Bioresource Technology 150 (2013) 266–270
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Fatty acid ethyl esters production in aqueous phase by the oleaginous yeast Rhodosporidium toruloides Guojie Jin a,c, Yixin Zhang a,c, Hongwei Shen a,b, Xiaobing Yang a, Haibo Xie a,b, Zongbao K. Zhao a,b,⇑ a
Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian 116023, PR China Dalian National Laboratory for Clean Energy, Dalian 116023, PR China c University of Chinese Academy of Sciences, Beijing 100049, PR China b
h i g h l i g h t s Fatty acid ethyl esters were produced in aqueous phase by the yeast Rhodosporidium toruloides. Up to 73% of cellular neutral glycerides were converted into fatty acid ethyl esters. Neutral glycerides were hydrolyzed to free fatty acids followed by esterification. Lipid droplets played important roles in the process.
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Article history: Received 30 July 2013 Received in revised form 5 October 2013 Accepted 7 October 2013 Available online 14 October 2013 Keywords: Biodiesel Fatty acid ethyl esters Aqueous phase conversion Lipid droplets Rhodosporidium toruloides
a b s t r a c t Fatty acid ethyl esters (FAEEs) are attractive biofuel molecules. Conventional FAEEs production process uses triglycerides and ethanol as feedstocks and is sensitive to water contents. In this work, we show that the oleaginous yeast Rhodosporidium toruloides cells are capable of converting lipids into FAEEs intracellularly in aqueous phase. Up to 73% of cellular neutral glycerides could be converted into FAEEs when lipid-rich cells were incubated for 84 h at 35 °C, pH 6.0 in a broth containing 10 vol% ethanol. It was found that neutral glycerides were first hydrolyzed to free fatty acids followed by esterification and that lipid droplets played important roles in the process. This new process provides a novel opportunity for integration of microbial lipid production technology with bioethanol fermentation for more efficient production of drop-in biofuels from renewable resources. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction With increasing transportation fuel demands and environmental concerns, biofuels have been recognized as important alternatives. Biodiesel, a typical form of liquid biofuel, has attracted considerable interest in recent years (Demirbas, 2007; Fjerbaek et al., 2009; Liang et al., 2013). Biodiesel consists of monoalkyl esters of long-chain fatty acids with short-chain alcohols, primarily methanol and ethanol, resulting in fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), respectively. In general, biodiesel is produced by transesterification of triglycerides (TAGs) with alcohols in the presence of different types of catalysts, such as bases, acids or enzymes (Cao et al., 2012; Fedosov et al., 2013; Ferella et al., 2010; Liu and Zhao, 2007; Liu et al., 2012). In these processes, water can facilitate hydrolysis of TAGs and in⇑ Corresponding author at: Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian 116023, PR China. Tel./fax: +86 411 84379211. E-mail address: [email protected] (Z.K. Zhao). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.10.023
crease the content of free fatty acids (FFAs). Base- and acid-catalyzed biodiesel production processes are particularly sensitive to water and require high-quality feedstock with a water content lower than 5 wt% (Atadashi et al., 2012). For those catalyzed by heterogeneous catalysts or lipases, biodiesel yields around 90% could be achieved for feedstocks with higher water contents (Tan et al., 2006; Yan et al., 2009). However, when water contents exceed 30%, biodiesel yields usually drop to below 60% (Atadashi et al., 2012). Because ethanol is less toxic than methanol and can be produced at large scale by fermentation from local and renewable agricultural resources, it is more appealing to make FAEEs as a sustainable biofuel. Efforts have been made for direct production of FAEEs by engineered cell factories. For example, recombinant Escherichia coli strains were constructed by expressing a wax ester synthase gene and ethanol-producing genes, such that FAEEs were successfully produced at a titre of 1.28 g/L after 72 h in the presence of exogenous fatty acids (Kalscheuer et al., 2006). When it was engineered to use the endogenous fatty acid pathway, E. coli
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could make 0.674 g/L FAEEs directly from simple sugars such as glucose (Steen et al., 2010). To avoid the toxicity of ethanol, a dynamic sensor-regulator system was developed to substantially improve strain stability as well as FAEEs titre to 1.5 g/L (Zhang et al., 2012). Similarly, the yeast Saccharomyces cerevisiae has also been explored for de novo production of FAEEs at a titer of 8.2 mg/L from glucose (Shi et al., 2012). However, the efficiency of direct FAEEs production by engineered cell factories remained low. It is tempting to develop a FAEEs production process in aqueous phase by using ethanol fermentation broth directly as the alcohol source. Ethanol fermentation broth normally contains less than 20% ethanol, plus water and byproducts such as acetaldehyde, glycerol and acetic acid. It has been known that the oleaginous yeast Rhodosporidium toruloides is a robust lipid producer as it can accumulate lipid over 70% with a titer of over 100 g/L (Li et al., 2007), and utilize lignocellulosic hydrolysates with excellent tolerance towards inhibitory compounds (Hu et al., 2009). Lipids in this yeast can be recovered easily by enzyme-assisted extraction (Jin et al., 2012). In this study, we show that R. toruloides cells are capable of converting lipids into FAEEs intracellularly in aqueous phase. This new process represents an efficient alternative approach to traditional technology using TAGs as the feedstock or engineered cell factories for FAEEs production. It also provides a novel opportunity for integration of microbial lipid production technology with bioethanol fermentation for more efficient production of drop-in biofuels from renewable resources. 