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Recycling biodiesel-derived glycerol by the oleaginous yeast Rhodosporidium toruloides Y4 through the two-stage lipid production process
Biochemical Engineering Journal 91 (2014) 86–91
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Recycling biodiesel-derived glycerol by the oleaginous yeast Rhodosporidium toruloides Y4 through the two-stage lipid production process Xiaobing Yang a,b,c , Guojie Jin b , Zhiwei Gong b , Hongwei Shen b , Fengwu Bai a,∗∗ , Zongbao Kent Zhao b,∗ a
Dalian University of Technology, Dalian 116024, PR China Dalian National Laboratory for Clean Energy, Dalian Institute of Chemical Physics, CAS, Dalian 116023, PR China c University of Chinese Academy of Sciences, Beijing 100049, PR China b
a r t i c l e
i n f o
Article history: Received 5 May 2014 Received in revised form 26 July 2014 Accepted 28 July 2014 Available online 5 August 2014 Keywords: Bioconversion Biodiesel Glycerol Microbial growth Optimization Oleaginous yeast
a b s t r a c t Direct utilization of crude glycerol, a major byproduct in biodiesel industry, becomes imperative, because its production has outpaced the demand recently. We demonstrated that the oleaginous yeast Rhodosporidium toruloides Y4 had a great capacity to convert glycerol into lipids with high yield using the two-stage production process. Significantly higher cell mass and lipid yield were observed when the media were made with synthetic crude glycerol than pure glycerol. The process achieved a lipid yield of 0.22 g g−1 glycerol, which was comparable with the lipid yield using glucose as the substrate. Lipid samples showed similar fatty acid compositional profiles to those of vegetable oils, suggesting that such microbial lipids were potential feedstock for biodiesel production. Our data provided an attractive route to integrate biodiesel production with microbial lipid technology for better resource efficiency and economical viability. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Concerns about the costs and supply of fossil resources and environmental deterioration have been driving our society to explore alternative transportation fuels. Biodiesel is a proven biofuel that can be dropped directly in the established infrastructure and blended legally with petroleum diesel in any percentages [1]. Our current technology for biodiesel production is depending on vegetable oils and animal fats, however, these resources are limited. Microbial lipids prepared from oleaginous yeasts, bacteria, and microalgae have been employed to produce biodiesel of comparable properties with those made from typical plant oils [2]. Besides, yeast lipids can be utilized as substitutes of high-added value lipids (e.g. cocoa-butter of other exotic fats equivalents) [3,4]. Microbial lipid technology has a number of advantages, such as shorter life cycle and without competing with agriculture for land use. Thus,
∗ Corresponding author. Tel.: +86 411 84379211; fax: +86 411 84379211. ∗∗ Corresponding author. Tel.: +86 411 84379066; fax: +86 411 84706329. E-mail addresses: [email protected] (F. Bai), [email protected], [email protected] (Z.K. Zhao). http://dx.doi.org/10.1016/j.bej.2014.07.015 1369-703X/© 2014 Elsevier B.V. All rights reserved.
microbial lipids are considered as potential feedstock for biodiesel [5,6]. It remains challenging to produce microbial lipids at large scale because costs for feedstock and product recovery are high. Attempts to use lignocellulosic biomass have been reported by a number of groups, but the overall efficiency has been low [7,8]. Crude glycerol is a major byproduct in biodiesel and oleochemical industries, and its production has outpaced the demand recently [9]. Because of the presence of impurities, such as salts, methanol, organic acids and heavy metals, it is difficult to find a direct utilization of crude glycerol in chemical industry. Although refined glycerol is valuable, the purification process is costly. In some European countries, crude glycerol is even simply treated as industrial wastewater. Glycerol has been explored as carbon source for the fermentative production of value-added products such as 1,3propanediol [10,11], citric acid [10], biopolymers [12] and succinic acid [13]. Microbial lipid production by fed-batch culture of oleaginous species on glycerol has also been known, but lipid yields usually remained lower than those with glucose as the carbon source [14–17]. We have recently developed the two-stage lipid production process [18,19]. In the first stage, cells are cultivated in a nutrientrich medium for propagation; and in the second stage, cells are
X. Yang et al. / Biochemical Engineering Journal 91 (2014) 86–91
A
87
Media with C–, N– and P–sources
Pre-cultures
Lipid-rich cells
B
Cells
Media with C–source only
Fig. 1. Microbial lipid production based on conventional (path A) and two-stage (path B) process.
