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α, ω-Dodecanedioic acid production by Candida viswanathii ipe-1 with co-utilization of wheat straw hydrolysates and n-dodecane
Bioresource Technology 243 (2017) 179–187
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a, x-Dodecanedioic acid production by Candida viswanathii ipe-1 with co-utilization of wheat straw hydrolysates and n-dodecane Weifeng Cao a, Bin Liu b, Jianquan Luo a, Junxiang Yin c, Yinhua Wan a,⇑ a
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China College of Food Science and Engineering, Qilu University of Technology, Jinan 250353, PR China c China National Center for Biotechnology Development, Beijing 100036, PR China b
h i g h l i g h t s Co-utilization of wheat straw hydrolysates and n-dodecane improved DC12 production. Glucose, xylose and sodium acetate are simultaneously assimilated by C. viswanathii. DC12 production can be increased by the addition of sodium acetate. Detoxification of the wheat straw hydrolysates is unnecessary for DC12 production.
a r t i c l e
i n f o
Article history: Received 17 April 2017 Received in revised form 13 June 2017 Accepted 14 June 2017 Available online 17 June 2017 Keywords: a, x-Dodecanedioic acid Candida viswanathii Wheat straw n-Dodecane
a b s t r a c t Candida viswanathii ipe-1 was used to produce a, x-dodecanedioic acid (DC12), which showed capability to ferment xylose and glucose simultaneously, while arabinose utilization was less efficient. A low concentration of furfural enhanced cell growth, and the addition of 4.0 g/L sodium acetate largely increased DC12 production. It indicated that detoxification of the wheat straw hydrolysates was not necessary for the biosynthesis of DC12. Based on the promising features of our strain, an efficient process was developed to produce DC12 from co-utilization of wheat straw hydrolysates and n-dodecane. Using this process, 129.7 g/L DC12 with a corresponding productivity of 1.13 gL1h1 could be produced, which was increased by 40.0% compared with a sole carbon of glucose. The improved DC12 yield by the coutilization of wheat straw hydrolysates and n-dodecane using C. viswanathii ipe-1 demonstrates the great potential of using biomass as a feedstock in the production of DC12. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
a, x-Dicarboxylic acids (DC) are versatile chemical intermediates with different chain length, generally represented as HOOC(R)n-COOH. These chemical compounds are used as raw materials for the preparation of perfumes, polymers, adhesives and macrolide antibiotics for many years and are well known polymer building block (Sailakshmi et al., 2013), and they are also considered as a promising alternative energy substrate for metabolism (Mingrone et al., 2013). DC can be produced in two ways: chemical synthesis and fermentation. The fermentation process is attractive because of its advantages of cost-efficient and greener process alternative. Nowadays there is a huge variety of microorganisms described for conversion of n-alkanes to the corresponding DC by utilizing hydrocarbons as carbon and energy source. Many organisms could ⇑ Corresponding author. E-mail address: [email protected] (Y. Wan). http://dx.doi.org/10.1016/j.biortech.2017.06.082 0960-8524/Ó 2017 Elsevier Ltd. All rights reserved.
achieve this transformation, including Cryptococus neoformans and Pseudomonas aeruginosa (Chan et al., 1991), Corynebacterium sp. (Broadway et al., 1992), Yarrowia lipolytica (Smit et al., 2005) as well as at least two strains of Candida, C. cloacae (Shiio and Uchio, 1971) and C. tropicalis (Liu et al., 2004; Yi and Rehm, 1982). The bioprocess engineering to enhance the production of DC could be divided into two steps. The first step included the growth of microorganisms with an easy utilizable carbon source to get high biomass concentrations. The second step was the DC production stage with adjusting pH to pH 8.0 and/or adding alkane based on requirements (Jiao et al., 2001; Liu et al., 2004). Usually, sucrose (Cao et al., 2006) and glucose (Picataggio et al., 1992) were used as the easy utilizable carbon source in the first step. Actually, abundant and renewable lignocellulosic materials could be used as suitable feedstocks for the production of lactic acid (Zhang et al., 2014), ethanol (Qi et al., 2010) and succinic acid (Zhao et al., 2016). Mixture of hexose and pentose sugars can be obtained by hydrolyzing lignocellulose. It was reported that Candida shehatae possessed a
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relaxed carbon catabolite repression mechanism, showing simultaneous consumption of glucose and xylose (Kim et al., 2010). Candida auringiensis, Candida succiphila and Candida sp. (YB-2248) possessed the ability to ferment L-arabinose while they could ferment D-xylose better (Dien et al., 1996). Candida viswanathii Y-E4 strain was able to use a wide range of substrates, especially C5 and C6 sugars as well as glycerol and hydrophobic substrates (Ayadi et al., 2016). Therefore, Candida may be capable of fermenting all the lignocellulose- released sugars for the economical production of DC. Usually, lignocellulosic hydrolysates obtained by sulfate pretreatment contained furans, acetate, and sulfuric acid (Palmqvist and Hahn-Hägerdal, 2000), and furfural and acetic acid are the main two inhibitors that retard microbial fermentation (Qi et al., 2010). However, high DC-producing mutants could achieve highlevel DC production by subterminal, terminal or diterminal oxidation of carbon atoms in the alkanes or fatty acids (Huf et al., 2011; Scheller et al., 1998). Therefore, the inhibitors in lignocellulosic hydrolysates may be not harmful to the DC production because DC producers may oxidate the inhibitors and further utilize them for cell growth. Among the DC, dodecanedioic acid (DC12) usually serves as a monomer of polyamides and some special nylons such as nylon 612 and nylon 1212 (Zheng et al., 2004). DC12 is also of interest as potential alternate fuel substrates in parenteral nutrition (Salinari et al., 2006). With growing demand for DC12, it is required to produce DC12 with low cost and large yield, and DC12 production with combined renewable lignocellulosic materials and n-dodecane may be a good choice. In the study reported here, an efficient process would be established to produce DC12 via co-utilization of wheat straw hydrolysates and n-dodecane by Candida viswanathii ipe-1. Effects of the main inhibitors from wheat straw hydrolysates on DC12 production would also be detected. 2. Materials and methods 2.1. Microorganism C. viswanathii ipe-1 (CGMCC No. 8824) used in this study was stored in China General Microbiological Culture Collection Center, Beijing, China. The strain was maintained on malt extract agar (MEA) (Beijing Aoboxing Bio-tech Co. LTD, China) slant at 4 °C. 2.2. Culture medium The seed culture medium contained 50 mL/L n-alkane, 8.0 g/L KH2PO4, 5.0 g/L yeast extract (Beijing Aoboxing Bio-tech Co. LTD, China), 1.5 g/L dry powder of corn steep liquor, 30.0 g/L sucrose and 3.0 g/L urea in deionized water. For the production of DC12 in the 7.5 L bioreactor (BioFioÒ110), the following fermentation medium was used: 8.0 g/L KH2PO4, 4.0 g/L yeast extract (Beijing Aoboxing Bio-tech Co. LTD, China), 1.5 g/L dry powder of corn steep liquor, 60.0 g/L sucrose, 4.0 g/L sodium acetate, 3.0 g/L KNO3, 1.0 g/ L NaCl, 0.5 g/L Twain 60 and 2.0 g/L urea in deionized water. After sterilization, 400 mL (i.e. 299.5 g) sterile n-dodecane was added to per liter fermentation medium. 2.3. Culture method In all fermentation experiments, the seed culture of the strain was prepared by inoculating cells grown on malt-extract agar into 500 mL Erlenmeyer flasks containing 100 mL of seed culture medium, followed by incubation at 30 °C for 2 days in the rotary shaker (HYG-A, Taicang Experimental Equipment Factory, China) at 180 rpm. The seed prepared for the bioreactor was cultured in four
