic pressure (osmol/kg)

Initial glycerol conc. (g/L) NaCla (g/L) Residual glycerol (g/L) Mannitol (g/L) Erythritol (g/L) Total polyols (g/L) x (g/L) Yp/s (g/g) Yx/s (g/g) rE/M (g/g)

3.20 ± 0.12

3.20 ± 0.09

3.21 ± 0.06

3.21 ± 0.05

50 75 1.69 ± 0.16 1.65 ± 0.23 25.0 ± 2.1 26.6 ± 1.6 2.95 ± 0.24 0.55 ± 0.02 0.059 ± 0.003 15.6 ± 0.2

100 50 1.84 ± 0.20 5.14 ± 0.48 47.3 ± 3.2 52.4 ± 2.7 5.94 ± 0.21 0.53 ± 0.01 0.060 ± 0.002 9.27 ± 0.25

150 25 1.89 ± 0.35 15.7 ± 1.7 64.8 ± 1.7 80.5 ± 3.8 9.46 ± 0.45 0.54 ± 0.03 0.063 ± 0.004 4.13 ± 0.12

200 0 10.3 ± 1.4 50.4 ± 2.6 55.7 ± 3.7 106.1 ± 4.4 16.0 ± 0.4 0.56 ± 0.02 0.084 ± 0.002 1.11 ± 0.06

Data are given as means ± standard deviations, n = 3. a NaCl was added at the beginning of the fermentation.

The resulting residual glycerol would complicate the downstream processing and purification of polyols. It is conceivable that appropriate concentration of salts as osmotic agents might offer the necessary osmotic stress for higher polyols yield. Consequently, complete conversion of glycerol to polyols would be attainable. This study was conducted in shake flasks containing 200 g/L of glycerol and various concentrations of NaCl as osmotic agents (Table 3). With the osmotic pressure increased from 3.21 to 4.92 osmol/kg, the biomass was reduced from 16.0 (without salt) to 7.8 g/L (with 50 g/L NaCl addition). Significant increases in total polyols yield and erythritol production have been achieved. However, mannitol production was more than 3-fold lower. The ratio of the mannitol and erythritol concentration was changed remarkably. The inhibitory effect on mannitol production was inconsistent with the reports about high glycerol concentration enhanced mannitol production (Chatzifragkou et al., 2011). The reasons might be that: (1) in erythritol fermentation, Y. lipolytica synthesized two kinds of polyol (erythritol and mannitol). The synergistic effect of the polyols could prevent damage caused by high osmotic pressure. However, in the literature, mannitol was the only polyol produced by the yeast to provide protection against osmotic stress. (2) The erythritol molecule is smaller than mannitol. The osmotic pressure generated by erythritol is much higher than mannitol at the same molarity. When exposed to high osmotic pressure, Y. lipolytica preferred to produce erythritol rather than mannitol to balance the osmotic pressure between inside and outside of the plasma membrane more effectively. In accordance with the results, high osmotic pressure induced Y. lipolytica to produce polyols while low osmotic pressure was more favorable for cell growth. Cell growth was significantly affected when salinity was higher than 30 g/L. However, mannitol

production was inhibited even at 10 g/L salinity. The Yp/s of total polyols reached to the maximum value of 0.64 g/g at the osmotic pressure of 4.17 osmol/kg but decreased with higher osmotic pressure. Thus, this osmotic pressure value was chosen for further testing. 3.4. Batch fermentation of polyols by adjusting osmotic pressures with NaCl In the 7.0 L batch fermenter, the effects of osmotic pressure on erythritol and mannitol production were examined. In both media not-supplemented and supplemented with 30 g/L NaCl were used to adjust the osmotic pressure of fermentation system to 3.21 and 4.17 osmol/kg, respectively. Time profiles of the polyol fermentation are shown in Fig. 1. The patterns of cell growth and polyols production were partially associated. All growth indices including the maximum cell mass and cell growth rates decreased as the initial osmotic pressure increased. The production and Yp/s of the total polyols at low and high initial osmotic pressure were 106.9 g/L, 0.53 g/g and 133.7 g/L, 0.68 g/g, respectively. At the initial osmotic pressure of 4.17 osmol/kg, 100.6 g/L of erythritol was obtained which was 47.0 g/L higher than that at 3.21 osmol/kg (53.6 g/L). The concentration of mannitol, the main by-product, decreased from 53.3 g/L to 33.5 g/L. The final mannitol concentration was higher than that in shake flask fermentation under the same osmotic pressure. The reason might be due to increasing biomass and consumption of glycerol as a result of better transfer of the oxygen. To investigate the effects of osmotic pressure on the relevant enzyme activities, the activities of two critical enzymes i.e., erythrose reductase (ER) and mannitol-1-phosphate dehydrogenase (M-1-PDH) involved in erythritol and mannitol biosynthesis were