2. Methods 2.1. Strain, media and chemicals R. toruloides Y4, a variant of R. toruloides AS 2.1389 obtained from the China General Microbiological Culture Collection Center, was grown in YPD liquid medium (containing 20 g/L glucose, 10 g/L yeast extract, and 10 g/L peptone, pH 6.0) at 30 °C and 200 rpm. The lipid production medium contained: 40 g/L glucose, 0.4 g/L KH2PO4, and 1.5 g/L MgSO47H2O, pH 6.0. After sterilized at 121 °C for 15 min, it was supplemented with 10 mL/L of a trace element solution contained (per liter): 4.0 g CaCl22H2O, 0.55 g FeSO47H2O, 0.52 g citric acidH2O, 0.10 g ZnSO47H2O, 0.076 g MnSO4H2O, and 100 lL 18 M H2SO4 (Wu et al., 2011). Lipid-rich culture was prepared according to a published procedure (Lin et al., 2011). R. toruloides Y4 cells were incubated in YPD liquid medium at 30 °C, 200 rpm for 24 h, collected by centrifugation, and washed twice with sterile 0.9 wt% NaCl solution. Subsequently, they were cultured in lipid production medium at 30 °C, 200 rpm until the glucose was exhausted. The final cell mass and lipid titer were 17.5 g/L and 7.3 g/L, respectively. Anhydrous ethanol (P99.8% purity) was obtained from Tianjin Hengxing Chemical Co., Ltd., Tianjin, China. Ethyl laurate (P99% purity), ethyl myristate (P98% purity), ethyl cis-9-Hexadecenoate (P95% purity), ethyl oleate (P98% purity), ethyl stearate (P99% purity), ethyl linolenate (P99% purity) and CDCl3 (99.8% D with 0.03% (vol/vol) tetramethylsilane (TMS)) were obtained from J&K Scientific Ltd., Beijing, China. Ethyl palmitate (P99% purity), ethyl linoleate (P99% purity), Nile red (chemical pure) and p-nitrobenzaldehyde (p-NBD, 98% purity) were obtained from Sigma, Shanghai, China. All other chemicals and reagents were analytical pure and obtained commercially. 2.2. FAEEs production by R. toruloides Y4 cells The typical procedure was performed as follows: aliquots of 6 mL of R. toruloides Y4 lipid-rich culture were mixed with 2 mL of 40 vol% ethanol solution in screw-capped vials sealed with para-
267
film. The reaction mixture was incubated at 35 °C, pH 6.0 and 200 rpm for 48 h. Ethanol concentration and water content of the reaction system were 10 vol% and 91.3 wt%, respectively. When one of these factors including temperature, pH, ethanol concentration and reaction time was investigated, the levels of others were held constant. 2.3. FAEEs production catalyzed by R. toruloides Y4 cell homogenates R. toruloides Y4 cells from 6 mL of lipid-rich culture were lyophilized, milled to powder and extracted by cold acetone (Gu et al., 2011). Extracts and cell residues were mixed again, dried by N2, and then resuspended with 6 mL of deionized water. Then the cell homogenates were mixed with 2 mL of 40 vol% ethanol solution and incubated at 35 °C, pH 6.0 and 200 rpm for 96 h. 2.4. Lipid extraction Cells were homogenized with glass beads and extracted with chloroform–methanol (2:1, vol/vol) at room temperature (Folch et al., 1957). The organic extracts were washed with 0.1 wt% NaCl solution, dried over anhydrous Na2SO4, and evaporated under reduced pressure to give the total lipids. 2.5. Lipid fractionation About 50 mg of the lipid sample was loaded on a silica gel column, eluted by 120 mL of 1,2-dichloroethane, 80 mL of 1,2dichloroethane/acetone (1:10, vol/vol), and 50 mL methanol in sequence (Wu et al., 2010). The solvents from each fraction were evaporated under reduced pressure. The fractions, in the order of elution, were neutral lipids (N), glycolipids plus sphingolipids (G + S), and phospholipids (P). The neutral lipids included TAGs, diglycerides (DAGs), monoglycerides (MAGs), FFAs and FAEEs. Lipid fractions were also analyzed by thin layer chromatography (TLC) using a mixture of n-hexane, diethyl ether and acetic acid (80:20:1, vol/vol/vol) as the developing agent (Nojima et al., 1995). 2.6. Analytical methods Glucose concentration was monitored with a SBA-50B glucose analyzer (Shandong Academy of Sciences, Jinan, China). The lipids were analyzed quantitatively by nuclear magnetic resonance (NMR) spectroscopy using a Bruker AVANCE III spectrometer (Bruker Co., Germany) operating at 400 MHz (Anderson and Franz, 2012). Lipids extracted from 8 mL of sample were dissolved in 1 mL of CDCl3 and transferred to an NMR tube. Spectra were recorded at room temperature with p-NBD as internal standard. NMR Data were processed using MestReNova 6.1 (Mestrelab Research, Spain). Proton chemical shifts were assigned as shown in Table 1. The amounts of fatty acyl group (nFA) in a lipid sample were quantified based on the amount of internal standard p-NBD (nNBD, 66.2 lmol) according to Eq. (1):
nFA ¼ ½2Ab =ðAf þ Ag Þ nNBD
ð1Þ
Ab, Af, and Ag are the integration areas associated with the proton of Hb in fatty acyl, Hf and Hg in p-NBD, respectively. The contents of the lipid components were determined by the ratio of the amounts of their fatty acyl group to those of the fatty acyl group of total lipids (TL). The equations for neutral lipid content are shown in Table 1. The contents of neutral lipids (N), glycolipids plus sphingolipids (G + S) and phospholipids (P) were calculated according to Eq. (2):
C i ¼ nFAðiÞ =nFAðTLÞ
ð2Þ
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Table 1 H NMR chemical shift assignments.