Erlenmeyer flasks each time. After culturing and mixing the seed culture broth, 200 mL of the mixture was transferred to a 2.7 L bioreactor (BioFioÒ110, NewBrunswick Scientific, USA) containing 1.0 L of the fermentation culture medium. The temperature, stirring speed and the aeration rate in the bioreactor were maintained at 30 °C, 800 rpm and 2 L/min, respectively. The fermentation process was divided into growth phase and conversion production phase. pH in culture medium was maintained at 5.0 for 18 h during the growth phase, and then raised to pH 8.0 during the conversion phase. Medium pH was automatically maintained at set points by the addition of 8 M NaOH and 5 M H2SO4. To investigate the utilization of sugars by C. viswanathii ipe-1, 60 g/L of glucose, sucrose, xylose or arabinose were sterilized separately, then respectively added to the sterilized fermentation culture medium. To investigate the effect of sodium acetate or furfural on the production of DC12, different concentrations of sodium acetate or furfural were sterilized separately, then added to the sterilized fermentation culture medium. The final fermentation volume before inoculation was 1.4 L. All fermentation experiments were performed at least twice, to ensure the trends observed were reproducible. The data presented in the figures are the average values with error bars. 2.4. Preparation of wheat straw hydrolysates The conditions for preparation of wheat straw hydrolysates were similar to those described elsewhere (Qi et al., 2010; Zhang et al., 2014). Briefly, wheat straw was cleaned, chopped, and then pretreated by 2% (w/v) sulfuric acid at a 10% (w/v) loading. The mixture was treated at 121 °C for 90 min, and the obtained slurry was filtrated to achieve liquid and solid fractions. Both liquid and solid fractions were collected and defined as dilute acid hydrolysates and water insoluble solids (WIS), respectively. The dilute acid hydrolysates were alkalified with calcium hydroxide to pH 10.0, and then the mixture was filtered with filter paper (No. 43, Whatman, UK). The filtrate was then acidified to pH 5.0 with sulfuric acid, followed by filtration with filter paper. The final filtrate was concentrated to a mixture containing 10.6 g/L sodium acetate, 33.1 g/L xylose, 1.79 g/L glucose, 6.12 g/L arabinose, 0.058 g/L HMF, 0.25 g/L furfural and 0.48 g/L total phenolics by vacuum evaporation at 60 °C, and the mixture was named as concentrated acid hydrolysates (CAH). For production of DC12 only with CAH, CAH was further concentrated to a mixture containing 101.2 g/L mixed sugars. The WIS (containing 59.96% cellulose) was hydrolyzed by commercial cellulase (Sunson Group Ningxia Enzyme Preparation Plant, China) at a 10% (w/v) solid loading. Enzyme loading was 20 FPU (filter paper activity units)/g cellulose. The enzymatic hydrolysis was carried out in a 7.5 L jar fermentor (BioFioÒ110, NewBrunswick Scientific, USA) at 50 °C, pH 5.0 and 200 rpm. Enzymatic hydrolysates were obtained by centrifuging at 5000g for 15 min. And then the obtained supernatant was concentrated by vacuum evaporation at 60 °C to glucose concentration of 92.0 g/L, and the mixture was named as concentrated enzymatic hydrolysates (CZH). Then the wheat straw hydrolysates were stored at 4 °C prior to use. 2.5. Analytical methods Biomass and DC12 concentrations were measured as described by Liu et al. (2004). Cell growth was determined by measuring of the dry weight of the cells. Samples (5 mL) were taken at regular intervals and centrifuged at 8000 rpm (6869g) for 10 min. The sediment was washed for three times with distilled water and dried at 90 °C. The dry residue was designated as biomass. DC12 concentrations in undiluted samples were measured by titration. A sample of 10 mL in a sealed tube, adjusted to pH 10 with several grains of NaOH, was boiled for 5 min to completely convert DC into sodium
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salts. After centrifugation at 4000 rpm (1717g) for 10 min at room temperature, a supernatant of 2 mL, diluted with 30 mL distilled water, was acidified to pH 2–3 with 2 M H2SO4 and boiled for 0.5 min to precipitate DC. After cooling to room temperature, the mixture was filtered through a filtration paper and washed with distilled water until the inorganic acid was fully removed. Then the precipitated DC was dissolved in 30 mL warmed ethanol (90%, v/v). DC concentration was determined by titration with 0.2 M NaOH as standard solution. Sucrose concentrations were determined by the phenol sulfuric acid method (Dubois et al., 1956). The concentrations of sodium acetate, glucose, xylose and arabinose were measured by a HPLC apparatus (Shimadzu LC20AT) equipped with an Aminex HP-X87H Ion Exclusion column using refractive index detector (RI). The process was performed at a temperature of 40 °C, a flow rate of 0.6 mL/min with 5 mmol/L H2SO4 as moving phase. 3. Results and discussion 3.1. Utilization of carbon sources by C. viswanathii ipe-1 for DC12 production As shown in Fig. 1, the C. viswanathii ipe-1 could assimilate ndodecane as sole carbon source for cell growth (9.6 g/L) and DC12 production (43.7 g/L). When an easily utilizable carbon source, such as sucrose, glucose or xylose, was added in the broth, the cells concentration and DC12 production increased significantly. However, compared with the pentose xylose, the fermentation kinetic was different when another pentose L-arabinose was used as carbon source. With the addition of L-arabinose, the cell growth increased by 24.0% with a decreasing DC12 production by 17.8% compared without exogenous carbon source addition. The utilizable performance of glucose, xylose and arabinose as substrates was similar to the biolipid production by C. viswanathii Y-E4 (Ayadi et al., 2016). Simultaneous assimilation of glucose and xylose was important to the utilization of lignocellulosic biomass and other raw materials as the carbon sources, in order to reduce the costs of microbial DC12 production. However, only 5.0 g/L Larabinose was consumed before 42 h when 60.0 g/L of sucrose, glucose or xylose was depleted. A large amount of residual arabinose in the broth after 42 h may be responsible to the decrease of DC12 production with the addition of 60 g/L arabinose case. Therefore, if the L-arabinose added could be depleted in the same period as other sugars, the DC12 production may be not affected. This assumption would be discussed in the next section. 3.2. Effect of mixed sugars on DC12 production It could be concluded that C. viswanathii ipe-1 could ferment xylose and glucose simultaneously, being better than L-arabinose (Fig. 1). In addition, the concentration of arabinose in wheat straw hydrolysates with dilute sulfuric acid pretreatment was low (i.e. 3.8 g arabinose/100 g wheat straw with 2% (w/v) sulfuric acid pretreatment) (Qi et al., 2010), while the concentration of xylose was 22.6 g/L. Therefore, the strain ipe-1 may be capable of fermenting all the lignocellulose-released sugars for the economical production of DC12. When the total sugar concentration was about 60 g/ L, a ratio of 29.7 g/L glucose, 24.7 g/L xylose and 5.1 g/L arabinose could be obtain from hydrolysates of wheat straw (Zhang et al., 2014). In order to evaluate the fermentation performance of C. viswanathii ipe-1 with the sugars from wheat straw, the modeling solution with the same sugars ratio as in the straw hydrolysates (Zhang et al., 2014) was used. Kinetics of DC12 production under different mixed sugar concentrations were described in Fig. 2. During the fermentations of 30, 45, and 60 g/L mixed sugars, the
Fig. 1. Utilization of carbon sources by C. viswanathii ipe-1 for DC12 production. The time courses refer to the effect of sugars on DC12 (A) and biomass (B), and the variation of sugars during the fermentation (C). Data are given as mean ± SD, n = 3.
obtained DC12 concentration were 52.9, 83.7 and 87.3 g/L, respectively. And the corresponding cell mass were 12.1, 15.5 and 16.8 g/L, respectively. The result showed that C. viswanathii ipe-1 could co-ferment the mixed sugar to DC12. The mechanism of mixed sugar utilization by Candida viswanathii ipe-1 may be described as follows: the strain ipe-1 has an ability of relaxed carbon catabolite repression and can simultaneously consume the mixed sugars, which was similar with the strain Candida shehatae (Kim et al., 2010). The glucose and xylose were assimilated simultaneously, while the metabolic rate of the strain for glucose was a
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Fig. 2. Kinetics of DC12 production with mixed sugars of glucose, xylose and arabinose under 4.0 g/L sodium acetate. The time courses refer to the mixed sugars of 30 g/L (A) and 45 g/L (B) and 60 g/L (C). The ratio of the sugars was the same as that in the straw hydrolysates (i.e. 29.72 g/L glucose: 24.69 g/L xylose: 5.14 g/L arabinose) reported by Zhang et al. (2014). Data are given as mean ± SD, n = 3.
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little faster than that for xylose. After glucose or xylose was depleted, arabinose began to be consumed for DC12 production. During the fermentation of 45 g/L mixed sugars, the residual arabinose concentration was 1.1 g/L at 42 h. Compared with the fermentation with 30 g/L mixed sugars, the DC12 production increased by 58% with 45 g/L mixed sugars (50% higher sugar concentration), implying that such low residual arabinose (1.1 g/L) may not affect DC12 production. Therefore, if the L-arabinose added could be almost depleted in the same period as other sugars, the DC12 production are not affected. However, during the fermentation of 60 g/ L mixed sugars, the residual arabinose concentration was 4.1 g/L at 42 h and only 1.0 g/L arabinose was metabolized in the first 42 h cultivation (Fig. 2C). The little improvement in DC12 production with increasing mixed sugars from 45 g/L to 60 g/L was possibly caused by the high concentration of residual arabinose (4.1 g/L) in the broth. Actually, only a few oleaginous types of yeast such as Y. lipolytica, C. tropicalis and L. starkey could use hydrophobic substrates via an special anabolic pathway (Beopoulos and Nicaud, 2012). C. viswanathii ipe-1 is probably one of them, because it has a specific mechanism for n-dodecane transport and DC12 production. When a high concentration of other carbon source exists in the conversion phase, the pathway for biosynthesis of DC12 will be disturbed.
