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Exploration of sodium lignosulphonate's effects on lipid production by Rhodosporidium toruloides
Process Biochemistry 50 (2015) 424–431
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Exploration of sodium lignosulphonate’s effects on lipid production by Rhodosporidium toruloides Jingyang Xu a,b,∗ , Wei Du b , Xuebing Zhao b , Dehua Liu b a b
Key Laboratory of Forensic Science and Technology, Zhejiang Police College, Hangzhou 310053, PR China Institute of Applied Chemistry, Department of Chemical Engineering, Tsinghua University, Beijing 100084, PR China
a r t i c l e
i n f o
Article history: Received 24 July 2014 Received in revised form 21 December 2014 Accepted 1 January 2015 Available online 17 January 2015 Keywords: Microbial lipid R. toruloides Surfactant Sodium lignosulphonate Oxygen transfer
a b s t r a c t Microbial oil is drawing increasing interest all over the world as an alternative non-food feedstock for biodiesel production. Rhodosporidium toruloides has been considered as a promising candidate to convert glycerol into microbial oils due to its good substrate adaptability, high intracellular lipid content, and the potential of co-production of some pigments. Interestingly, we have also found that sodium lignosulphonate (SL) could significantly enhance the cell growth and lipid accumulation of R. toruloides. In the present work, we investigated the possible mechanisms for the enhancement of lipid production by SL. It was found that SL could not be utilized by R. toruloides as a carbon source. No apparent changes on the morphology of cell membranes and the fatty acid profiles of microbial lipids were found when SL was added. Further experiments indicated that the volumetric oxygen transfer coefficient was increased by nearly 15% on average by adding 4 g/L SL, which might be the main mechanism for the improvement of lipid production. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, researchers have been increasingly interested in developing the potential of oleaginous microorganisms to produce microbial oil as an alternative non-food oil feedstock for biodiesel preparation [1,2]. Among those oleaginous microorganisms, Rhodosporidium toruloides is considered as a promising candidate due to its excellent capability of lipid accumulation, broad adaptability to various substrates, and the potential of co-production of some carotenoids [2–4]. Our previous study also proved that R. toruloides could well convert refined and crude glycerol into lipids, which showed great potential to utilize the biodiesel byproduct glycerol as a sustainable and inexpensive carbon source to produce microbial oil, and in turn the oil could be used as the biodiesel feedstock [5]. In order to obtain microbial oils in a more effective way, much effort has been focused on directing carbon fluxes toward lipid synthesis either through optimization of cultivation process or biotechnological approaches such as genetic engineering or metabolic engineering [2,6]. What is more, researchers noticed that some chemicals with the structure of surfactants could enhance
∗ Corresponding author at: Key Laboratory of Forensic Science and Technology, Zhejiang Police College, Hangzhou 310053, PR China. Tel.: +86 571 87787219. E-mail address: [email protected] (J. Xu). http://dx.doi.org/10.1016/j.procbio.2015.01.006 1359-5113/© 2015 Elsevier Ltd. All rights reserved.
lipid accumulation. For example, Saenge et al. found that the accumulation of lipid and carotenoids in R. glutinis was increased by supplementing a non-ionic surfactant Tween 20 [7]; Taoka et al. found that non-ionic surfactant Tween 80 could promote cell growth and lipid accumulation of T. aureum, and the oleic acid composition in lipid profiles increased. We have also investigated the effects of several commonly used surfactants, and found that some anionic surfactants could promote the lipid production process of R. toruloides. However, the exact mechanism of how surfactants affect microbial lipid production was not well understood. According to our previous study, it was interesting to find that sodium lignosulphonate (SL), a typical surfactant, showed positive effect in cultivating R. toruloides by increasing the biomass concentration, and was able to enhance enzymatic saccharification of lignocelluloses as well [8–10]. As we know, lignosulphonates are by-products of the biomass pulping processes. Since they are inexpensive, renewable and readily available [11], a valueadded utilization way of lignosulphonates can be beneficial to not only the biorefinery process, but also to the pulping industry. In the present work, we investigated for the first time the possible mechanism of SL on lipid production of R. toruloides, by considering the possibility of SL for being utilized as a carbon source, the effect of SL on cell membrane morphology, and the influence of SL on oxygen transfer in lipid accumulation process.
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2. Materials and methods
425
10 ◦ C/min, and then maintained for 6 min. The temperatures of the injector and detector were set at 245 ◦ C and 260 ◦ C, respectively.
2.1. Microorganism and chemicals 2.4. Transmission electron microscopy [17,18] R. toruloides AS 2. 1389 was obtained from China General Microbiological Culture Collection Center (CGMCC). For preparing inocula, the organism was pre-cultured overnight in YPG medium containing 15 g/L glycerol, 10 g/L yeast extract and 10 g/L peptone at 30 ◦ C and 200 rpm in an air-bath shaker. SL was purchased from Tokyo Chemical Industry Co., Ltd., which contains 5.0–7.0% methoxyl group. Other chemicals in all the experiments were purchased locally and were analytically pure.
Samples of fermentation broth (1 mL) from 2 to 4 days culture were centrifuged (3000 rpm, 2 min). The supernatant liquor was discarded, and the yeast cells were harvested and fixed with 200 L KMnO4 (1.5%, w/v). Samples were preserved in refrigerator at 4 ◦ C. They were then post-stained and cut by an ultramicrotome. The ultrathin sections were observed in the transmission electron microscopy (TEM) of Model H-7650B (Hitachi, Japan).
2.2. Fermentation process
2.5. Determination of kL a
Batch fermentation was conducted in 500 mL flasks with 100 mL liquid medium containing 50 g/L glycerol, 0.1 g/L (NH4 )2 SO4 , 0.75 g/L yeast extract, 0.4 g/L KH2 PO4 , and 1.5 g/L MgSO4 ·7H2 O. Before inoculation, the biomass concentration of the inocula was carefully controlled at 0.5–0.6 g/L. Each culture was performed by triplicate test.
Double-film theory was used to describe oxygen transfer processes, and the volumetric mass transfer coefficient kL a was used to represent the oxygen transfer efficiency. The dynamic method based on the dissolved oxygen electrode [19] was used for determining the kL a (s−1 , volumetric oxygen transfer coefficient). The oxygen concentration was monitored by an oxygen sensor (VISFERM DO ARC 225, Hamilton Corp.). Specifically, during the fermentation process in a 2-L bioreactor with an aeration condition of 0.5 vvm, aeration was ceased for a very short time at a certain moment, and the dissolved oxygen (DO, mg L−1 ) decreased linearly. Once aeration was recovered, the DO gradually increased again. kL a was further calculated using the following kinetic model. Where DO* (mg L−1 ) is the dissolved oxygen concentration at saturation in a gas–liquid equilibrium, Q is the respiration rate (mg g−1 s−1 ), X (g/L) is the biomass concentration.