Table 3 Determination of the optimal osmotic pressure in polyols batch production. Parameters

Initial osmotic pressure (osmol/kg) 3.21 ± 0.05

Initial glycerol conc. (g/L) NaCl (g/L) Residual glycerol (g/L) Mannitol (g/L) Erythritol (g/L) Total polyols (g/L) x (g/L) Yp/s (g/g) Yx/s (g/g) rE/M (g/g)

3.54 ± 0.04

3.89 ± 0.07

4.17 ± 0.12

4.6 ± 0.10

4.92 ± 0.11

10 3.1 ± 0.7 38.8 ± 2.0 78.8 ± 3.3 117.6 ± 2.8 15.5 ± 0.5 0.59 ± 0.02 0.079 ± 0.001 2.03 ± 0.10

20 11.5 ± 1.1 27.7 ± 2.3 86.7 ± 2.4 114.4 ± 3.2 11.9 ± 0.3 0.6 ± 0.01 0.063 ± 0.002 3.13 ± 0.13

30 20.5 ± 2.5 16.6 ± 1.5 98.9 ± 3.1 115.5 ± 2.4 8.8 ± 0.2 0.64 ± 0.02 0.049 ± 0.002 5.96 ± 0.09

40 34.0 ± 2.9 9.9 ± 0.8 89.4 ± 3.1 99.3 ± 2.8 7.9 ± 0.2 0.59 ± 0.02 0.048 ± 0.001 9.03 ± 0.20

50 37.6 ± 3.6 8.0 ± 0.6 87.7 ± 2.6 95.7 ± 2.0 7.8 ± 0.3 0.58 ± 0.01 0.048 ± 0.002 11.0 ± 0.1

200 0 10.3 ± 1.4 50.4 ± 2.6 55.7 ± 3.7 106.1 ± 4.4 16.0 ± 0.4 0.56 ± 0.02 0.084 ± 0.002 1.11 ± 0.06

Data are given as means ± standard deviations, n = 3.

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In addition, NADPH, generated in PPP, is the coenzyme of ER which reduces the erythrose to erythritol. There are many NADPH-dependent enzymes (e.g., glutathione reductase, thioredoxin reductase, and 3-oxoacyl reductase) which are responsible for cellular defense mechanisms against stresses (e.g., oxidative, osmotic, heat, and heavy metal stress) (Schneider et al., 1997). In this possible scenario, microorganisms generate sufficient NADPH for cell survival and maintaining the redox balance. Therefore, it is conceivable that yeast prefers to enhance the PPP activity and to diminish the EMP pathway when exposed to high osmotic pressure. Consequently, biomass and mannitol production are inhibited, while erythritol biosynthesis, via the PPP, is enhanced. Fig. 1. Effects of osmotic pressure on polyols production by Y. lipolytica with glycerol. The symbols represent: biomass (s), glycerol (h), erythritol (4), mannitol (r) in low osmotic pressure (3.21 osmol/kg); biomass (d), residual glycerol (j), erythritol (N), mannitol (.) in high osmotic pressure (4.17 osmol/kg). Each point represents the mean (n = 3) ± standard deviation.