1
Molecules TAGs
DAGs
Protons
Content equationsa
Chemical shift d (ppm)
–CH2–O– –CH–O–
4.40–4.20 (Ha); 4.20–4.00 5.31–5.21 (Hc)
–CH–O–
5.11–5.02 (Hd)
ðH0a Þ
C TAGs ¼ Ac =Ab 6
C DAGs ¼ Ad =Ab 4 MAGs
–CH–O–
3.92–3.88 (He)
C MAGs ¼ Ae =Ab 2 FAEEs
4.20–4.00 ðH00a Þ
–CH2–O–
C FAEEs ¼ ðA2 A1 Þ=Ab Fatty acyl p-NBD
–CH2–CO–
Hf
2.40–2.20 (Hb) 8.50–8.30 (Hf); 8.20–8.00 (Hg)
g
H
O O2N Hf
g
The region area Ai was corresponding to the proton of Hi except that A2 was corresponding to H0a and H00a , and A1 was corresponding to Ha.
The ‘i’ represents the lipid component N, G + S, or P. Ci and nFA(i) are the content and the amount of fatty acyl group of lipid component ‘i’, respectively. nFA(TL) is the amount of fatty acyl group of total lipids. To determine fatty acid composition, total lipids were transmethylated according to a published procedure (Li et al., 2007). FAMEs and FAEEs were analyzed using a 7890F gas chromatography instrument (Techcomp Scientific Instrument Co. Ltd., Shanghai, China) equipped with a cross-linked capillary free fatty acid phase (FFAP) column (30 m 0.32 mm 0.4 lm) and flame ionization detector. Operating conditions were as follows: N2 41 mL/min, injection port temperature 250 °C, oven temperature 190 °C and detector temperature 280 °C. Fatty acids were identified by comparison of their retention times with those of standard ones, quantified based on their respective peak areas and normalized. Aliquots of 20 lL of cell sample were mixed with 80 lL of deionized water and 5 lL of 10 lg/mL nile red, successively. The cells were stained for 10 s and analyzed (excitation at 515– 560 nm, emission at >590 nm) using Eclipse 80i fluorescence microscope (Nikon Co., Japan) (Greenspan et al., 1985). Images were taken with a charge coupled device (CCD) camera. All the experiments were carried out in triplicate and the results were expressed as mean ± standard deviation of independent experiments. Means were compared by using t-Test and the level of significance was set at P < 0.05. 3. Results and discussion 3.1. FAEEs production by R. toruloides Y4 To test whether R. toruloides Y4 could make FAEEs during the lipid production process, cells were cultured in the presence of up to 10 vol% ethanol in the medium. However, no FAEEs were observed, indicating that there was no FAEEs synthase or its activity was low at the lipid production stage. Alternatively, lipids may be protected from being converted into other esters by an unknown mechanism. Interestingly, when the lipid-rich culture broth was mixed with 10 vol% ethanol and incubated at 35 °C for 84 h, TLC analysis of the resulting lipids indicated the formation of a large amount of FAEEs
(Supplementary Fig. 1). It was clear that total lipids contained TAGs, FFAs, and a small amount of DAGs, MAGs and polar lipids. To our surprise, neutral glycerides including TAGs, DAGs and MAGs almost disappeared. It should be noted that formation of FAEEs was observed at similar efficiency when experiments were held at 30–40 °C, and pH 5–8 (data not shown). These results indicated that R. toruloides cells could convert neutral glycerides into FAEEs effectively in aqueous phase. However, cells could not be revived in YPD liquid medium. In an early study, biodiesel was produced using lipid-rich yeast cells as the feedstock directly (Liu and Zhao, 2007). This was done in the presence of an acid catalyst under anhydrous conditions. In the current study, however, yeast cells provided lipids as well as biocatalyst for transesterification reaction in aqueous phase. To attain more quantitative information of the FAEEs production process, product samples were analyzed by the proton NMR method (Supplementary Fig. 2). It was clear that NMR data were consistent with TLC results. Because of the presence of internal standard p-NBD, FAEEs and related species could be quantified according to equations shown in Table 1. Thus, FAEEs yield was 65.7% to total lipids for the sample. Compositions of total lipids
100 90
Lipid fraction contents (%)
a
H
A
B
80 70 60 50 40 30 20 10 0 Neutral lipids
Glycolipids plus sphingolipids
Phospholipids
Fig. 1. Compositions of lipid samples. (A) Total lipids from R. toruloides Y4 cells. (B) Samples obtained from R. toruloides Y4 cells treated for 84 h in the presence of 10 vol% ethanol at 35 °C, pH 6.0.
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G. Jin et al. / Bioresource Technology 150 (2013) 266–270 Table 2 Fatty acid compositions of the total lipid and FAEEs products. Lipids
Relative fatty acid content (%)
Total lipids FAEEsa a
C14:0
C16:0
C16:1
C18:0
C18:1
C18:2
C18:3
1.1 ± 0.0 1.2 ± 0.0
26.7 ± 0.2 25.7 ± 0.2
0.4 ± 0.0 0.4 ± 0.1
10.5 ± 0.2 9.4 ± 0.4
53.4 ± 0.3 54.9 ± 0.2
5.8 ± 0.1 6.3 ± 0.1
1.8 ± 0.0 1.9 ± 0.1
Samples were prepared in the presence of 10 vol% ethanol at 35 °C, pH 6.0 for 84 h.
TAGs, DAGs, MAGs, FAEEs and FFAs (%)
80
TAGs
DAGs
MAGs
FAEEs
were analyzed by GC, and the distribution of fatty acids had no significant differences (Table 2). Thus, there was no preference over any particular fatty acids during FAEEs formation.
FFAs
70 60
3.2. Time course of FAEEs production
50 40 30 20 10 0 0
20
40
60
80
100
Time (h) Fig. 2. Time course of FAEEs production in aqueous phase by R. toruloides in the presence of 10 vol% ethanol at 35 °C, pH 6.0.