2.3. Analytical methods Glycerol concentration was determined by a Shimadzu10AVP HPLC system (Shimadzu Corp., Japan) equipped with a RID10A refractive index detector, Aminex HPX-87H column (300 mm × 7.8 mm, Bio-Rad, USA) at 65 ◦ C with 5 mM H2 SO4 as the eluent at a flow rate of 0.8 mL/min. Ultraviolet–Visible spectrophotometry is a common method to characterize and measure the concentration of lignosulphonates [12–14]. In the present work, SL was added in the 100 mL medium at the beginning of fermentation, and the initial concentrations were up to 2 g/L and 4 g/L, respectively. Fermentation broth samples were taken from the cultivation every 24 h. The concentration of SL was determined by a UV-vis 8500 spectrophotometer (Shanghai Tianmei Instrument Co., Ltd., China). The concentration of SL has a linearity relation with the absorbance at 235 nm. One absorbance unit corresponds to 0.0558 g/L SL. The biomass concentration was expressed as the dry weight concentration of cell biomass. Samples of fermentation broth were centrifuged, washed with deioned water, and dried at 105 ◦ C to a constant weight. Intracellular lipids were extracted by acid-heating procedure [15] with mixture of chloroform and methanol (1:1, v/v) as the extractant. After solvent extraction, the chloroform phase was taken and washed with 0.1% NaCl. After the solvent washing and evaporation, the lipid extract was weighted. Cell biomass (CB ), lipid concentration (CL ) and lipid content (CnL ) are defined as follows, respectively: CB (g/L) =
dry weight of cell biomass (g) volume of fermentation broth (L)
CL (g/L) =
weight of lipid (g) volume of fermentation broth (L)
CnL (%) =
weight of lipid (g) × 100% weight of cell biomass (g)
Methanolysis was carried out following the procedures described by Li et al. [16]. Fatty acid compositions of the methyl ester were analyzed with a GC-14B gas chromatography (Shimadzu, Japan) equipped with a CP-FFAP CB capillary column (25 m × 0.32 m × 0.30 m) produced by Agilent. Heptadecanoic acid methyl ester was used as internal standard. The column temperature was kept at 180 ◦ C for 0.5 min and heated to 250 ◦ C at
dDO = KL a × (DO∗ − DO) − QO2 X dt OTR = KL a × (DO∗ − DO) In the SL–water system without inoculation of R. toruloides cells, SL was supplemented at 0 (control group), 0.3, 1.0, 1.5, 2.0, 2.5, 3.0, and 4.0 g/L respectively. Determination of kL a followed the same procedures, but the respiration rate item (QX) was zero. 3. Results and discussions 3.1. Effect of SL on lipid accumulation Lignosulphonate is a polymeric surfactant derived from lignin. It is a main by-product in sulfite pulping or pretreatment of biomass [20]. In our previous study, Zhao et al. have found that SL could somehow enhance cell growth of R. toruloides [8]. In the present work, we further investigated the effect of SL on lipid accumulation of R. toruloides. As shown in Fig. 1, the addition of 1–6 g/L SL resulted in increases of biomass and lipid accumulation. The most significant improvement was observed when 2–4 g/L SL was added. It is possible that SL as a salt might alter the ionic strength of the fermentation broth, thus affecting lipid accumulation. However, we had investigated the effects of salt (NaCl) on the growth and lipid production using R. toruloides. No significant effects were observed by adding NaCl with concentration below 4 g/L. In the present work, addition of 2–4 g/L SL led to sodium concentration of far below 4 g/L. Therefore, the positive effect caused by SL is not due to sodium. Other mechanisms should be considered. The effects of surfactants on lipid production have also been reported by other researchers. Saenge et al. found that the concentrations of lipid and carotenoid in R. glutinis were increased by supplementing Tween 20 [7], and they proposed that it was probably because Tween 20 influenced the physicochemical status of the culture medium and certain nutrients became easier to be taken by
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the physiochemical features of the fermenting liquor, and is able to promote the mass transfer including oxygen transfer. We will discuss the possible mechanisms later. 3.3. Effect of SL on the membrane morphology and lipid profiles of R. toruloides
Fig. 1. The effect of SL on biomass and lipid accumulation of R. toruloides. Symbol elements: (): CB ; (䊉): CL ; (): CnL .