resuspended in a medium contained carbon source without auxiliary nutrients for lipid production (Fig. 1, Path B). Because of the absence of auxiliary nutrients such as nitrogen and phosphorous sources, biosynthesis of protein and nucleic acids ceased, and cell propagation was inhibited. However, the conversion of carbon source into lipid was more efficient, leading to higher lipid yield and productivity [19]. We have further demonstrated the usefulness of this two-stage lipid production process for direct conversion of cellulose and lignocellulosic materials into lipids [8]. In this study, we applied the two-stage process for the conversion of crude glycerol into lipid by the oleaginous yeast Rhodosporidium toruloides Y4. This yeast strain is a robust lipid producer capable of accumulating lipids up to 76% of cell dry weight [19] and tolerating major inhibitors found in biomass hydrolysates [20]. Our data provided an attractive route to integrate biodiesel production with microbial lipid technology for better resource utilization efficiency and economical viability. 2. Materials and methods 2.1. Strain, reagents and media When R. toruloides AS 2.1389 was cultivated in cornstalk hydrolysates, a more robust strain R. toruloides Y4 was isolated [19]. It was maintained at 4 ◦ C on YPD agar slant containing (g L−1 ) glucose, 20; yeast extract, 10; peptone, 10 and agar powder 15. Compound 2-(N-morpholino)ethanesulfonic acid (MES) was purchased from Sigma. All other chemicals were of analytical grade and bought locally. The edible soy bean oil was from China Oil & Foodstuffs Corporation (China). Because the composition of crude glycerol varies and usually contains 60–90% glycerol, 5–6% salts and 4–6% methanol [10–12,16], the synthetic crude glycerol stock solution in this study was made by dissolving glycerol, methanol and K2 SO4 in distilled water to final concentrations of 60 wt%, 6 wt% and 6 wt%, respectively. The preculture medium contained (g L−1 ) glucose or glycerol, 20; yeast extract 10 and peptone 10, pH 6.0. To make lipid production media using pure glycerol, appropriate amounts of glycerol, K2 SO4 , K2 HPO4 and methanol were dissolved in distilled water or 50 mM MES buffer. To make lipid production media using synthetic crude glycerol, the stock solution was diluted to an appropriate glycerol concentration with distilled water or 50 mM MES. Initial pH was adjusted with 2 M NaOH when necessary. No sterilization was done for all lipid production media. Crude glycerol samples were also prepared in the lab. Briefly, a mixture of vegetable oil and 5% KOH methanol solution (v/v, 5/1) was treated at 65 ◦ C for 3 h. Then the reaction mixture was placed in a funnel overnight to ensure the separation of biodiesel from crude glycerol into two layers. The lower layer was evaporated, neutralized with H2 SO4 , and dilution with distilled water. This home-made crude glycerol solution contained: glycerol 50 g L−1 , K2 SO4 5 g L−1 , methanol 5 g L−1 , and others 6 g L−1 . 2.2. Lipid production All cultures were performed with 250-mL Erlenmeyer flasks on a rotary shaker. R. toruloides Y4 cells were incubated in the
preculture medium at 200 rpm, 30 ◦ C for 48 h, collected by centrifugation at 8000 × g for 5 min. The seed cells had a lipid content of 10%. Cells corresponding an initial concentration of 7.0 g L−1 were inoculated into lipid production media and incubated at 200 rpm, 30 ◦ C for 120 h, while the time intervals were 24 h for pH analysis. The control experiments were performed with 50 g L−1 synthetic crude glycerol under identical culture conditions unless otherwise specified. Cells were harvested by centrifugation at 8000 × g for 5 min, washed twice with distilled water (for experiments performed with crude glycerol, the cells were washed with ethanol once before water washes to remove the vegetable oil), and dried at 105 ◦ C overnight to a constant weight to obtain dry cell weight (DCW).