70
FAEEs yield (%)
60
To better understand the process of FAEEs production, the time course of neutral glycerides depletion as well as FAEEs and FFAs formation were investigated, and results are shown in Fig. 2. It was found that TAGs, DAGs and MAGs were depleted rapidly during the early stage, and dropped to a low level after 24 h. There appeared a burst for FFAs formation before 24 h, and the level then dropped over time to a final content of 20.0% at 84 h. FAEEs were produced continually over time up to 60 h, and then the formation rate was very slow. These results were consistent with a previous report that biodiesel production from plant oil was catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent (Kaieda et al., 1999). These data suggested that neutral glycerides were first hydrolyzed to FFAs, and then FFAs were esterified to FAEEs by R. toruloides. Since the rate of esterification was lower than that of the hydrolysis, FFAs were accumulated as intermediate products in the reaction mixture until neutral glycerides were exhausted.
50 40
3.3. Effect of lipid droplets
30
Yeast lipid droplets consist of a highly hydrophobic core formed from neutral lipids (TAGs and steryl esters (SEs)) surrounded by a phospholipid monolayer called lipid droplet membrane, in which a small number of proteins are embedded. Many of these proteins participate in lipid metabolism, such as phosphatidate and sterol synthesis, fatty acid activation, and TAGs and SEs synthesis/lipolysis (Athenstaedt et al., 1999; Ivashov et al., 2013; Zhu et al., 2012). For R. toruloides Y4, cells typically formed two lipid droplets located in the poles after cultured in lipid production media (Supplementary Fig. 3A). The lipid droplets are important for TAGs synthesis/lipolysis (Athenstaedt et al., 1999; Ivashov et al., 2013; Zhu et al., 2012). During the FAEEs production process, lipid droplets inflated to the center, but did not break (Supplementary Fig. 3B). So lipid droplets maintained their integrity and the membrane did not degrade. As shown in Fig. 1, the content of phospholipids did not change significantly after 84 h, which was supportive to the above observations. To demonstrate that the lipid droplets were important for FAEEs synthesis in aqueous phase, experiments
20 10 0
0
5
10
15
20
25
30
Ethanol concentration (vol%) Fig. 3. Effect of ethanol concentration on the FAEEs yield. Conversion conditions: 35 °C, pH 6.0 and 48 h.
were also analyzed by column chromatography. It was found that the contents of neutral lipids, glycolipids plus sphingolipids and phospholipids were 89.0%, 9.1% and 0.9%, respectively, for the sample at 0 h. There was little change in terms of compositional profile of lipids after 84 h (Fig. 1), indicating that only neutral lipids were involved in FAEEs production. So, FAEEs yield was 73.8% based on neutral lipids. Fatty acid compositions of total lipids and FAEEs
Fig. 4. Conceptive integration of microbial lipid technology and ethanol fermentation. The pretreatment of biomass includes chemical, physical and/or biological methods. The ethanol fermentation is especially the process producing higher than 10 vol% of ethanol.
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were done for 96 h with intact cells and cell homogenates of R. toruloides Y4. For intact cells, FAEEs and FFAs contents were 64.7% and 20.3%, while for cell homogenates, 23.5% and 10.5%, respectively. These data indicated that lipid droplets promoted the formation of both FAEEs and FFAs, but the formation of FAEEs was favored. Lipid droplets might provide a membrane structure to harbor enzymes for hydrolysis of neutral glycerides and esterification of FFAs, and a hydrophobic microenvironment for more efficient synthesis of FAEEs. It should be mentioned that high quality omics data about R. toruloides have been published recently (Zhu et al., 2012), which should promote further studies to unveil detailed mechanisms involved in converting glycerides into FAEEs intracellularly. 3.4. Effect of ethanol concentration The concentration of ethanol is one of the most important parameters in FAEEs production. While higher ethanol concentration may improve reaction rate and yield, it may also inhibit enzyme activities. As shown in Fig. 3, FAEEs yield increased with ethanol concentration increasing from 5 vol% to 10 vol%, but decreased when ethanol concentration was over 15 vol%. Currently, ethanol concentrations of fermentation industry are higher than 10 vol% by using very high gravity fermentation technology from renewable agriculture resources (Lim et al., 2013). Therefore, the ethanol fermentation broth can be directly used for FAEEs production without concentration process, which should reduce energy consumption and process costs. Furthermore, because ethanol was in large excess, residual stream after FAEEs production contained ethanol and reaction byproduct–glycerol, which could be returned to ethanol fermentation facility for reuse. So biodiesel production could be integrated with ethanol fermentation from renewable agriculture resources through this aqueous phase FAEEs production process by R. toruloides (Fig. 4). 4. Conclusion Results show that R. toruloides cells can effectively convert lipids into FAEEs intracellularly in aqueous phase. It was found that neutral glycerides were first hydrolyzed to FFAs followed by esterification and that lipid droplets played important roles in the process. This new process provides a novel opportunity for integration of microbial lipid technology with bioethanol fermentation for more efficient production of drop-in biofuels from renewable resources. Further study is required to improve the conversion efficiency. Acknowledgements This work was financially supported by the National Basic Research and Development Program of China (2011CB707405), the Knowledge Innovation Program of Chinese Academy of Sciences (KSCX2-EW-G-1-3), and the State Key Laboratory of Motor Vehicle Biofuel Technology (2013001). 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. 10.023. References Anderson, L.A., Franz, A.K., 2012. Real-time monitoring of transesterification by 1H NMR spectroscopy: catalyst comparison and improved calculation for biodiesel conversion. Energy Fuels 26, 6404–6410.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Fatty acid ethyl esters production in aqueous phase by the oleaginous yeast Rhodosporidium toruloides Guojie Jin a,c, Yixin Zhang a,c, Hongwei Shen a,b, Xiaobing Yang a, Haibo Xie a,b, Zongbao K. Zhao a,b,⇑ a
Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian 116023, PR China Dalian National Laboratory for Clean Energy, Dalian 116023, PR China c University of Chinese Academy of Sciences, Beijing 100049, PR China b
h i g h l i g h t s Fatty acid ethyl esters were produced in aqueous phase by the yeast Rhodosporidium toruloides. Up to 73% of cellular neutral glycerides were converted into fatty acid ethyl esters. Neutral glycerides were hydrolyzed to free fatty acids followed by esterification. Lipid droplets played important roles in the process.