the yeast cell. Taoka et al. found that non-ionic surfactant Tween 80 could promote cell growth and lipid accumulation of T. aureum, and the oleic acid composition in lipid profiles increased. They analyzed that probably Tween 80 was utilized as the carbon source, and might affect the activity of fatty acid synthases. Besides, it was also possible that Tween 80 interferes with the cell membrane permeability, and the cell was enhanced to uptake some essential components [21]. However, the effect of SL on oleaginous microorganisms has not been elucidated and further studies are needed to investigate the mechanism of how it should affect microbial lipid production. 3.2. Possibility of SL utilized as a carbon source for lipid accumulation As a lignin derivative, SL has aromatic rings and conjugated carbonyl groups, thus is able to absorb ultraviolet light. In the present work, Ultraviolet–visible spectrophotometry was used to measure the variation of SL concentration in the culture medium during the lipid production process. It was found that the concentration of SL did not vary throughout the cultivation time from 0 h to 207 h, which indicated that SL could not be uptaken and utilized by R. toruloides (Fig. 2). According to this result, we further suggested other possible effects of SL on R. toruloides. For instance, SL may interfere with the cell membrane and then enhance the uptake of some essential nutrients. Besides, SL may exert some influences on
We further investigated the effect of SL on the membrane morphology of R. toruloides. Some researchers pointed out that surfactants could affect the membrane permeability, thus it might be easier to uptake some components or excrete some intracellular substances out of the cell. By this means, microbial production might be promoted by enhanced nutrient uptake or decreased feedback inhibition [22,23]. However, supplementation has to be well regulated. Otherwise the surfactants may dissolve the cell membrane and result in cells losing the reproduction capability [24,25]. The variation in membrane morphology could be observed by Scanning Electron Microscope (SEM) or Transmission Electron Microscope (TEM). Wu et al. investigated the effect of Polyoxyethylene nonyl phenyl ether-10 on the morphology of K. pneumonia by TEM, and observed the plasmolysis and deformation of outer membranes [23]. Galabova et al. studied the effect of Triton X-100 on the morphology of Yarrowia lipolytica by SEM and TEM, who found that when the addition concentration was up to 0.1% (w/v), cell shapes were altered. A disruption of the cell wall in certain zones was observed, where cell walls folded, cell membranes (cell walls and cytoplasmic membranes) became thin, translucent and indistinct compared with controls [26]. In the present work, different addition concentrations ranged from 1 to 6 g/L were tested. We prepared ultrathin sections of R. toruloides cells for TEM observation, and observed the sections of yeast cells at different growth phases. The representative images are taken and shown in Fig. 3. As is shown in Fig. 3a, the cells of control group had intact cell membranes with distinct outer and inner membrane outlines. Cells of experimental groups with addition of SLs did not appear deformed, where smooth surface of membrane structures could still be observed. Although it is still possible that the fluidity of cell membranes was altered, which phenomenon could not be observed by TEM. At least, the present result indicated that SL did not have significant effect on the cell morphology of R. toruloides. We further analyzed the microbial lipids to study whether SL would affect the fatty acid profiles (Table 1). Fatty acid compositions play an essential role in the biophysical characteristics of cell membranes. The dependence of membrane fluidity on the extent of unsaturation of fatty acids is a well characterized phenomenon, which has been demonstrated in animals, fungi, yeasts, plants and bacteria [27,28]. In the yeast Rodosporidium, our research group also has found that the ability of cells to alter the degree of unsaturation in their lipid compositions is an important factor in cellular acclimatization to the environmental conditions. If there is a significant change in the fluidization of cellular membrane, the fatty acid compositions will show a difference. In the present study, the predominant fatty acids of R. toruloides lipids were palmitic acid, stearic acid, oleic acid, and linoleic acid, wherein palmitic acid and oleic acid accounted for about 70% of the total fatty acid compositions. Compared with the control experiment, the fatty acid compositions were not significantly changed when 2–6 g/L SLs were supplemented. This result implied that SL did not affect the fatty acid profiles of the cellular lipids. 3.4. Effect of SL on oxygen transfer
Fig. 2. The concentration of SL during the fermentation.
Cell culture is a typical multi-phase system, including the gas phase (O2 , CO2 ), solid phase (cells) and liquid phase (fermentation liquor), where mass transfer process between different
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Fig. 3. The effect of SL on the morphology of cell membranes. (a) Control; (b) 1 g/L SL; (c) 4 g/L SL (d) 6 g/L SL; Yeast Cells were sampled at 48 h. (e) Control; (f) 1 g/L SL; (g) 4 g/L SL (h) 6 g/L SL; Yeast Cells were sampled at 96 h.
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Table 1 The effect of SL on fatty acid profiles of R. toruloides lipid. SL (g/L)
C14:0
0 2 4 6
1.4 1.8 1.6 1.6
± ± ± ±
C16:0 0.0 0.1 0.1 0.1
25.8 27.4 27.5 28.5
± ± ± ±
C16:1 0.5 1.0 0.6 1.0
2.5 1.9 3.1 3.1
± ± ± ±
C18:0 0.1 0.2 0.1 0.1
phases can be a limiting factor for a bio-reaction process. For those aerobic microorganisms, as the terminal electron acceptor in respiration, oxygen is the most common and important composition in the gas phase. Moreover, oxygen transfer is one of the most important mass transfer processes in fermentations. Since oxygen has poor solubility in water, the low concentration difference between the gas–liquid phases resulted in a limited mass transfer.
12.5 11.9 11.9 11.9
± ± ± ±
C18:1 1.2 1.2 0.9 1.0
44.8 46.4 44.2 42.2
± ± ± ±
C18:2 0.5 1.9 1.1 1.2
10.6 8.8 9.5 10.5
± ± ± ±
C18:3 0.0 0.9 0.1 0.1
2.2 1.9 2.3 2.3
± ± ± ±
0.0 0.0 0.1 0.1
We found that oxygen supply condition in the fermentation broth was correlated to the cell growth (Fig. 4). Optimized oxygen supply condition could enhance the cell growth and lipid accumulation in R. toruloides. Otherwise, both the cell growth and lipid accumulation would be inhibited. The importance of aeration strategy has also been described in other literatures. Yen et al. reported the inhibited cell growth of R. glutinis under a poor oxygen aeration condition [29]. Li [30] and Zhao [31] et al. provided a high dissolved
Fig. 4. The effect of oxygen supply condition on cell growth and lipid production of R. toruloides. Oxygen supply conditions: (a) and (b) 1.1 vvm, 300 rpm; (c) and (d) 0.75 vvm, 270 rpm; (e) and (f) 0.5 vvm, 270 rpm. Symbol elements: (): CB ; (䊉): CL ; (): CnL ; (): DO.