2.3. Analysis methods Glycerol was quantified by ion chromatography (IC) on the Dionex ICS2500 system (Dionex, America) at 30 ◦ C with a flow rate at 1 mL/min using an analytical column CarboPac PA10 4 × 250 mm and the guard column CarboPac PA10 4 × 50 mm by isocratic elution with a mobile phase consisting of 18 mM NaOH. Under such conditions, the retention time for glycerol was 1.63 min. Methanol was determined using the alcohol oxidase method [21]. Cell density was determined at 600 nm with a JASCO V-530 UV-visible spectrophotometer (JASCO, Japan). Cellular lipid was extracted by a mixture of chloroform and methanol according to the reported procedure [19]. Briefly, 1.0 g of dry cell was treated with 4 M HCl at 78 ◦ C for 90 min and extracted twice with chloroform and methanol (1/1, v/v), the obtained organic layer were combined and dried using anhydrous Na2 SO4 , then the solvent was removed under vacuum conditions. The lipids were heated to a constant weight at 105 ◦ C. Lipid content was calculated as g lipid per g DCW. Lipid yield was defined as g lipid per g glycerol consumed. The yield of DCW was calculated as per g DCW per g glycerol consumed. Five milliliter of crude glycerol was sampled to determine residual lipids using the chloroform/methanol (1/1, v/v) extraction. To calculate DCW and lipid yields, the amounts of cell mass (7.0 g L−1 ) and lipid (0.7 g L−1 ) of the inocula cells were subtracted, respectively, from the total cell mass and lipid data of each experiment. Experimental data were based on four independent cultures, and were shown as mean ± SD. Lipid samples were transmethylated according to the published procedure with minor modification [19]. Briefly, wet cell pellets from 5 ml of culture were treated with 0.5 ml 5% KOH methanol solution at 65 ◦ C for 50 min, followed by the addition of 0.2 ml BF3 diethyl etherate and 0.5 ml methanol, refluxed for 10 min, cooled, diluted with distilled water and extracted with petroleum ether (bp 60–90 ◦ C). The organic layer was washed twice with distilled water and subjected to fatty acid compositional analysis. Fatty acids were profiled with a 7890F gas chromatography instrument (Techcomp Scientific Instrument Co. Ltd., Shanghai, China) equipped with a cross-linked capillary FFAP column (30 m × 0.32 mm × 0.4 mm) and flame ionization detector. Operating conditions: N2 carrier as 40 ml/min, injection port temperature 250 ◦ C, oven temperature 190 ◦ C and the detector temperature was 280 ◦ C. Fatty acids were identified by comparison of their retention times with those of
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standards (Sigma, USA) and quantified based on their respective peak areas and normalized.
A 12
3. Results and discussion
Control
7
9
11
9
3.2. The effect of pH on lipid production Culture pH has major effects on cell growth and product accumulation in biological processes. In addition, acid (H2 SO4 ) and base (KOH or NaOH) were utilized in the process of biodiesel production. If R. toruloides Y4 can tolerate a wide range of initial pH, there will be less chemicals consumption to control pH when the crude glycerol is employed. Thus, two sets of experiments were done to test the effect of pH on lipid production by R. toruloides Y4. Firstly, cells were inoculated in lipid production media of different initial pH values. Media pH dropped rapidly to about 3.3 within 24 h for all experiments (Fig. 2A). These results suggested that R. toruloides Y4 might initially produce some organic acids, such as oxalic acid, that brought culture pH to lower values. While the production of organic acids from glycerol has been demonstrated previously [22], oxalic acid concentration in this study was below 0.5 g L−1 , and thus only a minus amount of glycerol was converted into acids. The lipid content was slightly affected by higher initial pH while the cell mass and lipid yield were similar (Fig. 2B). Interestingly, other oleaginous yeasts have also been demonstrated to acidify the culture broth and be able to tolerate high initial pH values [24]. When the media were buffered with 50 mM MES to hold constant pH at a range of 3.7–6.5, there was no significant difference (Fig. 2C). Specifically, higher
6
3
0 0
20
40
60
80
100
120
Time (h)
B
Cell mass
Lipid content
Lipid yield
Cell mass(g/L), Lipid content (%) Lipid yield x100 (g/g)
40
30
20
10
0 Control
I7.0
I9.0
I11.0
pH
C
Cell mass
Lipid content
Lipid yield
40 Cell mass (g/L), Lipid content (%) Lipid yield x100 (g/g)
Two sets of experiments were done where lipid production media were made with pure glycerol or synthetic crude glycerol at different initial glycerol concentrations, and results are shown in Table 1. It was clear that cell mass and lipid titer were the highest when initial glycerol concentration was 100 g L−1 . When initial glycerol concentration increased further, cell mass and lipid titer decreased (Table 1). This was likely due to the inhibition of cell growth by an increased osmotic pressure [10,14]. Except for experiments with initial glycerol at 20 g L−1 , lipid contents were within the range of 34.2–42.5% for all other experiments. Because the inoculated cells had residual nitrogen sources, those experiments with 20 g L−1 initial glycerol might have lower carbon-to-nitrogen ratio than others with higher initial glycerol concentrations, and thus leading to low lipid contents. Lipid yields were very close to 0.20 g g−1 glycerol in the experiments with 50 g L−1 of initial glycerol, and were higher than 0.21 g g−1 glycerol for those where initial glycerol concentrations of the media were 100 g L−1 or 150 g L−1 (Table 1). The maximum net lipid yield of 0.22 g g−1 glycerol, comparable with lipid yields using glucose as the substrate [22,23], was achieved for the experiments using 150 g L−1 pure glycerol or 50 g L−1 crude glycerol (Table 1). The fact that high lipid yields were achieved across a wide concentration range of initial glycerol indicated that consumed glycerol was efficiently used for lipid biosynthesis. It was interesting to note that synthetic crude glycerol, at 50 g L−1 , gave better results than pure glycerol in terms of cell mass, lipid and lipid content. It should be noted that, for the experiment with synthetic crude glycerol at 50 g L−1 , the medium also contained 5.0 g L−1 methanol and 5.0 g L−1 K2 SO4 . Therefore, it seemed that the presence of low concentrations of methanol or K2 SO4 favored lipid production (vide infra). However, when glycerol concentrations were higher than 100 g L−1 , higher concentrations of methanol and K2 SO4 might inhibit lipid production due to toxicity or high osmotic pressure. Similar phenomena have been observed for lipid production recently [16].