a r t i c l e
i n f o
Article history: Received 30 July 2013 Received in revised form 5 October 2013 Accepted 7 October 2013 Available online 14 October 2013 Keywords: Biodiesel Fatty acid ethyl esters Aqueous phase conversion Lipid droplets Rhodosporidium toruloides
a b s t r a c t Fatty acid ethyl esters (FAEEs) are attractive biofuel molecules. Conventional FAEEs production process uses triglycerides and ethanol as feedstocks and is sensitive to water contents. In this work, we show that the oleaginous yeast Rhodosporidium toruloides cells are capable of converting lipids into FAEEs intracellularly in aqueous phase. Up to 73% of cellular neutral glycerides could be converted into FAEEs when lipid-rich cells were incubated for 84 h at 35 °C, pH 6.0 in a broth containing 10 vol% ethanol. It was found that neutral glycerides were first hydrolyzed to free fatty acids followed by esterification and that lipid droplets played important roles in the process. This new process provides a novel opportunity for integration of microbial lipid production technology with bioethanol fermentation for more efficient production of drop-in biofuels from renewable resources. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction With increasing transportation fuel demands and environmental concerns, biofuels have been recognized as important alternatives. Biodiesel, a typical form of liquid biofuel, has attracted considerable interest in recent years (Demirbas, 2007; Fjerbaek et al., 2009; Liang et al., 2013). Biodiesel consists of monoalkyl esters of long-chain fatty acids with short-chain alcohols, primarily methanol and ethanol, resulting in fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), respectively. In general, biodiesel is produced by transesterification of triglycerides (TAGs) with alcohols in the presence of different types of catalysts, such as bases, acids or enzymes (Cao et al., 2012; Fedosov et al., 2013; Ferella et al., 2010; Liu and Zhao, 2007; Liu et al., 2012). In these processes, water can facilitate hydrolysis of TAGs and in⇑ Corresponding author at: Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian 116023, PR China. Tel./fax: +86 411 84379211. E-mail address: [email protected] (Z.K. Zhao). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.10.023
crease the content of free fatty acids (FFAs). Base- and acid-catalyzed biodiesel production processes are particularly sensitive to water and require high-quality feedstock with a water content lower than 5 wt% (Atadashi et al., 2012). For those catalyzed by heterogeneous catalysts or lipases, biodiesel yields around 90% could be achieved for feedstocks with higher water contents (Tan et al., 2006; Yan et al., 2009). However, when water contents exceed 30%, biodiesel yields usually drop to below 60% (Atadashi et al., 2012). Because ethanol is less toxic than methanol and can be produced at large scale by fermentation from local and renewable agricultural resources, it is more appealing to make FAEEs as a sustainable biofuel. Efforts have been made for direct production of FAEEs by engineered cell factories. For example, recombinant Escherichia coli strains were constructed by expressing a wax ester synthase gene and ethanol-producing genes, such that FAEEs were successfully produced at a titre of 1.28 g/L after 72 h in the presence of exogenous fatty acids (Kalscheuer et al., 2006). When it was engineered to use the endogenous fatty acid pathway, E. coli
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could make 0.674 g/L FAEEs directly from simple sugars such as glucose (Steen et al., 2010). To avoid the toxicity of ethanol, a dynamic sensor-regulator system was developed to substantially improve strain stability as well as FAEEs titre to 1.5 g/L (Zhang et al., 2012). Similarly, the yeast Saccharomyces cerevisiae has also been explored for de novo production of FAEEs at a titer of 8.2 mg/L from glucose (Shi et al., 2012). However, the efficiency of direct FAEEs production by engineered cell factories remained low. It is tempting to develop a FAEEs production process in aqueous phase by using ethanol fermentation broth directly as the alcohol source. Ethanol fermentation broth normally contains less than 20% ethanol, plus water and byproducts such as acetaldehyde, glycerol and acetic acid. It has been known that the oleaginous yeast Rhodosporidium toruloides is a robust lipid producer as it can accumulate lipid over 70% with a titer of over 100 g/L (Li et al., 2007), and utilize lignocellulosic hydrolysates with excellent tolerance towards inhibitory compounds (Hu et al., 2009). Lipids in this yeast can be recovered easily by enzyme-assisted extraction (Jin et al., 2012). In this study, we show that R. toruloides cells are capable of converting lipids into FAEEs intracellularly in aqueous phase. This new process represents an efficient alternative approach to traditional technology using TAGs as the feedstock or engineered cell factories for FAEEs production. It also provides a novel opportunity for integration of microbial lipid production technology with bioethanol fermentation for more efficient production of drop-in biofuels from renewable resources. 