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Table 2 Effect of SL on KL a and DO* values. Stirring speed (rpm)
SL (g/L)
KL a (s−1 )
100
0 0.3 1.0 1.5 2.0 2.5 3.0 4.0
0.0005 0.0007 0.0013 0.0019 0.0006 0.0006 0.0007 0.0019
± ± ± ± ± ± ± ±
0.0000 0.0000 0.0000 0.0001 0.0000 0.0000 0.0000 0.0004
6.93 6.76 6.82 6.71 7.12 6.89 6.90 6.60
± ± ± ± ± ± ± ±
0.01 0.03 0.02 0.01 0.02 0.01 0.02 0.08
200
0 0.3 1.0 1.5 2.0 2.5 3.0 4.0
0.0021 0.0025 0.0022 0.0040 0.0032 0.0026 0.0032 0.0024
± ± ± ± ± ± ± ±
0.0001 0.0000 0.0001 0.0002 0.0001 0.0001 0.0002 0.0001
6.72 6.69 6.78 6.63 6.70 6.80 6.66 6.67
± ± ± ± ± ± ± ±
0.07 0.05 0.02 0.10 0.01 0.01 0.10 0.08
300
0 0.3 1.0 1.5 2.0 2.5 3.0 4.0
0.0025 0.0038 0.0039 0.0072 0.0088 0.0088 0.0054 0.0050
± ± ± ± ± ± ± ±
0.0001 0.0001 0.0002 0.0001 0.0004 0.0003 0.0002 0.0001
6.76 6.66 6.79 6.62 6.63 6.55 6.73 6.52
± ± ± ± ± ± ± ±
0.04 0.03 0.03 0.02 0.01 0.03 0.08 0.05
500
0 0.3 1.0 1.5 2.0 2.5 3.0 4.0
0.0101 0.0113 0.0114 0.0196 0.0157 0.0158 0.0151 0.0140
± ± ± ± ± ± ± ±
0.0001 0.0001 0.0004 0.0014 0.0002 0.0003 0.0001 0.0004
6.77 6.75 6.76 6.70 6.74 6.79 6.80 6.69
± ± ± ± ± ± ± ±
0.02 0.01 0.02 0.01 0.00 0.01 0.01 0.02
oxygen level of 40–50% to ensure a sufficient oxygen supply for R. toruloides Y4. As an aerobic oleaginous yeast, R. toruloides utilize oxygen in both the cell growth and lipid synthesis. Oxygen even plays a role in synthesis of unsaturated fatty acids, as an electron acceptor in the dehydrogenation step catalyzed by desaturases. It was also reported that some surfactants could decrease the average diameter of air bubbles by reducing the surface tension, and thus were able to increase the gas–liquid interfacial area and enhance the oxygen transfer between the gas and liquid phases [19]. To discuss the mechanism of how SL influences the cell growth and lipid accumulation of R. toruloides, we investigated the effect of SL on oxygen transfer in both SL–water system and lipid production system.
3.4.1. Effect of SL on oxygen transfer in SL–water system In a fermentation system, the media contains different components and the respiration of microorganisms makes the oxygen transfer complicated. For simplification, we first carried out experiments in deionized water system without inoculation, in which the supplementation of SL was the only variable and all other operation parameters were kept consistent. The kL as determined at stirring speeds of 100–500 rpm and under different SL addition conditions are shown in Table 2. According to the kL a and DO* values, DO* did not varied at different SL concentrations, which indicated that SL had no effect on the dissolved oxygen concentration in a liquid–gas equilibrium status. Increase of kL a value was correlated to the SL concentration, which implied that SL could enhance the oxygen transfer in water. We also noticed that at each stirring condition there was a peak range of kL a. Further increase of SL could not promote the oxygen transfer but decrease the kL a. Surfactants may affect kL a by increasing the interface area of oxygen transfer. However, excessive addition of
DO* (mg L−1 )
surfactants may impede the mobility of gas–liquid interface, and cause an inhibitive effect [19]. 3.4.2. Effect of SL on oxygen transfer in fermentation system Effect of SL on oxygen transfer in fermentation system was further investigated. For every 24 h, biomass concentration and oxygen consumption rate were tested, and the oxygen consumption rate per gram of biomass could be calculated accordingly. The results are summarized in Fig. 5 and Table 3. In the present work, since the seed cells were inoculated directly into the nitrogen limited batch media, both the cell growth and lipid accumulation were activated soon. As a result, oxygen consumption was significant in the beginning stage, as shown in Fig. 5. In the late stage of lipid production, oxygen consumption per gram of biomass decreased, which suggested a comparatively lower need of oxygen in the lipid synthesis process. However, the overall oxygen consumption was still of high rate due to the biomass growth. Comparing the experimental group supplemented with SL with the control one, we know that the overall oxygen consumption was higher for the experimental group, but the oxygen consumptions per gram of biomass were almost the same. Table 3 also implied that SL had a positive effect on the oxygen transfer coefficient and oxygen transfer rate. The kL a value was increased by about 15% on average. The above results indicated that SL indeed could enhance the cell growth and lipid accumulation of R. toruloides by promoting the oxygen transfer process. Such effect might be related to the physiochemical feature of SL and its behavior in fermentation media. SL has some special characteristics such as hydrophilicity, lipophilicity, absorbability and dispersibility [32–34]. It may decrease the air bubble diameter by reducing the surface tension, increase the interface area between the gas phase and liquid phase, or exert an influence on the mass transfer by affecting certain physiochemical
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Table 3 Effect of SL on oxygen transfer. KL a (s−1 )
Time (h)
OTR (mg L−1 S−1 )
Control 0 21 45 70 90 116 140 160
0.0121 0.0159 0.0073 0.0079 0.0071 0.0080 0.0079 0.0085
SL 4 g/L ± ± ± ± ± ± ± ±
0.0001 0.0005 0.0002 0.0004 0.0001 0.0004 0.0001 0.0002
0.0111 0.0209 0.0114 0.0083 0.0092 0.0073 0.0089 0.0098
Control ± ± ± ± ± ± ± ±
0.0003 0.0003 0.0007 0.0001 0.0006 0.0002 0.0002 0.0001
(a)
0.0206 0.0636 0.0394 0.0387 0.0341 0.0304 0.0284 0.0298
SL 4 g/L ± ± ± ± ± ± ± ±
0.0010 0.0029 0.0018 0.0021 0.0005 0.0016 0.0004 0.0009
0.0266 0.0815 0.0638 0.0415 0.0478 0.0328 0.0392 0.0382
± ± ± ± ± ± ± ±
0.0011 0.0033 0.0034 0.0005 0.0026 0.0012 0.0010 0.0012
Acknowledgments The authors express their gratitude to the support from Tsinghua University Initiative Support Program (20111081123, 20121080046) and “863” Project (2012AA052101).
References
(b)
Fig. 5. Oxygen consumption rate in lipid production process. (a) Oxygen consumption rate; (b) oxygen consumption rate per gram of biomass. Symbol elements: (): Control; (䊉): 4 g/L SL.
features [19]. It has been reported that some surfactants even could form “colloidal gas aphrons”, and aeration under such conditions could significantly enhance the volumetric oxygen transfer coefficient [35–37]. However, in an actual fermentation process, the media contains different kinds of components including inorganic compounds, organic compounds and microorganism cells, and the microorganisms are continuously exchange substances with the fermentation media. In such a complicated system, the microscopic behavior of SL deserves further exploration. 4. Conclusion SL could somehow promote the lipid production process. The concentration of SL in the fermentation media did not vary at different stages of lipid production, which suggested that SL could not be utilized by R. toruloides. The cell membrane of R. toruloides with supplementation of SL showed similar morphology with that of control cells, and the fatty acid profiles of the microbial lipids remained unchanged. It is probable that SL plays a positive role in oxygen transfer thus improving substrate utilization for lipid production.