3.3. Tolerance to the main hydrolysates inhibitors As described by Qi et al. (2010), acetic acid and furfural were the two main inhibitors in the dilute acid hydrolysates. Therefore, acetic acid and furfural were selected to test the susceptibility of C. viswanathii ipe-1 to the inhibitors. The results are showed in Tables 1 and 2, where the inhibitors were added at the initial stage of fermentation. When 0.5 g/L furfural was added in the broth, cell mass increased in the growth phase and reached 17.3 g/L at 18 h (Table 1). The cell mass in the final fermentation (114 h) also increased when 0.5 g/L furfural was added in the broth, but no further increase of cell mass was observed when the concentration of furfural was above 0.5 g/L. The inhibition effect of furfural on cell growth became significant when the concentration of furfural was above 0.5 g/L, while the inhibition effect of furfural on DC12 production was negligible at the furfural concentrations from 0.5 to 1.25 g/L. When the concentration of furfural reached 4.0 g/L, the cell growth was completely inhibited in the growth phase without glucose consumption (Fig. 3A). After entering into the conversion production phase, the cell growth was recovered and the cell mass reached its maximal level of 20.3 g/L at 90 h, however only 26.2 g/L of DC12 was obtained. The repressed DC12 production may be caused by the low cell mass at the beginning of the conversion production phase. In order to clarify the inhibition mechanism of furfural, 4.0 g/L of furfural was added at the beginning of the conversion production phase at 18 h after obtaining 13.2 g/L of cell mass (Fig. 3B). At the end of fermentation, 22.4 g/L of DC12 and 14.7 g/L of cell mass were obtained, which was respectively
Table 1 Effect of furfural on the cell growth and DC12 production with the addition of 4.0 g/L sodium acetate.a Furfural (g/L)
Biomass18h (g/L)
Biomass114h (g/L)
DC12
0 0.5 1.25 2.0 3.0 4.0
14.6 ± 0.83 17.3 ± 0.97 14.0 ± 0.79 13.0 ± 0.81 5.9 ± 0.42 1.5 ± 0.08
14.1 ± 0.92 17.6 ± 0.83 17.8 ± 0.91 17.9 ± 0.74 16.9 ± 0.71 18.6 ± 0.87
92.6 ± 3.97 93.5 ± 4.01 90.6 ± 3.75 75.4 ± 2.63 54.4 ± 2.04 26.2 ± 1.13
114h
(g/L)
a Growth phase was ended at 18 h, and fermentation was ended at 114 h. Data are given as mean ± SD, n = 3.
Table 2 Effect of acetate on the cell growth and DC12 production without furfural addition.a
a
Sodium acetate (g/L)
Biomass18h (g/L)
Biomass114h (g/L)
DC12
0 2.0 4.0 7.0 10.0
12.9 ± 0.87 13.5 ± 0.92 14.6 ± 0.83 14.9 ± 0.78 15.4 ± 0.98
16.5 ± 0.91 15.9 ± 0.85 14.1 ± 0.92 16.4 ± 0.73 16.9 ± 0.94
55.8 ± 2.84 63.8 ± 2.81 92.6 ± 3.97 66.7 ± 3.12 63.9 ± 3.06
114h
(g/L)
Data are given as mean ± SD, n = 3.
decreased by 14.5% and 21.0% compared with that in Fig. 3A. The results indicated that the furfural with high concentration inhibited not only cell growth but also DC12 production. However, furfural may be not a problem for DC12 production using lignocellulose hydrolysates because the concentration of furfural was low (0.7 g/L) with the method as described by Qi et al. (2010), and detoxification of dilute acid hydrolysates of lignocellulose was extensively reported (Jönsson and Martín, 2016). Detoxification of dilute acid hydrolysates of lignocellulose with lime was optimized and total furans and phenolic compounds were largely reduced with overliming, while acetic acid levels were unchanged (Martinez et al., 2001). Microbial oil was also successfully produced by oleaginous yeasts using the hydrolysate, which was obtained from wheat straw treated by dilute sulfuric acid and following calcium hydroxide pretreatment (Yu et al., 2011). Therefore, the dilute acid hydrolysates of lignocellulose could be detoxified with lime to avoid the negative effects of furfural on DC12 production (see Table 3). As listed in Table 2, sodium acetate showed a significant positive effect on cell growth and DC12 production with strain ipe-1 when the concentration of sodium acetate added was below 4.0 g/L. However, when the concentration of sodium acetate exceeded 4.0 g/L, DC12 production was strongly inhibited. The same phenomenon was also reported for ethanol production by S. cerevisiae (Palmqvist and Hahn-Hägerdal, 2000). As shown in Table 2, the highest DC12 production of 92.6 g/L with 14.6 g/L cell mass was obtained when 4.0 g/L sodium acetate was added at the initial stage of fermentation. However, as seen in Fig. 3C, when 4.0 g/L sodium acetate was added at the beginning of conversion production phase (no sodium acetate addition at the initial stage of fermentation), DC12 production was decreased by 10.9% compared with control experiment (no addition of sodium acetate during the whole fermentation). Therefore, the high residual sodium acetate in the conversion production phase resulted in a decreasing production of DC12, while this negative effect was not observed in the case with 4.0 g/L sodium acetate addition at the initial stage of fermentation. Moreover, it had been confirmed that oxygen atoms derived from molecular oxygen for inserting into two terminals of alkane was a key step for DC biosynthesis (Ji et al., 2013), and the molecular oxygen could enter into the cytosol through the respiration of the strain ipe-1. The respiration rate could be enhanced by the extracellular low acetate concentration (Hueting and Tempest, 1977). Under pH 5.0, undissociated acetic acid is liposoluble and can diffuse across the plasma membrane into cytosol (Palmqvist and Hahn-Hägerdal, 2000). Therefore, adding low concentration acetate could improve DC12 production. 3.4. DC12 production by co-utilization of wheat straw hydrolysate and n-dodecane Based on the promising features of strain ipe-1, an efficient process was developed to produce DC12 by co-utilization of wheat straw hydrolysate and n-dodecane. Wheat straw hydrolysate was used as an easily utilizable carbon source to get high biomass concentrations in the growth phase, and n-dodecane was used as the
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Fig. 3. Effect of furfural and acetate on cell growth and DC12 production under different adding time. The time courses refer to 4.0 g/L furfural added at the initial culture time (A), 4.0 g/L furfural added at 18 h (B) and 4.0 g/L sodium acetate added at 18 h (C). Data are given as mean ± SD, n = 3.
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W. Cao et al. / Bioresource Technology 243 (2017) 179–187 Table 3 Comparison of DC12 production with different substrates by different strains. Cmax: maximal product concentration, Rmax: maximal production rate of DC12. Substrate Dodecane, Dodecane Dodecane, Dodecane, Dodecane, Dodecane,
Glucose Sucrose Glucose 5-aminolevulinic acid, thiourea wheat straw hydrolysates
Cmax (g/L)
Rmax g/(Lh)
Strain
Ref.
140 0.3 166 8 0.159 129.7
0.9 0.0025 1.38 0.056 0.013 1.13
C. tropicalis H5343 Corynebacterium sp. ATCC19067 C. tropicalis CGMCC 356 Y. lipolytica MTLY37 E. coli REx C. viswanathii ipe-1
Picataggio et al. (1992) Broadway et al. (1993) Liu et al. (2004) Smit et al. (2005) Sathesh-Prabu and Lee (2015) This study
substance to biosynthesis DC12 by the strain ipe-1 in the conversion production phase. Wheat straw hydrolysate was prepared as described in Section 2.4. Kinetics of DC12 production with 45.0 g/ L total sugars mixed with 302 mL concentrated acidic hydrolysates (CAH) and 373 mL concentrated enzymatic hydrolysates (CZH) (Fig. 4A) and 60.0 g/L total sugars mixed with 403 mL CAH and 497 mL CZH (Fig. 4B) are showed in Fig. 4. For the fermentation of 45 g/L mixed sugars (Fig. 4A), a cell mass of 12.9 g/L was obtained at 18 h and then the cell mass increased to 20.7 g/L with
a corresponding DC12 production of 106.7 g/L at the end of the fermentation (114 h). Compared with those in Fig. 2B, the final cell mass and DC12 production increased by 33.6% and 27.5%, respectively. For the fermentation of 60 g/L mixed sugars (Fig. 4B), the cell mass reached 19.2 g/L at 18 h, and then increased gradually to 24.2 g/L with a corresponding DC12 production of 129.7 g/L at the end of the fermentation (114 h). Compared with those in Fig. 2C, the final cell mass and DC12 production increased by 44.0% and 48.6%, respectively. Meanwhile, the strain ipe-1 exhib-
Fig. 4. Kinetics of DC12 production with wheat straw hydrolysates. The time courses refer to the 45.0 g/L total sugars mixed with 302 mL concentrated acidic hydrolysates (CAH) and 373 mL concentrated enzymatic hydrolysates (CZH) (A) and 60.0 g/L total sugars mixed with 403 mL CAH and 497 mL CZH (B). The exact constitutions of CAH and CZH are in the Section 2.4. Data are given as mean ± SD, n = 2.