2. Methods 2.1. Strain, media and chemicals R. toruloides Y4, a variant of R. toruloides AS 2.1389 obtained from the China General Microbiological Culture Collection Center, was grown in YPD liquid medium (containing 20 g/L glucose, 10 g/L yeast extract, and 10 g/L peptone, pH 6.0) at 30 °C and 200 rpm. The lipid production medium contained: 40 g/L glucose, 0.4 g/L KH2PO4, and 1.5 g/L MgSO47H2O, pH 6.0. After sterilized at 121 °C for 15 min, it was supplemented with 10 mL/L of a trace element solution contained (per liter): 4.0 g CaCl22H2O, 0.55 g FeSO47H2O, 0.52 g citric acidH2O, 0.10 g ZnSO47H2O, 0.076 g MnSO4H2O, and 100 lL 18 M H2SO4 (Wu et al., 2011). Lipid-rich culture was prepared according to a published procedure (Lin et al., 2011). R. toruloides Y4 cells were incubated in YPD liquid medium at 30 °C, 200 rpm for 24 h, collected by centrifugation, and washed twice with sterile 0.9 wt% NaCl solution. Subsequently, they were cultured in lipid production medium at 30 °C, 200 rpm until the glucose was exhausted. The final cell mass and lipid titer were 17.5 g/L and 7.3 g/L, respectively. Anhydrous ethanol (P99.8% purity) was obtained from Tianjin Hengxing Chemical Co., Ltd., Tianjin, China. Ethyl laurate (P99% purity), ethyl myristate (P98% purity), ethyl cis-9-Hexadecenoate (P95% purity), ethyl oleate (P98% purity), ethyl stearate (P99% purity), ethyl linolenate (P99% purity) and CDCl3 (99.8% D with 0.03% (vol/vol) tetramethylsilane (TMS)) were obtained from J&K Scientific Ltd., Beijing, China. Ethyl palmitate (P99% purity), ethyl linoleate (P99% purity), Nile red (chemical pure) and p-nitrobenzaldehyde (p-NBD, 98% purity) were obtained from Sigma, Shanghai, China. All other chemicals and reagents were analytical pure and obtained commercially. 2.2. FAEEs production by R. toruloides Y4 cells The typical procedure was performed as follows: aliquots of 6 mL of R. toruloides Y4 lipid-rich culture were mixed with 2 mL of 40 vol% ethanol solution in screw-capped vials sealed with para-
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film. The reaction mixture was incubated at 35 °C, pH 6.0 and 200 rpm for 48 h. Ethanol concentration and water content of the reaction system were 10 vol% and 91.3 wt%, respectively. When one of these factors including temperature, pH, ethanol concentration and reaction time was investigated, the levels of others were held constant. 2.3. FAEEs production catalyzed by R. toruloides Y4 cell homogenates R. toruloides Y4 cells from 6 mL of lipid-rich culture were lyophilized, milled to powder and extracted by cold acetone (Gu et al., 2011). Extracts and cell residues were mixed again, dried by N2, and then resuspended with 6 mL of deionized water. Then the cell homogenates were mixed with 2 mL of 40 vol% ethanol solution and incubated at 35 °C, pH 6.0 and 200 rpm for 96 h. 2.4. Lipid extraction Cells were homogenized with glass beads and extracted with chloroform–methanol (2:1, vol/vol) at room temperature (Folch et al., 1957). The organic extracts were washed with 0.1 wt% NaCl solution, dried over anhydrous Na2SO4, and evaporated under reduced pressure to give the total lipids. 2.5. Lipid fractionation About 50 mg of the lipid sample was loaded on a silica gel column, eluted by 120 mL of 1,2-dichloroethane, 80 mL of 1,2dichloroethane/acetone (1:10, vol/vol), and 50 mL methanol in sequence (Wu et al., 2010). The solvents from each fraction were evaporated under reduced pressure. The fractions, in the order of elution, were neutral lipids (N), glycolipids plus sphingolipids (G + S), and phospholipids (P). The neutral lipids included TAGs, diglycerides (DAGs), monoglycerides (MAGs), FFAs and FAEEs. Lipid fractions were also analyzed by thin layer chromatography (TLC) using a mixture of n-hexane, diethyl ether and acetic acid (80:20:1, vol/vol/vol) as the developing agent (Nojima et al., 1995). 2.6. Analytical methods Glucose concentration was monitored with a SBA-50B glucose analyzer (Shandong Academy of Sciences, Jinan, China). The lipids were analyzed quantitatively by nuclear magnetic resonance (NMR) spectroscopy using a Bruker AVANCE III spectrometer (Bruker Co., Germany) operating at 400 MHz (Anderson and Franz, 2012). Lipids extracted from 8 mL of sample were dissolved in 1 mL of CDCl3 and transferred to an NMR tube. Spectra were recorded at room temperature with p-NBD as internal standard. NMR Data were processed using MestReNova 6.1 (Mestrelab Research, Spain). Proton chemical shifts were assigned as shown in Table 1. The amounts of fatty acyl group (nFA) in a lipid sample were quantified based on the amount of internal standard p-NBD (nNBD, 66.2 lmol) according to Eq. (1):
nFA ¼ ½2Ab =ðAf þ Ag Þ nNBD
ð1Þ
Ab, Af, and Ag are the integration areas associated with the proton of Hb in fatty acyl, Hf and Hg in p-NBD, respectively. The contents of the lipid components were determined by the ratio of the amounts of their fatty acyl group to those of the fatty acyl group of total lipids (TL). The equations for neutral lipid content are shown in Table 1. The contents of neutral lipids (N), glycolipids plus sphingolipids (G + S) and phospholipids (P) were calculated according to Eq. (2):
C i ¼ nFAðiÞ =nFAðTLÞ
ð2Þ
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Table 1 H NMR chemical shift assignments.