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Exploration of sodium lignosulphonate’s effects on lipid production by Rhodosporidium toruloides Jingyang Xu a,b,∗ , Wei Du b , Xuebing Zhao b , Dehua Liu b a b
Key Laboratory of Forensic Science and Technology, Zhejiang Police College, Hangzhou 310053, PR China Institute of Applied Chemistry, Department of Chemical Engineering, Tsinghua University, Beijing 100084, PR China
a r t i c l e
i n f o
Article history: Received 24 July 2014 Received in revised form 21 December 2014 Accepted 1 January 2015 Available online 17 January 2015 Keywords: Microbial lipid R. toruloides Surfactant Sodium lignosulphonate Oxygen transfer
a b s t r a c t Microbial oil is drawing increasing interest all over the world as an alternative non-food feedstock for biodiesel production. Rhodosporidium toruloides has been considered as a promising candidate to convert glycerol into microbial oils due to its good substrate adaptability, high intracellular lipid content, and the potential of co-production of some pigments. Interestingly, we have also found that sodium lignosulphonate (SL) could significantly enhance the cell growth and lipid accumulation of R. toruloides. In the present work, we investigated the possible mechanisms for the enhancement of lipid production by SL. It was found that SL could not be utilized by R. toruloides as a carbon source. No apparent changes on the morphology of cell membranes and the fatty acid profiles of microbial lipids were found when SL was added. Further experiments indicated that the volumetric oxygen transfer coefficient was increased by nearly 15% on average by adding 4 g/L SL, which might be the main mechanism for the improvement of lipid production. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, researchers have been increasingly interested in developing the potential of oleaginous microorganisms to produce microbial oil as an alternative non-food oil feedstock for biodiesel preparation [1,2]. Among those oleaginous microorganisms, Rhodosporidium toruloides is considered as a promising candidate due to its excellent capability of lipid accumulation, broad adaptability to various substrates, and the potential of co-production of some carotenoids [2–4]. Our previous study also proved that R. toruloides could well convert refined and crude glycerol into lipids, which showed great potential to utilize the biodiesel byproduct glycerol as a sustainable and inexpensive carbon source to produce microbial oil, and in turn the oil could be used as the biodiesel feedstock [5]. In order to obtain microbial oils in a more effective way, much effort has been focused on directing carbon fluxes toward lipid synthesis either through optimization of cultivation process or biotechnological approaches such as genetic engineering or metabolic engineering [2,6]. What is more, researchers noticed that some chemicals with the structure of surfactants could enhance
∗ Corresponding author at: Key Laboratory of Forensic Science and Technology, Zhejiang Police College, Hangzhou 310053, PR China. Tel.: +86 571 87787219. E-mail address: [email protected] (J. Xu). http://dx.doi.org/10.1016/j.procbio.2015.01.006 1359-5113/© 2015 Elsevier Ltd. All rights reserved.
lipid accumulation. For example, Saenge et al. found that the accumulation of lipid and carotenoids in R. glutinis was increased by supplementing a non-ionic surfactant Tween 20 [7]; Taoka et al. found that non-ionic surfactant Tween 80 could promote cell growth and lipid accumulation of T. aureum, and the oleic acid composition in lipid profiles increased. We have also investigated the effects of several commonly used surfactants, and found that some anionic surfactants could promote the lipid production process of R. toruloides. However, the exact mechanism of how surfactants affect microbial lipid production was not well understood. According to our previous study, it was interesting to find that sodium lignosulphonate (SL), a typical surfactant, showed positive effect in cultivating R. toruloides by increasing the biomass concentration, and was able to enhance enzymatic saccharification of lignocelluloses as well [8–10]. As we know, lignosulphonates are by-products of the biomass pulping processes. Since they are inexpensive, renewable and readily available [11], a valueadded utilization way of lignosulphonates can be beneficial to not only the biorefinery process, but also to the pulping industry. In the present work, we investigated for the first time the possible mechanism of SL on lipid production of R. toruloides, by considering the possibility of SL for being utilized as a carbon source, the effect of SL on cell membrane morphology, and the influence of SL on oxygen transfer in lipid accumulation process.
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2. Materials and methods
425
10 ◦ C/min, and then maintained for 6 min. The temperatures of the injector and detector were set at 245 ◦ C and 260 ◦ C, respectively.
2.1. Microorganism and chemicals 2.4. Transmission electron microscopy [17,18] R. toruloides AS 2. 1389 was obtained from China General Microbiological Culture Collection Center (CGMCC). For preparing inocula, the organism was pre-cultured overnight in YPG medium containing 15 g/L glycerol, 10 g/L yeast extract and 10 g/L peptone at 30 ◦ C and 200 rpm in an air-bath shaker. SL was purchased from Tokyo Chemical Industry Co., Ltd., which contains 5.0–7.0% methoxyl group. Other chemicals in all the experiments were purchased locally and were analytically pure.
Samples of fermentation broth (1 mL) from 2 to 4 days culture were centrifuged (3000 rpm, 2 min). The supernatant liquor was discarded, and the yeast cells were harvested and fixed with 200 L KMnO4 (1.5%, w/v). Samples were preserved in refrigerator at 4 ◦ C. They were then post-stained and cut by an ultramicrotome. The ultrathin sections were observed in the transmission electron microscopy (TEM) of Model H-7650B (Hitachi, Japan).
2.2. Fermentation process
2.5. Determination of kL a
Batch fermentation was conducted in 500 mL flasks with 100 mL liquid medium containing 50 g/L glycerol, 0.1 g/L (NH4 )2 SO4 , 0.75 g/L yeast extract, 0.4 g/L KH2 PO4 , and 1.5 g/L MgSO4 ·7H2 O. Before inoculation, the biomass concentration of the inocula was carefully controlled at 0.5–0.6 g/L. Each culture was performed by triplicate test.
Double-film theory was used to describe oxygen transfer processes, and the volumetric mass transfer coefficient kL a was used to represent the oxygen transfer efficiency. The dynamic method based on the dissolved oxygen electrode [19] was used for determining the kL a (s−1 , volumetric oxygen transfer coefficient). The oxygen concentration was monitored by an oxygen sensor (VISFERM DO ARC 225, Hamilton Corp.). Specifically, during the fermentation process in a 2-L bioreactor with an aeration condition of 0.5 vvm, aeration was ceased for a very short time at a certain moment, and the dissolved oxygen (DO, mg L−1 ) decreased linearly. Once aeration was recovered, the DO gradually increased again. kL a was further calculated using the following kinetic model. Where DO* (mg L−1 ) is the dissolved oxygen concentration at saturation in a gas–liquid equilibrium, Q is the respiration rate (mg g−1 s−1 ), X (g/L) is the biomass concentration.