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ited a strong ability to metabolize furfural. During the fermentation of 60 g/L mixed sugars, furfural (0.10 g/L) was completely metabolized at 18 h (Fig. 4B) without any loss of biomass. Similarly, other yeasts Saccharomyces cerevisiae (Villa et al., 1992) and C. ligniaria (López et al., 2004) also have been reported to be able to metabolize furfural. It was also found that the strain ipe-1 could assimilate glucose, xylose and sodium acetate simultaneously (Fig. 4), which was different from C. langeronii cultivated in hemicellulosic hydrolysate of sugar cane bagasse for the production of single cell protein (Nigam, 2000). The utilizations of D-xylose, Larabinose, and acetic acid by C. langeronii were delayed due to the presence of glucose, and after glucose depletion the other carbon sources were utilized. Therefore, the strain ipe-1 demonstrated a great potential of using biomass as a feedstock to costeffectively produce DC12. In addition, when the total sugars from wheat straw hydrolysate increased from 45 g/L to 60 g/L (Fig. 4), the DC12 production and cell mass respectively increased by 21.6% and 16.9%, which was different from those with simulated solution in Fig. 2. It is speculated that cell grew more quickly in the growth phase with 60 g/L sugars from wheat straw hydrolysates, resulting in more consumption of L-arabinose and sodium acetate, which mitigated the negative effect of L-arabinose and sodium acetate residuals on the DC12 biosynthesis with n-dodecane in the conversion phase. Regarding the enhanced cell growth in the growth phase, there are two possible reasons, the Ca2+ introduced from detoxification of dilute acid hydrolysates with lime, and the cellulase used as an exogenous carbon source for cell growth. In order to clarify the effect of sodium acetate, Ca2+ and cellulase on the DC12 production, these chemicals were added in the culture medium in the initial stage of fermentation. It can be seen in Fig. 5A that the DC12 pro-
duction decreased when the concentration of sodium acetate increased to 5.0 g/L equal to that in 403 mL CAH, which was expected from the results in Table 2. However, cell growth and DC12 production increased when Ca2+ was introduced in the medium (Fig. 5B), which respectively increased by 24.8% and 10.3% compared with those in Fig. 5A. Ca2+ was also found to have the most significant effect on growth of C. viswanathii in other cases (de Almeida et al., 2013; Kamble et al., 2005; Soni et al., 2007). The increases in both cell growth of C. viswanathii PBR2 as well as carbonyl reductase production were observed when Ca2+ concentration increased from 1 to 4 mM (Soni et al., 2006). Furthermore, when the cellulase solution was co-added as the fermentation substance (Fig. 5C), the cell mass did not further increase, and the DC12 production was also similar with that in Fig. 5B. Therefore, DC12 production could be enhanced by the exogenous Ca2+, and other factors hydrolyzed from wheat straw might also contribute to the enhancement of DC12 production. Meanwhile, it also indicated that complete removal of the socalled toxic compounds from the wheat straw hydrolysates was not necessary. Pereira et al. (2011) also revealed that complete removal of toxic compounds from the fermentation medium was not necessary to obtain efficient conversion of xylose to xylitol by C. guilliermondii. Another strain C. tropicalis 2838 could utilize nonseparated straw hydrolyzates prepared by mild acid hydrolysis of milled straw for production of fodder mixtures, and its cells exhibited satisfactory growth rate and high yield coefficient (Volfová et al., 1979). In addition, the production of DC12 only with CAH was carried out and the results are shown in Fig. 5D, since a suitable concentration of exogenous Ca2+ and sodium acetate from CAH could enhance DC12 production. The initial concentration of furfural was 0.36 g/L, which was completely metabolized within
Fig. 5. Effect of sodium acetate, CaSO4 and cellulase on DC12 production. The time courses refer to (A) the addition of sodium acetate, (B) the addition of sodium acetate and CaSO4, (C) the addition of sodium acetate, CaSO4 and cellulose and (D) 60.0 g/L total sugars from concentrated CAH. The added concentrations of sodium acetate, CaSO4 and cellulase were the same as those in the hydrolysates, and the exact constitutions of CAH and CZH are in the Section 2.4. For production of DC12 only with CAH, CAH was further concentrated to a mixture containing 101.2 g/L mixed sugars. Data are given as mean ± SD, n = 2.
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18 h. However, the initial concentration of sodium acetate was 15.5 g/L, and 3.0 g/L sodium acetate was still remained at 18 h. As a result, the final cell growth was increased by 29.8% compared with those in Fig. 5C, while the DC12 concentration was decreased by 2.4%, which was consistent to the results in Table 2 and Fig. 3B. 4. Conclusions Glucose, xylose and acetate could be simultaneously assimilated by C. viswanathii ipe-1, while arabinose utilization was less efficient. Wheat straw hydrolysate was used as an easily utilizable carbon source to get high biomass concentrations in the growth phase, and n-dodecane was utilized to synthesize DC12 in the conversion phase. Using this process, DC12 production increased by 40.0% compared with the sole carbon of glucose. Meanwhile, the complete removal of the inhibitors from the wheat straw hydrolysates was not necessary. Therefore, the costs of DC12 production could be reduced by using lignocellulosic biomass as the carbon sources. Acknowledgements The authors thank the National High Technology Research and Development Program of China (No. 2015AA021002 and No. 2014AA021005), and the National Natural Science Foundation of China (No. 21406240) for the financial supports. References Ayadi, I., Kamoun, O., Trigui-Lahiani, H., Hdiji, A., Gargouri, A., Belghith, H., Guerfali, M., 2016. Single cell oil production from a newly isolated Candida viswanathii YE4 and agro-industrial by-products valorization. J. Ind. Microbiol. Biotechnol. 43, 901–914. Beopoulos, A., Nicaud, J.-M., 2012. Yeast: a new oil producer? OCL 19, 22–28. Broadway, N.M., Dickinson, F.M., Ratledge, C., 1992. Long-chain acyl-CoA ester intermediates of beta-oxidation of mono-and di-carboxylic fatty acids by extracts of Corynebacterium sp. strain 7E1C. Biochem. J. 285, 117–122. Broadway, N.M., Dickinson, F.M., Ratledge, C., 1993. The enzymology of dicarboxylic acid formation by Curynebacterium sp. strain 7E1C grown on n-alkanes. J. Gen. Microbiol. 139, 1337–1344. Cao, Z., Gao, H., Liu, M., Jiao, P., 2006. Engineering the acetyl-CoA transportation system of Candida tropicalis enhances the production of dicarboxylic acid. Biotechnol. J. 1, 68–74. Chan, E.C., Kuo, J., Lin, H.P., Mou, D.G., 1991. Stimulation of n-alkane conversion to dicarboxylic acid by organic-solvent-and detergent-treated microbes. Appl. Microbiol. Biotechnol. 34, 772–777. De Almeida, A.F., Tauk-Tornisielo, S.M., Carmona, E.C., 2013. Acid lipase from Candida viswanathii: production, biochemical properties, and potential application. Biomed. Res. Int. 2013. http://dx.doi.org/10.1155/2013/435818. Dien, B.S., Kurtzman, C.P., Saha, B.C., Bothast, R.J., 1996. Screening forl-arabinose fermenting yeasts. Appl. Biochem. Biotechnol. 57, 233–242. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.t., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Hueting, S., Tempest, D.W., 1977. Influence of acetate on the growth of Candida utilis in continuous culture. Arch. Microbiol. 115, 73–78. Huf, S., Krügener, S., Hirth, T., Rupp, S., Zibek, S., 2011. Biotechnological synthesis of long-chain dicarboxylic acids as building blocks for polymers. Eur. J. Lipid Sci. Technol. 113, 548–561. Ji, Y., Mao, G., Wang, Y., Bartlam, M., 2013. Structural insights into diversity and nalkane biodegradation mechanisms of alkane hydroxylases. Front. Microbiol. 4, 1–13. Jiao, P., Huang, Y., Li, S., Hua, Y., Cao, Z., 2001. Effects and mechanisms of H2O2 on production of dicarboxylic acid. Biotechnol. Bioeng. 75, 456–462. Jönsson, L.J., Martín, C., 2016. Pretreatment of lignocellulose: formation of inhibitory by-products and strategies for minimizing their effects. Bioresour. Technol. 199, 103–112.