assayed (data was not shown). Intracellular crude extract was prepared from the Y. lipolytica CICC 1675 cells at 48 h of fermentation. The specific activity of ER was elevated at high osmotic pressure, which was almost twice higher than that at low osmotic pressure. However, the specific activity of M-1-PDH exhibited the opposite trend. The result is quite similar to that the expression of ER in Candida magnoliae was up regulated under the conditions of osmotic and salt stress generated by a high concentration of sugar, KCl, and NaCl (Park et al., 2011). There were few reports about M-1-PDH activity affected by osmotic pressure or salinity, but Iwamoto et al. (2003) found that Km of M-1-PDH increased eight times by the addition of 200 mmol/L NaCl in the red alga Caloglossa continua. It is clear that the erythritol and mannitol distribution in the polyols mixture were highly dependent on the osmotic pressure. Notable increase in erythritol production and a reduction in mannitol production were observed with the initial osmotic pressure increasing. The reasons were postulated as: Cell growth associated with mannitol production was inhibited by high osmotic pressure, which has been demonstrated in Torulopsis versatilis (Onishi and Suzuki, 1968). However, erythritol was mainly accumulated after the exponential phase. There was no direct relationship between the biomass and maximum erythritol concentration. The reason was that the cell division and expansion were inhibited under osmotic stress, leading to increase in the specific carbohydrate concentration per cell unit and increase cellular lifespan, which reduce the metabolic energy level of primary metabolism and shift the energy to secondary metabolite production (Kim et al., 2001). On the other hand, the flux of the carbon source in biosynthetic pathways of polyols was changed. When exposed to high osmotic stress, microorganisms induced many molecular, biochemical and physiological changes, known as ‘‘osmotic stress response’’ (Blomberg and Adler, 1992). In yeasts, the most pronounced response was enhanced intracellular accumulation of polyhydroxy alcohols such as glycerol, erythritol, mannitol to maintain cytoplasmic water activity at an equal level with the surrounding environment (Lages et al., 1999). Significant increase of erythritol production can be explained that the yeasts need more erythritol to counteract the outflow of water from intracellular to extracellular under high osmotic stress. Consequently, more carbon sources flowed to the pentose phosphate pathway (PPP) in which erythritol was generated rather than the Embden–Meyerhof–Parnas (EMP) pathway in which mainly mannitol was produced.

3.5. Fed-batch fermentation with constant osmotic pressure at the exponential phase In accordance with the above experimental results, high initial osmotic pressure benefited the biosynthesis of erythritol and reduced mannitol production. However, later on during the fermentation, osmotic pressure dropped with the consumption of glycerol. To determine if erythritol production would be enhanced with stabilized osmotic pressure during cell growth phase, a set of fed-batch fermentations with controlled osmotic pressure were carried out. In these experiments, the decreasing trend of osmotic pressure was continuous and predictable, and different value of osmotic pressure was detectable. Glycerol was used not only as carbon source but also as osmotic regulator, added quantitatively into the culture broth at 6–12 h time interval to maintain the osmotic pressure at the preset constant value (3.29, 4.26 and 4.96 osmol/ kg) until the end of the exponential phase. The amount of feeding glycerol was calculated based on Eq. (5). Time profiles of the fermentations at various osmotic pressures are shown in Fig. 2A–C. The results indicated that osmotic pressure played a vital role in polyols production. With increasing osmotic pressure, relatively higher final erythritol concentrations up to 154.6 g/L was obtained at the osmotic pressure of 4.96 osmol/kg, while 133.7 g/L and 92.5 g/L erythritol were obtained at 4.26 osmol/kg and 3.29 osmol/kg, respectively. As the fermentation time was very long (192 h), the productivity of erythritol at 4.96 osmol/kg was the lowest (0.81 g/L/h). However, the highest value (0.93 g/L/h) was achieved at 4.26 osmol/kg of osmotic pressure. To analyze the kinetics of the fed-batch fermentations, three parameters, l, qery and dpery/dt as shown in Fig. 2D–F, were calculated based on the data in Fig. 2A–C. The l almost had the same values at the osmotic pressure of 3.29 osmol/kg and 4.26 osmol/kg. The results indicated no inhibitory effect on cell proliferation under the osmotic pressure of 4.26 osmol/kg. In Fig. 2E, qery had similar tendency and increased with higher osmotic pressure. All of the maximum values appeared at the same time (almost at 8 h). Consequently, higher osmotic pressure was better for erythritol production. However, cell growth was inhibited under extremely high osmotic pressure (4.96 osmol/kg), which led to the decrease of erythritol productivity. Therefore, dpery/dt was introduced to estimate the rate of erythritol formation. As shown in Fig. 2F, it was better to maintain a high erythritol formation rate at the initial osmotic pressure of 4.26 osmol/ kg and reached the highest value of 1.05 g/L/h, subsequently dropped down because of the decrease of osmotic pressure. However, after 132 h, the osmotic pressure of 4.96 osmol/kg was beneficial for erythritol production with a high value of dpery/dt. It is concluded that higher concentration, yield and productivity of erythritol could not be obtained simultaneously by maintaining appropriately high osmotic pressure throughout the entire fermentation process.