2.3. Analytical methods Glycerol concentration was determined by a Shimadzu10AVP HPLC system (Shimadzu Corp., Japan) equipped with a RID10A refractive index detector, Aminex HPX-87H column (300 mm × 7.8 mm, Bio-Rad, USA) at 65 ◦ C with 5 mM H2 SO4 as the eluent at a flow rate of 0.8 mL/min. Ultraviolet–Visible spectrophotometry is a common method to characterize and measure the concentration of lignosulphonates [12–14]. In the present work, SL was added in the 100 mL medium at the beginning of fermentation, and the initial concentrations were up to 2 g/L and 4 g/L, respectively. Fermentation broth samples were taken from the cultivation every 24 h. The concentration of SL was determined by a UV-vis 8500 spectrophotometer (Shanghai Tianmei Instrument Co., Ltd., China). The concentration of SL has a linearity relation with the absorbance at 235 nm. One absorbance unit corresponds to 0.0558 g/L SL. The biomass concentration was expressed as the dry weight concentration of cell biomass. Samples of fermentation broth were centrifuged, washed with deioned water, and dried at 105 ◦ C to a constant weight. Intracellular lipids were extracted by acid-heating procedure [15] with mixture of chloroform and methanol (1:1, v/v) as the extractant. After solvent extraction, the chloroform phase was taken and washed with 0.1% NaCl. After the solvent washing and evaporation, the lipid extract was weighted. Cell biomass (CB ), lipid concentration (CL ) and lipid content (CnL ) are defined as follows, respectively: CB (g/L) =
dry weight of cell biomass (g) volume of fermentation broth (L)
CL (g/L) =
weight of lipid (g) volume of fermentation broth (L)
CnL (%) =
weight of lipid (g) × 100% weight of cell biomass (g)
Methanolysis was carried out following the procedures described by Li et al. [16]. Fatty acid compositions of the methyl ester were analyzed with a GC-14B gas chromatography (Shimadzu, Japan) equipped with a CP-FFAP CB capillary column (25 m × 0.32 m × 0.30 m) produced by Agilent. Heptadecanoic acid methyl ester was used as internal standard. The column temperature was kept at 180 ◦ C for 0.5 min and heated to 250 ◦ C at
dDO = KL a × (DO∗ − DO) − QO2 X dt OTR = KL a × (DO∗ − DO) In the SL–water system without inoculation of R. toruloides cells, SL was supplemented at 0 (control group), 0.3, 1.0, 1.5, 2.0, 2.5, 3.0, and 4.0 g/L respectively. Determination of kL a followed the same procedures, but the respiration rate item (QX) was zero. 3. Results and discussions 3.1. Effect of SL on lipid accumulation Lignosulphonate is a polymeric surfactant derived from lignin. It is a main by-product in sulfite pulping or pretreatment of biomass [20]. In our previous study, Zhao et al. have found that SL could somehow enhance cell growth of R. toruloides [8]. In the present work, we further investigated the effect of SL on lipid accumulation of R. toruloides. As shown in Fig. 1, the addition of 1–6 g/L SL resulted in increases of biomass and lipid accumulation. The most significant improvement was observed when 2–4 g/L SL was added. It is possible that SL as a salt might alter the ionic strength of the fermentation broth, thus affecting lipid accumulation. However, we had investigated the effects of salt (NaCl) on the growth and lipid production using R. toruloides. No significant effects were observed by adding NaCl with concentration below 4 g/L. In the present work, addition of 2–4 g/L SL led to sodium concentration of far below 4 g/L. Therefore, the positive effect caused by SL is not due to sodium. Other mechanisms should be considered. The effects of surfactants on lipid production have also been reported by other researchers. Saenge et al. found that the concentrations of lipid and carotenoid in R. glutinis were increased by supplementing Tween 20 [7], and they proposed that it was probably because Tween 20 influenced the physicochemical status of the culture medium and certain nutrients became easier to be taken by
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the physiochemical features of the fermenting liquor, and is able to promote the mass transfer including oxygen transfer. We will discuss the possible mechanisms later. 3.3. Effect of SL on the membrane morphology and lipid profiles of R. toruloides
Fig. 1. The effect of SL on biomass and lipid accumulation of R. toruloides. Symbol elements: (): CB ; (䊉): CL ; (): CnL .