1
Molecules TAGs
DAGs
Protons
Content equationsa
Chemical shift d (ppm)
–CH2–O– –CH–O–
4.40–4.20 (Ha); 4.20–4.00 5.31–5.21 (Hc)
–CH–O–
5.11–5.02 (Hd)
ðH0a Þ
C TAGs ¼ Ac =Ab 6
C DAGs ¼ Ad =Ab 4 MAGs
–CH–O–
3.92–3.88 (He)
C MAGs ¼ Ae =Ab 2 FAEEs
4.20–4.00 ðH00a Þ
–CH2–O–
C FAEEs ¼ ðA2 A1 Þ=Ab Fatty acyl p-NBD
–CH2–CO–
Hf
2.40–2.20 (Hb) 8.50–8.30 (Hf); 8.20–8.00 (Hg)
g
H
O O2N Hf
g
The region area Ai was corresponding to the proton of Hi except that A2 was corresponding to H0a and H00a , and A1 was corresponding to Ha.
The ‘i’ represents the lipid component N, G + S, or P. Ci and nFA(i) are the content and the amount of fatty acyl group of lipid component ‘i’, respectively. nFA(TL) is the amount of fatty acyl group of total lipids. To determine fatty acid composition, total lipids were transmethylated according to a published procedure (Li et al., 2007). FAMEs and FAEEs were analyzed using a 7890F gas chromatography instrument (Techcomp Scientific Instrument Co. Ltd., Shanghai, China) equipped with a cross-linked capillary free fatty acid phase (FFAP) column (30 m 0.32 mm 0.4 lm) and flame ionization detector. Operating conditions were as follows: N2 41 mL/min, injection port temperature 250 °C, oven temperature 190 °C and detector temperature 280 °C. Fatty acids were identified by comparison of their retention times with those of standard ones, quantified based on their respective peak areas and normalized. Aliquots of 20 lL of cell sample were mixed with 80 lL of deionized water and 5 lL of 10 lg/mL nile red, successively. The cells were stained for 10 s and analyzed (excitation at 515– 560 nm, emission at >590 nm) using Eclipse 80i fluorescence microscope (Nikon Co., Japan) (Greenspan et al., 1985). Images were taken with a charge coupled device (CCD) camera. All the experiments were carried out in triplicate and the results were expressed as mean ± standard deviation of independent experiments. Means were compared by using t-Test and the level of significance was set at P < 0.05. 3. Results and discussion 3.1. FAEEs production by R. toruloides Y4 To test whether R. toruloides Y4 could make FAEEs during the lipid production process, cells were cultured in the presence of up to 10 vol% ethanol in the medium. However, no FAEEs were observed, indicating that there was no FAEEs synthase or its activity was low at the lipid production stage. Alternatively, lipids may be protected from being converted into other esters by an unknown mechanism. Interestingly, when the lipid-rich culture broth was mixed with 10 vol% ethanol and incubated at 35 °C for 84 h, TLC analysis of the resulting lipids indicated the formation of a large amount of FAEEs
(Supplementary Fig. 1). It was clear that total lipids contained TAGs, FFAs, and a small amount of DAGs, MAGs and polar lipids. To our surprise, neutral glycerides including TAGs, DAGs and MAGs almost disappeared. It should be noted that formation of FAEEs was observed at similar efficiency when experiments were held at 30–40 °C, and pH 5–8 (data not shown). These results indicated that R. toruloides cells could convert neutral glycerides into FAEEs effectively in aqueous phase. However, cells could not be revived in YPD liquid medium. In an early study, biodiesel was produced using lipid-rich yeast cells as the feedstock directly (Liu and Zhao, 2007). This was done in the presence of an acid catalyst under anhydrous conditions. In the current study, however, yeast cells provided lipids as well as biocatalyst for transesterification reaction in aqueous phase. To attain more quantitative information of the FAEEs production process, product samples were analyzed by the proton NMR method (Supplementary Fig. 2). It was clear that NMR data were consistent with TLC results. Because of the presence of internal standard p-NBD, FAEEs and related species could be quantified according to equations shown in Table 1. Thus, FAEEs yield was 65.7% to total lipids for the sample. Compositions of total lipids
100 90
Lipid fraction contents (%)
a
H
A
B
80 70 60 50 40 30 20 10 0 Neutral lipids
Glycolipids plus sphingolipids
Phospholipids
Fig. 1. Compositions of lipid samples. (A) Total lipids from R. toruloides Y4 cells. (B) Samples obtained from R. toruloides Y4 cells treated for 84 h in the presence of 10 vol% ethanol at 35 °C, pH 6.0.
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G. Jin et al. / Bioresource Technology 150 (2013) 266–270 Table 2 Fatty acid compositions of the total lipid and FAEEs products. Lipids
Relative fatty acid content (%)
Total lipids FAEEsa a
C14:0
C16:0
C16:1
C18:0
C18:1
C18:2
C18:3
1.1 ± 0.0 1.2 ± 0.0
26.7 ± 0.2 25.7 ± 0.2
0.4 ± 0.0 0.4 ± 0.1
10.5 ± 0.2 9.4 ± 0.4
53.4 ± 0.3 54.9 ± 0.2
5.8 ± 0.1 6.3 ± 0.1
1.8 ± 0.0 1.9 ± 0.1
Samples were prepared in the presence of 10 vol% ethanol at 35 °C, pH 6.0 for 84 h.
TAGs, DAGs, MAGs, FAEEs and FFAs (%)
80
TAGs
DAGs
MAGs
FAEEs
were analyzed by GC, and the distribution of fatty acids had no significant differences (Table 2). Thus, there was no preference over any particular fatty acids during FAEEs formation.
FFAs
70 60
3.2. Time course of FAEEs production
50 40 30 20 10 0 0
20
40
60
80
100
Time (h) Fig. 2. Time course of FAEEs production in aqueous phase by R. toruloides in the presence of 10 vol% ethanol at 35 °C, pH 6.0.