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Mingrone, G., Castagneto-Gissey, L., Macé, K., 2013. Use of dicarboxylic acids in type 2 diabetes. Br. J. Clin. Pharmacol. 75, 671–676. Nigam, J., 2000. Cultivation of Candida langeronii in sugar cane bagasse hemicellulosic hydrolyzate for the production of single cell protein. World J. Microbiol. Biotechnol. 16, 367–372. Palmqvist, E., Hahn-Hägerdal, B., 2000. Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour. Technol. 74, 25–33. Pereira, R.S., Mussatto, S.I., Roberto, I.C., 2011. Inhibitory action of toxic compounds present in lignocellulosic hydrolysates on xylose to xylitol bioconversion by Candida guilliermondii. J. Ind. Microbiol. Biotechnol. 38, 71–78. Picataggio, S., Rohrer, T., Deanda, K., Lanning, D., Reynolds, R., Mielenz, J., Eirich, L.D., 1992. Metabolic engineering of Candida tropicalis for the production of longchain dicarboxylic acids. Nat. Biotechnol. 10, 894–898. Qi, B., Chen, X., Wan, Y., 2010. Pretreatment of wheat straw by nonionic surfactantassisted dilute acid for enhancing enzymatic hydrolysis and ethanol production. Bioresour. Technol. 101, 4875–4883. Sailakshmi, G., Mitra, T., Chatterjee, S., Gnanamani, A., 2013. Engineering chitosan using a, x-dicarboxylic acids—an approach to improve the mechanical strength and thermal stability. J. Biomater. Nanobiotechnol. 4, 151–164. Salinari, S., Bertuzzi, A., Gandolfi, A., Greco, A.V., Scarfone, A., Manco, M., Mingrone, G., 2006. Dodecanedioic acid overcomes metabolic inflexibility in type 2 diabetic subjects. Am. J. Physiol. Endocrinol. Metab. 291, E1051–E1058. Sathesh-Prabu, C., Lee, S.K., 2015. Production of long-chain a, x-dicarboxylic acids by engineered Escherichia coli from renewable fatty Acids and plant oils. J. Agric. Food Chem. 63, 8199–8208. Scheller, U., Zimmer, T., Becher, D., Schauer, F., Schunck, W.-H., 1998. Oxygenation cascade in conversion of n-alkanes to a, x-dioic acids catalyzed by cytochrome P45052A3. J. Biol. Chem. 273, 32528–32534. Shiio, I., Uchio, R., 1971. Microbial production of long-chain dicarboxylic acids from n-alkanes: Part I. Screening and properties of microorganisms producing dicarboxylic acids. Agric. Biol. Chem. 35, 2033–2042. Smit, M.S., Mokgoro, M.M., Setati, E., Nicaud, J.M., 2005. a, x-Dicarboxylic acid accumulation by acyl-CoA oxidase deficient mutants of Yarrowia lipolytica. Biotechnol. Lett. 27, 859–864. Soni, P., Prasad, G.S., Banerjee, U.C., 2006. Optimization of physicochemical parameters for the enhancement of carbonyl reductase production by Candida viswanathii. Bioprocess Biosyst. Eng. 29, 149–156. Soni, P., Singh, M., Kamble, A.L., Banerjee, U.C., 2007. Response surface optimization of the critical medium components for carbonyl reductase production by Candida viswanathii MTCC 5158. Bioresour. Technol. 98, 829–833. Villa, G.P., Bartroli, R., López, R., Guerra, M., Enrique, M., Peñas, M., Rodríquez, E., Redondo, D., Jglesias, I., Díaz, M., 1992. 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a, x-Dodecanedioic acid production by Candida viswanathii ipe-1 with co-utilization of wheat straw hydrolysates and n-dodecane Weifeng Cao a, Bin Liu b, Jianquan Luo a, Junxiang Yin c, Yinhua Wan a,⇑ a
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China College of Food Science and Engineering, Qilu University of Technology, Jinan 250353, PR China c China National Center for Biotechnology Development, Beijing 100036, PR China b
h i g h l i g h t s Co-utilization of wheat straw hydrolysates and n-dodecane improved DC12 production. Glucose, xylose and sodium acetate are simultaneously assimilated by C. viswanathii. DC12 production can be increased by the addition of sodium acetate. Detoxification of the wheat straw hydrolysates is unnecessary for DC12 production.
a r t i c l e
i n f o
Article history: Received 17 April 2017 Received in revised form 13 June 2017 Accepted 14 June 2017 Available online 17 June 2017 Keywords: a, x-Dodecanedioic acid Candida viswanathii Wheat straw n-Dodecane
a b s t r a c t Candida viswanathii ipe-1 was used to produce a, x-dodecanedioic acid (DC12), which showed capability to ferment xylose and glucose simultaneously, while arabinose utilization was less efficient. A low concentration of furfural enhanced cell growth, and the addition of 4.0 g/L sodium acetate largely increased DC12 production. It indicated that detoxification of the wheat straw hydrolysates was not necessary for the biosynthesis of DC12. Based on the promising features of our strain, an efficient process was developed to produce DC12 from co-utilization of wheat straw hydrolysates and n-dodecane. Using this process, 129.7 g/L DC12 with a corresponding productivity of 1.13 gL1h1 could be produced, which was increased by 40.0% compared with a sole carbon of glucose. The improved DC12 yield by the coutilization of wheat straw hydrolysates and n-dodecane using C. viswanathii ipe-1 demonstrates the great potential of using biomass as a feedstock in the production of DC12. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
a, x-Dicarboxylic acids (DC) are versatile chemical intermediates with different chain length, generally represented as HOOC(R)n-COOH. These chemical compounds are used as raw materials for the preparation of perfumes, polymers, adhesives and macrolide antibiotics for many years and are well known polymer building block (Sailakshmi et al., 2013), and they are also considered as a promising alternative energy substrate for metabolism (Mingrone et al., 2013). DC can be produced in two ways: chemical synthesis and fermentation. The fermentation process is attractive because of its advantages of cost-efficient and greener process alternative. Nowadays there is a huge variety of microorganisms described for conversion of n-alkanes to the corresponding DC by utilizing hydrocarbons as carbon and energy source. Many organisms could ⇑ Corresponding author. E-mail address: [email protected] (Y. Wan). http://dx.doi.org/10.1016/j.biortech.2017.06.082 0960-8524/Ó 2017 Elsevier Ltd. All rights reserved.
achieve this transformation, including Cryptococus neoformans and Pseudomonas aeruginosa (Chan et al., 1991), Corynebacterium sp. (Broadway et al., 1992), Yarrowia lipolytica (Smit et al., 2005) as well as at least two strains of Candida, C. cloacae (Shiio and Uchio, 1971) and C. tropicalis (Liu et al., 2004; Yi and Rehm, 1982). The bioprocess engineering to enhance the production of DC could be divided into two steps. The first step included the growth of microorganisms with an easy utilizable carbon source to get high biomass concentrations. The second step was the DC production stage with adjusting pH to pH 8.0 and/or adding alkane based on requirements (Jiao et al., 2001; Liu et al., 2004). Usually, sucrose (Cao et al., 2006) and glucose (Picataggio et al., 1992) were used as the easy utilizable carbon source in the first step. Actually, abundant and renewable lignocellulosic materials could be used as suitable feedstocks for the production of lactic acid (Zhang et al., 2014), ethanol (Qi et al., 2010) and succinic acid (Zhao et al., 2016). Mixture of hexose and pentose sugars can be obtained by hydrolyzing lignocellulose. It was reported that Candida shehatae possessed a
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relaxed carbon catabolite repression mechanism, showing simultaneous consumption of glucose and xylose (Kim et al., 2010). Candida auringiensis, Candida succiphila and Candida sp. (YB-2248) possessed the ability to ferment L-arabinose while they could ferment D-xylose better (Dien et al., 1996). Candida viswanathii Y-E4 strain was able to use a wide range of substrates, especially C5 and C6 sugars as well as glycerol and hydrophobic substrates (Ayadi et al., 2016). Therefore, Candida may be capable of fermenting all the lignocellulose- released sugars for the economical production of DC. Usually, lignocellulosic hydrolysates obtained by sulfate pretreatment contained furans, acetate, and sulfuric acid (Palmqvist and Hahn-Hägerdal, 2000), and furfural and acetic acid are the main two inhibitors that retard microbial fermentation (Qi et al., 2010). However, high DC-producing mutants could achieve highlevel DC production by subterminal, terminal or diterminal oxidation of carbon atoms in the alkanes or fatty acids (Huf et al., 2011; Scheller et al., 1998). Therefore, the inhibitors in lignocellulosic hydrolysates may be not harmful to the DC production because DC producers may oxidate the inhibitors and further utilize them for cell growth. Among the DC, dodecanedioic acid (DC12) usually serves as a monomer of polyamides and some special nylons such as nylon 612 and nylon 1212 (Zheng et al., 2004). DC12 is also of interest as potential alternate fuel substrates in parenteral nutrition (Salinari et al., 2006). With growing demand for DC12, it is required to produce DC12 with low cost and large yield, and DC12 production with combined renewable lignocellulosic materials and n-dodecane may be a good choice. In the study reported here, an efficient process would be established to produce DC12 via co-utilization of wheat straw hydrolysates and n-dodecane by Candida viswanathii ipe-1. Effects of the main inhibitors from wheat straw hydrolysates on DC12 production would also be detected. 2. Materials and methods 2.1. Microorganism C. viswanathii ipe-1 (CGMCC No. 8824) used in this study was stored in China General Microbiological Culture Collection Center, Beijing, China. The strain was maintained on malt extract agar (MEA) (Beijing Aoboxing Bio-tech Co. LTD, China) slant at 4 °C. 2.2. Culture medium The seed culture medium contained 50 mL/L n-alkane, 8.0 g/L KH2PO4, 5.0 g/L yeast extract (Beijing Aoboxing Bio-tech Co. LTD, China), 1.5 g/L dry powder of corn steep liquor, 30.0 g/L sucrose and 3.0 g/L urea in deionized water. For the production of DC12 in the 7.5 L bioreactor (BioFioÒ110), the following fermentation medium was used: 8.0 g/L KH2PO4, 4.0 g/L yeast extract (Beijing Aoboxing Bio-tech Co. LTD, China), 1.5 g/L dry powder of corn steep liquor, 60.0 g/L sucrose, 4.0 g/L sodium acetate, 3.0 g/L KNO3, 1.0 g/ L NaCl, 0.5 g/L Twain 60 and 2.0 g/L urea in deionized water. After sterilization, 400 mL (i.e. 299.5 g) sterile n-dodecane was added to per liter fermentation medium. 2.3. Culture method In all fermentation experiments, the seed culture of the strain was prepared by inoculating cells grown on malt-extract agar into 500 mL Erlenmeyer flasks containing 100 mL of seed culture medium, followed by incubation at 30 °C for 2 days in the rotary shaker (HYG-A, Taicang Experimental Equipment Factory, China) at 180 rpm. The seed prepared for the bioreactor was cultured in four