the yeast cell. Taoka et al. found that non-ionic surfactant Tween 80 could promote cell growth and lipid accumulation of T. aureum, and the oleic acid composition in lipid profiles increased. They analyzed that probably Tween 80 was utilized as the carbon source, and might affect the activity of fatty acid synthases. Besides, it was also possible that Tween 80 interferes with the cell membrane permeability, and the cell was enhanced to uptake some essential components [21]. However, the effect of SL on oleaginous microorganisms has not been elucidated and further studies are needed to investigate the mechanism of how it should affect microbial lipid production. 3.2. Possibility of SL utilized as a carbon source for lipid accumulation As a lignin derivative, SL has aromatic rings and conjugated carbonyl groups, thus is able to absorb ultraviolet light. In the present work, Ultraviolet–visible spectrophotometry was used to measure the variation of SL concentration in the culture medium during the lipid production process. It was found that the concentration of SL did not vary throughout the cultivation time from 0 h to 207 h, which indicated that SL could not be uptaken and utilized by R. toruloides (Fig. 2). According to this result, we further suggested other possible effects of SL on R. toruloides. For instance, SL may interfere with the cell membrane and then enhance the uptake of some essential nutrients. Besides, SL may exert some influences on
We further investigated the effect of SL on the membrane morphology of R. toruloides. Some researchers pointed out that surfactants could affect the membrane permeability, thus it might be easier to uptake some components or excrete some intracellular substances out of the cell. By this means, microbial production might be promoted by enhanced nutrient uptake or decreased feedback inhibition [22,23]. However, supplementation has to be well regulated. Otherwise the surfactants may dissolve the cell membrane and result in cells losing the reproduction capability [24,25]. The variation in membrane morphology could be observed by Scanning Electron Microscope (SEM) or Transmission Electron Microscope (TEM). Wu et al. investigated the effect of Polyoxyethylene nonyl phenyl ether-10 on the morphology of K. pneumonia by TEM, and observed the plasmolysis and deformation of outer membranes [23]. Galabova et al. studied the effect of Triton X-100 on the morphology of Yarrowia lipolytica by SEM and TEM, who found that when the addition concentration was up to 0.1% (w/v), cell shapes were altered. A disruption of the cell wall in certain zones was observed, where cell walls folded, cell membranes (cell walls and cytoplasmic membranes) became thin, translucent and indistinct compared with controls [26]. In the present work, different addition concentrations ranged from 1 to 6 g/L were tested. We prepared ultrathin sections of R. toruloides cells for TEM observation, and observed the sections of yeast cells at different growth phases. The representative images are taken and shown in Fig. 3. As is shown in Fig. 3a, the cells of control group had intact cell membranes with distinct outer and inner membrane outlines. Cells of experimental groups with addition of SLs did not appear deformed, where smooth surface of membrane structures could still be observed. Although it is still possible that the fluidity of cell membranes was altered, which phenomenon could not be observed by TEM. At least, the present result indicated that SL did not have significant effect on the cell morphology of R. toruloides. We further analyzed the microbial lipids to study whether SL would affect the fatty acid profiles (Table 1). Fatty acid compositions play an essential role in the biophysical characteristics of cell membranes. The dependence of membrane fluidity on the extent of unsaturation of fatty acids is a well characterized phenomenon, which has been demonstrated in animals, fungi, yeasts, plants and bacteria [27,28]. In the yeast Rodosporidium, our research group also has found that the ability of cells to alter the degree of unsaturation in their lipid compositions is an important factor in cellular acclimatization to the environmental conditions. If there is a significant change in the fluidization of cellular membrane, the fatty acid compositions will show a difference. In the present study, the predominant fatty acids of R. toruloides lipids were palmitic acid, stearic acid, oleic acid, and linoleic acid, wherein palmitic acid and oleic acid accounted for about 70% of the total fatty acid compositions. Compared with the control experiment, the fatty acid compositions were not significantly changed when 2–6 g/L SLs were supplemented. This result implied that SL did not affect the fatty acid profiles of the cellular lipids. 3.4. Effect of SL on oxygen transfer
Fig. 2. The concentration of SL during the fermentation.
Cell culture is a typical multi-phase system, including the gas phase (O2 , CO2 ), solid phase (cells) and liquid phase (fermentation liquor), where mass transfer process between different
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Fig. 3. The effect of SL on the morphology of cell membranes. (a) Control; (b) 1 g/L SL; (c) 4 g/L SL (d) 6 g/L SL; Yeast Cells were sampled at 48 h. (e) Control; (f) 1 g/L SL; (g) 4 g/L SL (h) 6 g/L SL; Yeast Cells were sampled at 96 h.
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Table 1 The effect of SL on fatty acid profiles of R. toruloides lipid. SL (g/L)
C14:0
0 2 4 6
1.4 1.8 1.6 1.6
± ± ± ±
C16:0 0.0 0.1 0.1 0.1
25.8 27.4 27.5 28.5
± ± ± ±
C16:1 0.5 1.0 0.6 1.0
2.5 1.9 3.1 3.1
± ± ± ±
C18:0 0.1 0.2 0.1 0.1
phases can be a limiting factor for a bio-reaction process. For those aerobic microorganisms, as the terminal electron acceptor in respiration, oxygen is the most common and important composition in the gas phase. Moreover, oxygen transfer is one of the most important mass transfer processes in fermentations. Since oxygen has poor solubility in water, the low concentration difference between the gas–liquid phases resulted in a limited mass transfer.
12.5 11.9 11.9 11.9
± ± ± ±
C18:1 1.2 1.2 0.9 1.0
44.8 46.4 44.2 42.2
± ± ± ±
C18:2 0.5 1.9 1.1 1.2
10.6 8.8 9.5 10.5
± ± ± ±
C18:3 0.0 0.9 0.1 0.1
2.2 1.9 2.3 2.3
± ± ± ±
0.0 0.0 0.1 0.1
We found that oxygen supply condition in the fermentation broth was correlated to the cell growth (Fig. 4). Optimized oxygen supply condition could enhance the cell growth and lipid accumulation in R. toruloides. Otherwise, both the cell growth and lipid accumulation would be inhibited. The importance of aeration strategy has also been described in other literatures. Yen et al. reported the inhibited cell growth of R. glutinis under a poor oxygen aeration condition [29]. Li [30] and Zhao [31] et al. provided a high dissolved
Fig. 4. The effect of oxygen supply condition on cell growth and lipid production of R. toruloides. Oxygen supply conditions: (a) and (b) 1.1 vvm, 300 rpm; (c) and (d) 0.75 vvm, 270 rpm; (e) and (f) 0.5 vvm, 270 rpm. Symbol elements: (): CB ; (䊉): CL ; (): CnL ; (): DO.
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Table 2 Effect of SL on KL a and DO* values. Stirring speed (rpm)
SL (g/L)
KL a (s−1 )
100
0 0.3 1.0 1.5 2.0 2.5 3.0 4.0
0.0005 0.0007 0.0013 0.0019 0.0006 0.0006 0.0007 0.0019
± ± ± ± ± ± ± ±
0.0000 0.0000 0.0000 0.0001 0.0000 0.0000 0.0000 0.0004
6.93 6.76 6.82 6.71 7.12 6.89 6.90 6.60
± ± ± ± ± ± ± ±
0.01 0.03 0.02 0.01 0.02 0.01 0.02 0.08
200
0 0.3 1.0 1.5 2.0 2.5 3.0 4.0
0.0021 0.0025 0.0022 0.0040 0.0032 0.0026 0.0032 0.0024
± ± ± ± ± ± ± ±
0.0001 0.0000 0.0001 0.0002 0.0001 0.0001 0.0002 0.0001
6.72 6.69 6.78 6.63 6.70 6.80 6.66 6.67
± ± ± ± ± ± ± ±
0.07 0.05 0.02 0.10 0.01 0.01 0.10 0.08
300
0 0.3 1.0 1.5 2.0 2.5 3.0 4.0
0.0025 0.0038 0.0039 0.0072 0.0088 0.0088 0.0054 0.0050
± ± ± ± ± ± ± ±
0.0001 0.0001 0.0002 0.0001 0.0004 0.0003 0.0002 0.0001
6.76 6.66 6.79 6.62 6.63 6.55 6.73 6.52
± ± ± ± ± ± ± ±
0.04 0.03 0.03 0.02 0.01 0.03 0.08 0.05
500
0 0.3 1.0 1.5 2.0 2.5 3.0 4.0
0.0101 0.0113 0.0114 0.0196 0.0157 0.0158 0.0151 0.0140
± ± ± ± ± ± ± ±
0.0001 0.0001 0.0004 0.0014 0.0002 0.0003 0.0001 0.0004
6.77 6.75 6.76 6.70 6.74 6.79 6.80 6.69
± ± ± ± ± ± ± ±
0.02 0.01 0.02 0.01 0.00 0.01 0.01 0.02
oxygen level of 40–50% to ensure a sufficient oxygen supply for R. toruloides Y4. As an aerobic oleaginous yeast, R. toruloides utilize oxygen in both the cell growth and lipid synthesis. Oxygen even plays a role in synthesis of unsaturated fatty acids, as an electron acceptor in the dehydrogenation step catalyzed by desaturases. It was also reported that some surfactants could decrease the average diameter of air bubbles by reducing the surface tension, and thus were able to increase the gas–liquid interfacial area and enhance the oxygen transfer between the gas and liquid phases [19]. To discuss the mechanism of how SL influences the cell growth and lipid accumulation of R. toruloides, we investigated the effect of SL on oxygen transfer in both SL–water system and lipid production system.