70
FAEEs yield (%)
60
To better understand the process of FAEEs production, the time course of neutral glycerides depletion as well as FAEEs and FFAs formation were investigated, and results are shown in Fig. 2. It was found that TAGs, DAGs and MAGs were depleted rapidly during the early stage, and dropped to a low level after 24 h. There appeared a burst for FFAs formation before 24 h, and the level then dropped over time to a final content of 20.0% at 84 h. FAEEs were produced continually over time up to 60 h, and then the formation rate was very slow. These results were consistent with a previous report that biodiesel production from plant oil was catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent (Kaieda et al., 1999). These data suggested that neutral glycerides were first hydrolyzed to FFAs, and then FFAs were esterified to FAEEs by R. toruloides. Since the rate of esterification was lower than that of the hydrolysis, FFAs were accumulated as intermediate products in the reaction mixture until neutral glycerides were exhausted.
50 40
3.3. Effect of lipid droplets
30
Yeast lipid droplets consist of a highly hydrophobic core formed from neutral lipids (TAGs and steryl esters (SEs)) surrounded by a phospholipid monolayer called lipid droplet membrane, in which a small number of proteins are embedded. Many of these proteins participate in lipid metabolism, such as phosphatidate and sterol synthesis, fatty acid activation, and TAGs and SEs synthesis/lipolysis (Athenstaedt et al., 1999; Ivashov et al., 2013; Zhu et al., 2012). For R. toruloides Y4, cells typically formed two lipid droplets located in the poles after cultured in lipid production media (Supplementary Fig. 3A). The lipid droplets are important for TAGs synthesis/lipolysis (Athenstaedt et al., 1999; Ivashov et al., 2013; Zhu et al., 2012). During the FAEEs production process, lipid droplets inflated to the center, but did not break (Supplementary Fig. 3B). So lipid droplets maintained their integrity and the membrane did not degrade. As shown in Fig. 1, the content of phospholipids did not change significantly after 84 h, which was supportive to the above observations. To demonstrate that the lipid droplets were important for FAEEs synthesis in aqueous phase, experiments
20 10 0
0
5
10
15
20
25
30
Ethanol concentration (vol%) Fig. 3. Effect of ethanol concentration on the FAEEs yield. Conversion conditions: 35 °C, pH 6.0 and 48 h.
were also analyzed by column chromatography. It was found that the contents of neutral lipids, glycolipids plus sphingolipids and phospholipids were 89.0%, 9.1% and 0.9%, respectively, for the sample at 0 h. There was little change in terms of compositional profile of lipids after 84 h (Fig. 1), indicating that only neutral lipids were involved in FAEEs production. So, FAEEs yield was 73.8% based on neutral lipids. Fatty acid compositions of total lipids and FAEEs
Fig. 4. Conceptive integration of microbial lipid technology and ethanol fermentation. The pretreatment of biomass includes chemical, physical and/or biological methods. The ethanol fermentation is especially the process producing higher than 10 vol% of ethanol.
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were done for 96 h with intact cells and cell homogenates of R. toruloides Y4. For intact cells, FAEEs and FFAs contents were 64.7% and 20.3%, while for cell homogenates, 23.5% and 10.5%, respectively. These data indicated that lipid droplets promoted the formation of both FAEEs and FFAs, but the formation of FAEEs was favored. Lipid droplets might provide a membrane structure to harbor enzymes for hydrolysis of neutral glycerides and esterification of FFAs, and a hydrophobic microenvironment for more efficient synthesis of FAEEs. It should be mentioned that high quality omics data about R. toruloides have been published recently (Zhu et al., 2012), which should promote further studies to unveil detailed mechanisms involved in converting glycerides into FAEEs intracellularly. 3.4. Effect of ethanol concentration The concentration of ethanol is one of the most important parameters in FAEEs production. While higher ethanol concentration may improve reaction rate and yield, it may also inhibit enzyme activities. As shown in Fig. 3, FAEEs yield increased with ethanol concentration increasing from 5 vol% to 10 vol%, but decreased when ethanol concentration was over 15 vol%. Currently, ethanol concentrations of fermentation industry are higher than 10 vol% by using very high gravity fermentation technology from renewable agriculture resources (Lim et al., 2013). Therefore, the ethanol fermentation broth can be directly used for FAEEs production without concentration process, which should reduce energy consumption and process costs. Furthermore, because ethanol was in large excess, residual stream after FAEEs production contained ethanol and reaction byproduct–glycerol, which could be returned to ethanol fermentation facility for reuse. So biodiesel production could be integrated with ethanol fermentation from renewable agriculture resources through this aqueous phase FAEEs production process by R. toruloides (Fig. 4). 4. Conclusion Results show that R. toruloides cells can effectively convert lipids into FAEEs intracellularly in aqueous phase. It was found that neutral glycerides were first hydrolyzed to FFAs followed by esterification and that lipid droplets played important roles in the process. This new process provides a novel opportunity for integration of microbial lipid technology with bioethanol fermentation for more efficient production of drop-in biofuels from renewable resources. Further study is required to improve the conversion efficiency. Acknowledgements This work was financially supported by the National Basic Research and Development Program of China (2011CB707405), the Knowledge Innovation Program of Chinese Academy of Sciences (KSCX2-EW-G-1-3), and the State Key Laboratory of Motor Vehicle Biofuel Technology (2013001). 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. 10.023. References Anderson, L.A., Franz, A.K., 2012. Real-time monitoring of transesterification by 1H NMR spectroscopy: catalyst comparison and improved calculation for biodiesel conversion. Energy Fuels 26, 6404–6410.
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