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Fig. 2. A–C: Time profiles of biomass, glycerol, erythritol, mannitol for Y. lipolytica at different initial osmotic pressure. (A) 3.29 osmol/kg; (B) 4.26 osmol/kg; (C) 4.96 osmol/ kg. The symbols represented: biomass (s), glycerol (h), erythritol (N), mannitol (.), and osmotic pressure (—). D–F: Comparison of the kinetic parameters in erythritol fermentation by Y. lipolytica at different initial osmotic pressure. 3.29 osmol/kg (dashed line); 4.26 osmol/kg (solid line); 4.96 osmol/kg (dashed and dot line). (D) Specific cell growth rate (l); (E) specific erythritol formation rate (qery); and (F) erythritol formation rate (dpery/dt). Each point represents the mean (n = 3) ± standard deviation.

3.6. Two-stage osmotic pressure control strategy for higher erythritol productivity Based on the analysis of l, qery and dpery/dt, a proper two-stage osmotic pressure control fed-batch fermentation strategy was proposed from the kinetic stand point. In the stage I, the osmotic pressure was maintained at 4.25 osmol/kg by feeding glycerol in the first 96 h, and then, the fermentation process was continued without feeding glycerol until 132 h. Subsequently, in the stage II, the osmotic pressure was switched to 4.94 osmol/kg until the end of the fermentation. The time course of this strategy for erythritol production is shown in Fig. 3. The relatively low osmotic pressure resulted in rapid cell growth and glycerol consumption. The

increase of osmotic pressure generated by feeding glycerol promoted erythritol biosynthesis with a high productivity. Ultimately, the maximum concentration of erythritol reached 194.3 g/L representing erythritol productivity of 0.95 g/L/h, and 25.7% and 2.2% improvements in concentration and productivity of erythritol were achieved over the best result with the one-stage osmotic pressure control strategy (Table 4). This is the highest value in erythritol production from glycerol comparing with that in published reports (Rymowicz et al., 2009; Tomaszewska et al., 2012). Although the fermentation time was long, the erythritol productivity was comparable. Furthermore, the resulting by-product, mannitol, was produced at a low level (36.8 g/L). This strategy was successfully proven to enhance erythritol production significantly.

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Table 4 Comparison of parameters in fed-batch production of erythritol by Y. lipolytica under various osmotic pressure control strategies. Parameters

Dry cell weight (g/L) Erythritol (g/L) Mannitol (g/L) Erythritol productivity (g/L/h) Erythritol yield (g/g) Fermentation time (h)b a b

Increment (%)a

Initial osmotic pressure (osmol/kg) 3.29 ± 0.05

4.26 ± 0.04

4.96 ± 0.07

4.25 ± 0.05 (0–96 h), 4.94 ± 0.13 (after 132 h)

27.0 ± 1.2 92.5 ± 3.4 41.2 ± 3.6 0.77 ± 0.03 0.35 ± 0.02 120

24.0 ± 1.8 133.7 ± 4.8 30.2 ± 2.0 0.93 ± 0.02 0.50 ± 0.03 144

18.9 ± 1.9 154.6 ± 5.1 25.1 ± 4.8 0.81 ± 0.03 0.56 ± 0.02 192

23.6 ± 2.5 194.3 ± 5.5 36.8 ± 5.8 0.95 ± 0.03 0.49 ± 0.02 204

– 25.7 10.7 2.2 – –

Compared with the best result controlled by single-osmotic pressure. Fermentation time was defined as the time when the glycerol was exhausted.