3.4.1. Effect of SL on oxygen transfer in SL–water system In a fermentation system, the media contains different components and the respiration of microorganisms makes the oxygen transfer complicated. For simplification, we first carried out experiments in deionized water system without inoculation, in which the supplementation of SL was the only variable and all other operation parameters were kept consistent. The kL as determined at stirring speeds of 100–500 rpm and under different SL addition conditions are shown in Table 2. According to the kL a and DO* values, DO* did not varied at different SL concentrations, which indicated that SL had no effect on the dissolved oxygen concentration in a liquid–gas equilibrium status. Increase of kL a value was correlated to the SL concentration, which implied that SL could enhance the oxygen transfer in water. We also noticed that at each stirring condition there was a peak range of kL a. Further increase of SL could not promote the oxygen transfer but decrease the kL a. Surfactants may affect kL a by increasing the interface area of oxygen transfer. However, excessive addition of
DO* (mg L−1 )
surfactants may impede the mobility of gas–liquid interface, and cause an inhibitive effect [19]. 3.4.2. Effect of SL on oxygen transfer in fermentation system Effect of SL on oxygen transfer in fermentation system was further investigated. For every 24 h, biomass concentration and oxygen consumption rate were tested, and the oxygen consumption rate per gram of biomass could be calculated accordingly. The results are summarized in Fig. 5 and Table 3. In the present work, since the seed cells were inoculated directly into the nitrogen limited batch media, both the cell growth and lipid accumulation were activated soon. As a result, oxygen consumption was significant in the beginning stage, as shown in Fig. 5. In the late stage of lipid production, oxygen consumption per gram of biomass decreased, which suggested a comparatively lower need of oxygen in the lipid synthesis process. However, the overall oxygen consumption was still of high rate due to the biomass growth. Comparing the experimental group supplemented with SL with the control one, we know that the overall oxygen consumption was higher for the experimental group, but the oxygen consumptions per gram of biomass were almost the same. Table 3 also implied that SL had a positive effect on the oxygen transfer coefficient and oxygen transfer rate. The kL a value was increased by about 15% on average. The above results indicated that SL indeed could enhance the cell growth and lipid accumulation of R. toruloides by promoting the oxygen transfer process. Such effect might be related to the physiochemical feature of SL and its behavior in fermentation media. SL has some special characteristics such as hydrophilicity, lipophilicity, absorbability and dispersibility [32–34]. It may decrease the air bubble diameter by reducing the surface tension, increase the interface area between the gas phase and liquid phase, or exert an influence on the mass transfer by affecting certain physiochemical
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Table 3 Effect of SL on oxygen transfer. KL a (s−1 )
Time (h)
OTR (mg L−1 S−1 )
Control 0 21 45 70 90 116 140 160
0.0121 0.0159 0.0073 0.0079 0.0071 0.0080 0.0079 0.0085
SL 4 g/L ± ± ± ± ± ± ± ±
0.0001 0.0005 0.0002 0.0004 0.0001 0.0004 0.0001 0.0002
0.0111 0.0209 0.0114 0.0083 0.0092 0.0073 0.0089 0.0098
Control ± ± ± ± ± ± ± ±
0.0003 0.0003 0.0007 0.0001 0.0006 0.0002 0.0002 0.0001
(a)
0.0206 0.0636 0.0394 0.0387 0.0341 0.0304 0.0284 0.0298
SL 4 g/L ± ± ± ± ± ± ± ±
0.0010 0.0029 0.0018 0.0021 0.0005 0.0016 0.0004 0.0009
0.0266 0.0815 0.0638 0.0415 0.0478 0.0328 0.0392 0.0382
± ± ± ± ± ± ± ±
0.0011 0.0033 0.0034 0.0005 0.0026 0.0012 0.0010 0.0012
Acknowledgments The authors express their gratitude to the support from Tsinghua University Initiative Support Program (20111081123, 20121080046) and “863” Project (2012AA052101).
References
(b)
Fig. 5. Oxygen consumption rate in lipid production process. (a) Oxygen consumption rate; (b) oxygen consumption rate per gram of biomass. Symbol elements: (): Control; (䊉): 4 g/L SL.
features [19]. It has been reported that some surfactants even could form “colloidal gas aphrons”, and aeration under such conditions could significantly enhance the volumetric oxygen transfer coefficient [35–37]. However, in an actual fermentation process, the media contains different kinds of components including inorganic compounds, organic compounds and microorganism cells, and the microorganisms are continuously exchange substances with the fermentation media. In such a complicated system, the microscopic behavior of SL deserves further exploration. 4. Conclusion SL could somehow promote the lipid production process. The concentration of SL in the fermentation media did not vary at different stages of lipid production, which suggested that SL could not be utilized by R. toruloides. The cell membrane of R. toruloides with supplementation of SL showed similar morphology with that of control cells, and the fatty acid profiles of the microbial lipids remained unchanged. It is probable that SL plays a positive role in oxygen transfer thus improving substrate utilization for lipid production.
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