Erlenmeyer flasks each time. After culturing and mixing the seed culture broth, 200 mL of the mixture was transferred to a 2.7 L bioreactor (BioFioÒ110, NewBrunswick Scientific, USA) containing 1.0 L of the fermentation culture medium. The temperature, stirring speed and the aeration rate in the bioreactor were maintained at 30 °C, 800 rpm and 2 L/min, respectively. The fermentation process was divided into growth phase and conversion production phase. pH in culture medium was maintained at 5.0 for 18 h during the growth phase, and then raised to pH 8.0 during the conversion phase. Medium pH was automatically maintained at set points by the addition of 8 M NaOH and 5 M H2SO4. To investigate the utilization of sugars by C. viswanathii ipe-1, 60 g/L of glucose, sucrose, xylose or arabinose were sterilized separately, then respectively added to the sterilized fermentation culture medium. To investigate the effect of sodium acetate or furfural on the production of DC12, different concentrations of sodium acetate or furfural were sterilized separately, then added to the sterilized fermentation culture medium. The final fermentation volume before inoculation was 1.4 L. All fermentation experiments were performed at least twice, to ensure the trends observed were reproducible. The data presented in the figures are the average values with error bars. 2.4. Preparation of wheat straw hydrolysates The conditions for preparation of wheat straw hydrolysates were similar to those described elsewhere (Qi et al., 2010; Zhang et al., 2014). Briefly, wheat straw was cleaned, chopped, and then pretreated by 2% (w/v) sulfuric acid at a 10% (w/v) loading. The mixture was treated at 121 °C for 90 min, and the obtained slurry was filtrated to achieve liquid and solid fractions. Both liquid and solid fractions were collected and defined as dilute acid hydrolysates and water insoluble solids (WIS), respectively. The dilute acid hydrolysates were alkalified with calcium hydroxide to pH 10.0, and then the mixture was filtered with filter paper (No. 43, Whatman, UK). The filtrate was then acidified to pH 5.0 with sulfuric acid, followed by filtration with filter paper. The final filtrate was concentrated to a mixture containing 10.6 g/L sodium acetate, 33.1 g/L xylose, 1.79 g/L glucose, 6.12 g/L arabinose, 0.058 g/L HMF, 0.25 g/L furfural and 0.48 g/L total phenolics by vacuum evaporation at 60 °C, and the mixture was named as concentrated acid hydrolysates (CAH). For production of DC12 only with CAH, CAH was further concentrated to a mixture containing 101.2 g/L mixed sugars. The WIS (containing 59.96% cellulose) was hydrolyzed by commercial cellulase (Sunson Group Ningxia Enzyme Preparation Plant, China) at a 10% (w/v) solid loading. Enzyme loading was 20 FPU (filter paper activity units)/g cellulose. The enzymatic hydrolysis was carried out in a 7.5 L jar fermentor (BioFioÒ110, NewBrunswick Scientific, USA) at 50 °C, pH 5.0 and 200 rpm. Enzymatic hydrolysates were obtained by centrifuging at 5000g for 15 min. And then the obtained supernatant was concentrated by vacuum evaporation at 60 °C to glucose concentration of 92.0 g/L, and the mixture was named as concentrated enzymatic hydrolysates (CZH). Then the wheat straw hydrolysates were stored at 4 °C prior to use. 2.5. Analytical methods Biomass and DC12 concentrations were measured as described by Liu et al. (2004). Cell growth was determined by measuring of the dry weight of the cells. Samples (5 mL) were taken at regular intervals and centrifuged at 8000 rpm (6869g) for 10 min. The sediment was washed for three times with distilled water and dried at 90 °C. The dry residue was designated as biomass. DC12 concentrations in undiluted samples were measured by titration. A sample of 10 mL in a sealed tube, adjusted to pH 10 with several grains of NaOH, was boiled for 5 min to completely convert DC into sodium
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salts. After centrifugation at 4000 rpm (1717g) for 10 min at room temperature, a supernatant of 2 mL, diluted with 30 mL distilled water, was acidified to pH 2–3 with 2 M H2SO4 and boiled for 0.5 min to precipitate DC. After cooling to room temperature, the mixture was filtered through a filtration paper and washed with distilled water until the inorganic acid was fully removed. Then the precipitated DC was dissolved in 30 mL warmed ethanol (90%, v/v). DC concentration was determined by titration with 0.2 M NaOH as standard solution. Sucrose concentrations were determined by the phenol sulfuric acid method (Dubois et al., 1956). The concentrations of sodium acetate, glucose, xylose and arabinose were measured by a HPLC apparatus (Shimadzu LC20AT) equipped with an Aminex HP-X87H Ion Exclusion column using refractive index detector (RI). The process was performed at a temperature of 40 °C, a flow rate of 0.6 mL/min with 5 mmol/L H2SO4 as moving phase. 3. Results and discussion 3.1. Utilization of carbon sources by C. viswanathii ipe-1 for DC12 production As shown in Fig. 1, the C. viswanathii ipe-1 could assimilate ndodecane as sole carbon source for cell growth (9.6 g/L) and DC12 production (43.7 g/L). When an easily utilizable carbon source, such as sucrose, glucose or xylose, was added in the broth, the cells concentration and DC12 production increased significantly. However, compared with the pentose xylose, the fermentation kinetic was different when another pentose L-arabinose was used as carbon source. With the addition of L-arabinose, the cell growth increased by 24.0% with a decreasing DC12 production by 17.8% compared without exogenous carbon source addition. The utilizable performance of glucose, xylose and arabinose as substrates was similar to the biolipid production by C. viswanathii Y-E4 (Ayadi et al., 2016). Simultaneous assimilation of glucose and xylose was important to the utilization of lignocellulosic biomass and other raw materials as the carbon sources, in order to reduce the costs of microbial DC12 production. However, only 5.0 g/L Larabinose was consumed before 42 h when 60.0 g/L of sucrose, glucose or xylose was depleted. A large amount of residual arabinose in the broth after 42 h may be responsible to the decrease of DC12 production with the addition of 60 g/L arabinose case. Therefore, if the L-arabinose added could be depleted in the same period as other sugars, the DC12 production may be not affected. This assumption would be discussed in the next section. 3.2. Effect of mixed sugars on DC12 production It could be concluded that C. viswanathii ipe-1 could ferment xylose and glucose simultaneously, being better than L-arabinose (Fig. 1). In addition, the concentration of arabinose in wheat straw hydrolysates with dilute sulfuric acid pretreatment was low (i.e. 3.8 g arabinose/100 g wheat straw with 2% (w/v) sulfuric acid pretreatment) (Qi et al., 2010), while the concentration of xylose was 22.6 g/L. Therefore, the strain ipe-1 may be capable of fermenting all the lignocellulose-released sugars for the economical production of DC12. When the total sugar concentration was about 60 g/ L, a ratio of 29.7 g/L glucose, 24.7 g/L xylose and 5.1 g/L arabinose could be obtain from hydrolysates of wheat straw (Zhang et al., 2014). In order to evaluate the fermentation performance of C. viswanathii ipe-1 with the sugars from wheat straw, the modeling solution with the same sugars ratio as in the straw hydrolysates (Zhang et al., 2014) was used. Kinetics of DC12 production under different mixed sugar concentrations were described in Fig. 2. During the fermentations of 30, 45, and 60 g/L mixed sugars, the
Fig. 1. Utilization of carbon sources by C. viswanathii ipe-1 for DC12 production. The time courses refer to the effect of sugars on DC12 (A) and biomass (B), and the variation of sugars during the fermentation (C). Data are given as mean ± SD, n = 3.
obtained DC12 concentration were 52.9, 83.7 and 87.3 g/L, respectively. And the corresponding cell mass were 12.1, 15.5 and 16.8 g/L, respectively. The result showed that C. viswanathii ipe-1 could co-ferment the mixed sugar to DC12. The mechanism of mixed sugar utilization by Candida viswanathii ipe-1 may be described as follows: the strain ipe-1 has an ability of relaxed carbon catabolite repression and can simultaneously consume the mixed sugars, which was similar with the strain Candida shehatae (Kim et al., 2010). The glucose and xylose were assimilated simultaneously, while the metabolic rate of the strain for glucose was a
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Fig. 2. Kinetics of DC12 production with mixed sugars of glucose, xylose and arabinose under 4.0 g/L sodium acetate. The time courses refer to the mixed sugars of 30 g/L (A) and 45 g/L (B) and 60 g/L (C). The ratio of the sugars was the same as that in the straw hydrolysates (i.e. 29.72 g/L glucose: 24.69 g/L xylose: 5.14 g/L arabinose) reported by Zhang et al. (2014). Data are given as mean ± SD, n = 3.