The higher osmotic pressure and the lower pH (3.0) have many advantages in erythritol production. It reduces by-products formation and the need for pH neutralizing agents such as NaOH which reduces cost of downstream processing and alleviate environmental pollution. Furthermore, high osmotic pressure could protect Y. lipolytica from other contaminating microorganisms, which is a significant advantage for industrial fermentation production. 4. Conclusion

Fig. 3. Time profiles of biomass, glycerol, erythritol, mannitol formation by Y. lipolytica with glycerol as carbon source using the two-stage osmotic pressure control strategy. The symbols represented: biomass (s), glycerol (h), erythritol (N), mannitol (.), and osmotic pressure (—). Each point represents the mean (n = 5) ± standard deviation.

Glycerol has been shown to be the better carbon source for erythritol production than glucose under the same fermentation condition in Y. lipolytica (Rymowicz et al., 2009). However, the regulation of the biosynthesis of erythritol and mannitol from glycerol is currently not well understood (Rywinska et al., 2009). The possible routes are summarized in Fig. 4 (Supplementary data). Erythritol and mannitol are produced through different biosynthetic pathways. As for glycerol, the metabolic pathway is initiated by glycerol kinase and a mitochondrial glycerol-3-phosphate dehydrogenase in yeast such as Saccharomyces cerevisiae (Gancedo et al., 1968). Dihydroxyacetone phosphate is formed which is converted to glyceraldehydes-3-phosphate and, subsequently via the gluconeogenesis pathway to fructose-6-phosphate. For mannitol production, fructose-6-phosphate is converted to mannitol-1phosphate by M-1-PDH which is the key enzyme for mannitol biosynthesis. For erythritol production, glucose-6-phosphate, generated by the gluconeogenesis pathway, is converted to ribulose-5-phosphate via the pentose phosphate pathway. Ribulose-5-phosphate is considered to be an important precursor for producing polyols such as arabitol, xylitol, and erythritol (Bernard et al., 1981). It is then converted to ribose-5-phosphate and xylulose-5-phosphate. Sedoheptulose-7-phospate and glyceraldehydes-3-phospate are generated by the transketolase. Sawada et al. (2009) proved that high transketolase activity is essential to produce abundant intermediates which may be a critical requirement for high erythritol productivity in Trichosporonoides megachiliensis. Erythritol is obtained from D-erythrose-4-phospthate after dephosphorylation and reduction reactions with NADPH. Therefore, good erythritol yields from such process requires tightly controlled high intracellular NAD(P)H/NAD+ ratio.

A two-stage osmotic pressure control fed-batch fermentation process was developed for efficient erythritol fermentation production by Y. lipolytica. Glycerol was not only the substrate but also the osmotic regulator. The maximum erythritol production yield reached 194.3 g/L with the productivity of 0.95 g/L/h. The results indicate that osmotic pressure was successfully used as a controlling parameter to manipulate the culture environment for polyols production. This novel strategy conceivably can be applied to the other industrial biotechnological process to achieve high product concentration, low by-products formation and high productivity simultaneously. Acknowledgements This work was supported by the National Natural Science Foundation of China (31171640, 31301408), National High-tech R&D Program of China (2012AA021505, 2012AA021201), National Key Technologies R&D Program of China (2011BAD23B04), and Program for the Wuxi Bio-Agriculture Entrepreneurial Leader (130 Plan). The authors would like to thank these organizations for financial supports. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013.10. 031. References Andre, A., Chatzifragkou, A., Diamantopoulou, P., Sarris, D., Philippoussis, A., Galiotou-Panayotou, M., Komaitis, M., Papanikolaou, S., 2009. Biotechnological conversions of bio-diesel-derived crude glycerol by Yarrowia lipolytica strains. Eng. Life Sci. 9, 468–478. Bernard, E.M., Christiansen, K.J., Tsang, S., Kiehn, T.E., Armstrong, D., 1981. Rate of arabinitol production by pathogenic yeast species. J. Clin. Microbiol. 14, 189– 194. Blomberg, A., Adler, L., 1992. Physiology of osmotolerance in fungi. Adv. Microb. Physiol. 33, 145–212. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Chatzifragkou, A., Makri, A., Belka, A., Bellou, S., Mavrou, M., Mastoridou, M., Mystrioti, P., Onjaro, G., Aggelis, G., Papanikolaou, S., 2011. Biotechnological conversions of biodiesel derived waste glycerol by yeast and fungal species. Energy 36, 1097–1108.

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