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little faster than that for xylose. After glucose or xylose was depleted, arabinose began to be consumed for DC12 production. During the fermentation of 45 g/L mixed sugars, the residual arabinose concentration was 1.1 g/L at 42 h. Compared with the fermentation with 30 g/L mixed sugars, the DC12 production increased by 58% with 45 g/L mixed sugars (50% higher sugar concentration), implying that such low residual arabinose (1.1 g/L) may not affect DC12 production. Therefore, if the L-arabinose added could be almost depleted in the same period as other sugars, the DC12 production are not affected. However, during the fermentation of 60 g/ L mixed sugars, the residual arabinose concentration was 4.1 g/L at 42 h and only 1.0 g/L arabinose was metabolized in the first 42 h cultivation (Fig. 2C). The little improvement in DC12 production with increasing mixed sugars from 45 g/L to 60 g/L was possibly caused by the high concentration of residual arabinose (4.1 g/L) in the broth. Actually, only a few oleaginous types of yeast such as Y. lipolytica, C. tropicalis and L. starkey could use hydrophobic substrates via an special anabolic pathway (Beopoulos and Nicaud, 2012). C. viswanathii ipe-1 is probably one of them, because it has a specific mechanism for n-dodecane transport and DC12 production. When a high concentration of other carbon source exists in the conversion phase, the pathway for biosynthesis of DC12 will be disturbed.
3.3. Tolerance to the main hydrolysates inhibitors As described by Qi et al. (2010), acetic acid and furfural were the two main inhibitors in the dilute acid hydrolysates. Therefore, acetic acid and furfural were selected to test the susceptibility of C. viswanathii ipe-1 to the inhibitors. The results are showed in Tables 1 and 2, where the inhibitors were added at the initial stage of fermentation. When 0.5 g/L furfural was added in the broth, cell mass increased in the growth phase and reached 17.3 g/L at 18 h (Table 1). The cell mass in the final fermentation (114 h) also increased when 0.5 g/L furfural was added in the broth, but no further increase of cell mass was observed when the concentration of furfural was above 0.5 g/L. The inhibition effect of furfural on cell growth became significant when the concentration of furfural was above 0.5 g/L, while the inhibition effect of furfural on DC12 production was negligible at the furfural concentrations from 0.5 to 1.25 g/L. When the concentration of furfural reached 4.0 g/L, the cell growth was completely inhibited in the growth phase without glucose consumption (Fig. 3A). After entering into the conversion production phase, the cell growth was recovered and the cell mass reached its maximal level of 20.3 g/L at 90 h, however only 26.2 g/L of DC12 was obtained. The repressed DC12 production may be caused by the low cell mass at the beginning of the conversion production phase. In order to clarify the inhibition mechanism of furfural, 4.0 g/L of furfural was added at the beginning of the conversion production phase at 18 h after obtaining 13.2 g/L of cell mass (Fig. 3B). At the end of fermentation, 22.4 g/L of DC12 and 14.7 g/L of cell mass were obtained, which was respectively
Table 1 Effect of furfural on the cell growth and DC12 production with the addition of 4.0 g/L sodium acetate.a Furfural (g/L)
Biomass18h (g/L)
Biomass114h (g/L)
DC12
0 0.5 1.25 2.0 3.0 4.0
14.6 ± 0.83 17.3 ± 0.97 14.0 ± 0.79 13.0 ± 0.81 5.9 ± 0.42 1.5 ± 0.08
14.1 ± 0.92 17.6 ± 0.83 17.8 ± 0.91 17.9 ± 0.74 16.9 ± 0.71 18.6 ± 0.87
92.6 ± 3.97 93.5 ± 4.01 90.6 ± 3.75 75.4 ± 2.63 54.4 ± 2.04 26.2 ± 1.13
114h
(g/L)
a Growth phase was ended at 18 h, and fermentation was ended at 114 h. Data are given as mean ± SD, n = 3.
Table 2 Effect of acetate on the cell growth and DC12 production without furfural addition.a
a
Sodium acetate (g/L)
Biomass18h (g/L)
Biomass114h (g/L)
DC12
0 2.0 4.0 7.0 10.0
12.9 ± 0.87 13.5 ± 0.92 14.6 ± 0.83 14.9 ± 0.78 15.4 ± 0.98
16.5 ± 0.91 15.9 ± 0.85 14.1 ± 0.92 16.4 ± 0.73 16.9 ± 0.94
55.8 ± 2.84 63.8 ± 2.81 92.6 ± 3.97 66.7 ± 3.12 63.9 ± 3.06
114h
(g/L)
Data are given as mean ± SD, n = 3.
decreased by 14.5% and 21.0% compared with that in Fig. 3A. The results indicated that the furfural with high concentration inhibited not only cell growth but also DC12 production. However, furfural may be not a problem for DC12 production using lignocellulose hydrolysates because the concentration of furfural was low (0.7 g/L) with the method as described by Qi et al. (2010), and detoxification of dilute acid hydrolysates of lignocellulose was extensively reported (Jönsson and Martín, 2016). Detoxification of dilute acid hydrolysates of lignocellulose with lime was optimized and total furans and phenolic compounds were largely reduced with overliming, while acetic acid levels were unchanged (Martinez et al., 2001). Microbial oil was also successfully produced by oleaginous yeasts using the hydrolysate, which was obtained from wheat straw treated by dilute sulfuric acid and following calcium hydroxide pretreatment (Yu et al., 2011). Therefore, the dilute acid hydrolysates of lignocellulose could be detoxified with lime to avoid the negative effects of furfural on DC12 production (see Table 3). As listed in Table 2, sodium acetate showed a significant positive effect on cell growth and DC12 production with strain ipe-1 when the concentration of sodium acetate added was below 4.0 g/L. However, when the concentration of sodium acetate exceeded 4.0 g/L, DC12 production was strongly inhibited. The same phenomenon was also reported for ethanol production by S. cerevisiae (Palmqvist and Hahn-Hägerdal, 2000). As shown in Table 2, the highest DC12 production of 92.6 g/L with 14.6 g/L cell mass was obtained when 4.0 g/L sodium acetate was added at the initial stage of fermentation. However, as seen in Fig. 3C, when 4.0 g/L sodium acetate was added at the beginning of conversion production phase (no sodium acetate addition at the initial stage of fermentation), DC12 production was decreased by 10.9% compared with control experiment (no addition of sodium acetate during the whole fermentation). Therefore, the high residual sodium acetate in the conversion production phase resulted in a decreasing production of DC12, while this negative effect was not observed in the case with 4.0 g/L sodium acetate addition at the initial stage of fermentation. Moreover, it had been confirmed that oxygen atoms derived from molecular oxygen for inserting into two terminals of alkane was a key step for DC biosynthesis (Ji et al., 2013), and the molecular oxygen could enter into the cytosol through the respiration of the strain ipe-1. The respiration rate could be enhanced by the extracellular low acetate concentration (Hueting and Tempest, 1977). Under pH 5.0, undissociated acetic acid is liposoluble and can diffuse across the plasma membrane into cytosol (Palmqvist and Hahn-Hägerdal, 2000). Therefore, adding low concentration acetate could improve DC12 production. 3.4. DC12 production by co-utilization of wheat straw hydrolysate and n-dodecane Based on the promising features of strain ipe-1, an efficient process was developed to produce DC12 by co-utilization of wheat straw hydrolysate and n-dodecane. Wheat straw hydrolysate was used as an easily utilizable carbon source to get high biomass concentrations in the growth phase, and n-dodecane was used as the
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Fig. 3. Effect of furfural and acetate on cell growth and DC12 production under different adding time. The time courses refer to 4.0 g/L furfural added at the initial culture time (A), 4.0 g/L furfural added at 18 h (B) and 4.0 g/L sodium acetate added at 18 h (C). Data are given as mean ± SD, n = 3.
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W. Cao et al. / Bioresource Technology 243 (2017) 179–187 Table 3 Comparison of DC12 production with different substrates by different strains. Cmax: maximal product concentration, Rmax: maximal production rate of DC12. Substrate Dodecane, Dodecane Dodecane, Dodecane, Dodecane, Dodecane,
Glucose Sucrose Glucose 5-aminolevulinic acid, thiourea wheat straw hydrolysates
Cmax (g/L)
Rmax g/(Lh)
Strain
Ref.
140 0.3 166 8 0.159 129.7
0.9 0.0025 1.38 0.056 0.013 1.13
C. tropicalis H5343 Corynebacterium sp. ATCC19067 C. tropicalis CGMCC 356 Y. lipolytica MTLY37 E. coli REx C. viswanathii ipe-1
Picataggio et al. (1992) Broadway et al. (1993) Liu et al. (2004) Smit et al. (2005) Sathesh-Prabu and Lee (2015) This study
substance to biosynthesis DC12 by the strain ipe-1 in the conversion production phase. Wheat straw hydrolysate was prepared as described in Section 2.4. Kinetics of DC12 production with 45.0 g/ L total sugars mixed with 302 mL concentrated acidic hydrolysates (CAH) and 373 mL concentrated enzymatic hydrolysates (CZH) (Fig. 4A) and 60.0 g/L total sugars mixed with 403 mL CAH and 497 mL CZH (Fig. 4B) are showed in Fig. 4. For the fermentation of 45 g/L mixed sugars (Fig. 4A), a cell mass of 12.9 g/L was obtained at 18 h and then the cell mass increased to 20.7 g/L with
a corresponding DC12 production of 106.7 g/L at the end of the fermentation (114 h). Compared with those in Fig. 2B, the final cell mass and DC12 production increased by 33.6% and 27.5%, respectively. For the fermentation of 60 g/L mixed sugars (Fig. 4B), the cell mass reached 19.2 g/L at 18 h, and then increased gradually to 24.2 g/L with a corresponding DC12 production of 129.7 g/L at the end of the fermentation (114 h). Compared with those in Fig. 2C, the final cell mass and DC12 production increased by 44.0% and 48.6%, respectively. Meanwhile, the strain ipe-1 exhib-
Fig. 4. Kinetics of DC12 production with wheat straw hydrolysates. The time courses refer to the 45.0 g/L total sugars mixed with 302 mL concentrated acidic hydrolysates (CAH) and 373 mL concentrated enzymatic hydrolysates (CZH) (A) and 60.0 g/L total sugars mixed with 403 mL CAH and 497 mL CZH (B). The exact constitutions of CAH and CZH are in the Section 2.4. Data are given as mean ± SD, n = 2.
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ited a strong ability to metabolize furfural. During the fermentation of 60 g/L mixed sugars, furfural (0.10 g/L) was completely metabolized at 18 h (Fig. 4B) without any loss of biomass. Similarly, other yeasts Saccharomyces cerevisiae (Villa et al., 1992) and C. ligniaria (López et al., 2004) also have been reported to be able to metabolize furfural. It was also found that the strain ipe-1 could assimilate glucose, xylose and sodium acetate simultaneously (Fig. 4), which was different from C. langeronii cultivated in hemicellulosic hydrolysate of sugar cane bagasse for the production of single cell protein (Nigam, 2000). The utilizations of D-xylose, Larabinose, and acetic acid by C. langeronii were delayed due to the presence of glucose, and after glucose depletion the other carbon sources were utilized. Therefore, the strain ipe-1 demonstrated a great potential of using biomass as a feedstock to costeffectively produce DC12. In addition, when the total sugars from wheat straw hydrolysate increased from 45 g/L to 60 g/L (Fig. 4), the DC12 production and cell mass respectively increased by 21.6% and 16.9%, which was different from those with simulated solution in Fig. 2. It is speculated that cell grew more quickly in the growth phase with 60 g/L sugars from wheat straw hydrolysates, resulting in more consumption of L-arabinose and sodium acetate, which mitigated the negative effect of L-arabinose and sodium acetate residuals on the DC12 biosynthesis with n-dodecane in the conversion phase. Regarding the enhanced cell growth in the growth phase, there are two possible reasons, the Ca2+ introduced from detoxification of dilute acid hydrolysates with lime, and the cellulase used as an exogenous carbon source for cell growth. In order to clarify the effect of sodium acetate, Ca2+ and cellulase on the DC12 production, these chemicals were added in the culture medium in the initial stage of fermentation. It can be seen in Fig. 5A that the DC12 pro-
duction decreased when the concentration of sodium acetate increased to 5.0 g/L equal to that in 403 mL CAH, which was expected from the results in Table 2. However, cell growth and DC12 production increased when Ca2+ was introduced in the medium (Fig. 5B), which respectively increased by 24.8% and 10.3% compared with those in Fig. 5A. Ca2+ was also found to have the most significant effect on growth of C. viswanathii in other cases (de Almeida et al., 2013; Kamble et al., 2005; Soni et al., 2007). The increases in both cell growth of C. viswanathii PBR2 as well as carbonyl reductase production were observed when Ca2+ concentration increased from 1 to 4 mM (Soni et al., 2006). Furthermore, when the cellulase solution was co-added as the fermentation substance (Fig. 5C), the cell mass did not further increase, and the DC12 production was also similar with that in Fig. 5B. Therefore, DC12 production could be enhanced by the exogenous Ca2+, and other factors hydrolyzed from wheat straw might also contribute to the enhancement of DC12 production. Meanwhile, it also indicated that complete removal of the socalled toxic compounds from the wheat straw hydrolysates was not necessary. Pereira et al. (2011) also revealed that complete removal of toxic compounds from the fermentation medium was not necessary to obtain efficient conversion of xylose to xylitol by C. guilliermondii. Another strain C. tropicalis 2838 could utilize nonseparated straw hydrolyzates prepared by mild acid hydrolysis of milled straw for production of fodder mixtures, and its cells exhibited satisfactory growth rate and high yield coefficient (Volfová et al., 1979). In addition, the production of DC12 only with CAH was carried out and the results are shown in Fig. 5D, since a suitable concentration of exogenous Ca2+ and sodium acetate from CAH could enhance DC12 production. The initial concentration of furfural was 0.36 g/L, which was completely metabolized within
Fig. 5. Effect of sodium acetate, CaSO4 and cellulase on DC12 production. The time courses refer to (A) the addition of sodium acetate, (B) the addition of sodium acetate and CaSO4, (C) the addition of sodium acetate, CaSO4 and cellulose and (D) 60.0 g/L total sugars from concentrated CAH. The added concentrations of sodium acetate, CaSO4 and cellulase were the same as those in the hydrolysates, and the exact constitutions of CAH and CZH are in the Section 2.4. For production of DC12 only with CAH, CAH was further concentrated to a mixture containing 101.2 g/L mixed sugars. Data are given as mean ± SD, n = 2.
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18 h. However, the initial concentration of sodium acetate was 15.5 g/L, and 3.0 g/L sodium acetate was still remained at 18 h. As a result, the final cell growth was increased by 29.8% compared with those in Fig. 5C, while the DC12 concentration was decreased by 2.4%, which was consistent to the results in Table 2 and Fig. 3B. 4. Conclusions Glucose, xylose and acetate could be simultaneously assimilated by C. viswanathii ipe-1, while arabinose utilization was less efficient. Wheat straw hydrolysate was used as an easily utilizable carbon source to get high biomass concentrations in the growth phase, and n-dodecane was utilized to synthesize DC12 in the conversion phase. Using this process, DC12 production increased by 40.0% compared with the sole carbon of glucose. Meanwhile, the complete removal of the inhibitors from the wheat straw hydrolysates was not necessary. Therefore, the costs of DC12 production could be reduced by using lignocellulosic biomass as the carbon sources. Acknowledgements The authors thank the National High Technology Research and Development Program of China (No. 2015AA021002 and No. 2014AA021005), and the National Natural Science Foundation of China (No. 21406240) for the financial supports. References Ayadi, I., Kamoun, O., Trigui-Lahiani, H., Hdiji, A., Gargouri, A., Belghith, H., Guerfali, M., 2016. Single cell oil production from a newly isolated Candida viswanathii YE4 and agro-industrial by-products valorization. J. Ind. Microbiol. Biotechnol. 43, 901–914. Beopoulos, A., Nicaud, J.-M., 2012. Yeast: a new oil producer? OCL 19, 22–28. Broadway, N.M., Dickinson, F.M., Ratledge, C., 1992. Long-chain acyl-CoA ester intermediates of beta-oxidation of mono-and di-carboxylic fatty acids by extracts of Corynebacterium sp. strain 7E1C. Biochem. J. 285, 117–122. Broadway, N.M., Dickinson, F.M., Ratledge, C., 1993. The enzymology of dicarboxylic acid formation by Curynebacterium sp. strain 7E1C grown on n-alkanes. J. Gen. Microbiol. 139, 1337–1344. Cao, Z., Gao, H., Liu, M., Jiao, P., 2006. Engineering the acetyl-CoA transportation system of Candida tropicalis enhances the production of dicarboxylic acid. Biotechnol. J. 1, 68–74. Chan, E.C., Kuo, J., Lin, H.P., Mou, D.G., 1991. Stimulation of n-alkane conversion to dicarboxylic acid by organic-solvent-and detergent-treated microbes. Appl. Microbiol. Biotechnol. 34, 772–777. De Almeida, A.F., Tauk-Tornisielo, S.M., Carmona, E.C., 2013. Acid lipase from Candida viswanathii: production, biochemical properties, and potential application. Biomed. Res. Int. 2013. http://dx.doi.org/10.1155/2013/435818. Dien, B.S., Kurtzman, C.P., Saha, B.C., Bothast, R.J., 1996. Screening forl-arabinose fermenting yeasts. Appl. Biochem. Biotechnol. 57, 233–242. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.t., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Hueting, S., Tempest, D.W., 1977. Influence of acetate on the growth of Candida utilis in continuous culture. Arch. Microbiol. 115, 73–78. Huf, S., Krügener, S., Hirth, T., Rupp, S., Zibek, S., 2011. Biotechnological synthesis of long-chain dicarboxylic acids as building blocks for polymers. Eur. J. Lipid Sci. Technol. 113, 548–561. Ji, Y., Mao, G., Wang, Y., Bartlam, M., 2013. Structural insights into diversity and nalkane biodegradation mechanisms of alkane hydroxylases. Front. Microbiol. 4, 1–13. Jiao, P., Huang, Y., Li, S., Hua, Y., Cao, Z., 2001. Effects and mechanisms of H2O2 on production of dicarboxylic acid. Biotechnol. Bioeng. 75, 456–462. Jönsson, L.J., Martín, C., 2016. Pretreatment of lignocellulose: formation of inhibitory by-products and strategies for minimizing their effects. Bioresour. Technol. 199, 103–112.
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