Initial pH
3.1. The effect of initial glycerol concentration on lipid production
30
20
10
0 Control
M3.7
M5.5 pH
M6.0
M6.5
Fig. 2. Effects of pH on lipid production by R. toruloides Y4. Synthetic crude glycerol (50 g L−1 glycerol, 5 g L−1 K2 SO4 and 5 g L−1 methanol) was used, and the cultures were held at 30 ◦ C, 200 rpm for 120 h. (A) The evolution of media pH over time. (B) Results of lipid production at different initial pH values. (C) Results of lipid production where media pH was maintained with 50 mM MES.
X. Yang et al. / Biochemical Engineering Journal 91 (2014) 86–91
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Table 1 The effect of initial glycerol concentration on lipid production by the two-stage process.a Glycerol (g L−1 )
Glycerol quality
Total cell mass (g L−1 )
20
Pure Crude Pure Crude Crudeb Pure Crude Pure Crude Pure Crude
11.7 11.7 17.7 19.4 24.9 21.1 20.3 18.5 16.6 16.3 13.3
50
100 150 200 a b
± ± ± ± ± ± ± ± ± ± ±
0.5 1.4 1.3 1.4 0.5 0.2 2.4 0.5 2.2 0.1 1.4
Lipid (g L−1 ) 2.3 2.5 6.2 6.9 12.2 8.5 8.6 7.6 6.6 6.8 5.6
± ± ± ± ± ± ± ± ± ± ±
0.4 0.3 0.2 0.6 0.2 1.1 0.7 0.6 0.3 0.3 0.2
Lipid content (%) 19.7 21.6 34.8 35.4 48.9 40.3 42.5 41.1 40.4 39.6 41.5
± ± ± ± ± ± ± ± ± ± ±
3.3 0.3 2.2 0.7 0.6 4.7 2.3 3.8 3.0 4.9 4.0
Lipid yield (g 100 g−1 ) 16.3 14.8 19.3 19.7 22.0 21.9 21.8 22.2 21.8 18.4 18.5
± ± ± ± ± ± ± ± ± ± ±
1.8 1.4 0.6 1.1 0.1 0.5 1.6 0.5 1.5 2.1 0.3
Glycerol used (g L−1 ) 12.9 18.3 30.6 36.8 50.0 38.0 42.2 35.7 29.0 35.9 27.3
± ± ± ± ± ± ± ± ± ± ±
2.3 1.5 0.5 2.1 0.0 2.5 2.6 0.7 0.5 1.3 2.2
DCW yield (g 100 g−1 ) 36.4 26.1 32.7 33.6 35.8 37.3 31.6 32.9 33.1 26.1 23.0
± ± ± ± ± ± ± ± ± ± ±
4.0 8.5 0.6 2.3 1.1 5.5 6.1 1.3 1.7 1.8 4.2
Pure glycerol was used, and the culture conditions were 30 ◦ C, 200 rpm, 120 h, pH 5.5. Crude glycerol was home-made from the base-catalyzed biodiesel production process.
initial pH resulted in slightly lower cell mass and lipid yield but similar lipid content. The highest cell mass and lipid content of 22.2 g L−1 and 40%, respectively, were achieved at pH 6.0. The highest lipid yield of 0.21 g g−1 , however, was observed at pH 3.7. These results demonstrated that R. toruloides Y4 could maintain good capacity for the conversion of glycerol into lipid at a relatively broad range of initial pH. 3.3. The effect of loading volume on lipid production Since lipid synthesis is an aerobic process, dissolved oxygen should be a crucial factor. To evaluate the effect of aeration on lipid production, cultures were done in 250-mL flasks with different media volumes, and results are shown in Fig. 3. It was obvious that cell mass, lipid content and lipid yield were decreasing when the media volume increased from 20 mL to 50 mL. The highest cell mass, lipid content and net lipid yield were 24.3 g L−1 , 47.3% and 0.22 g g−1 , respectively, obtained at the loading volume of 20 mL. Compared with results of the loading volume of 50 mL, significantly higher cell mass (17.4% increase) and lipid content (24.4% increase) were obtained. The net lipid yield was 0.22 g g−1 if lipids from the seed cells, which contained 8–10% lipid, were subtracted. These results suggested that oxygen supply was limiting, and that oxygen-transfer should be intensified to further improve lipid productivity. Detailed biochemical analysis of lipid biosynthesis indicated that the production of 1 mole of trioleoylglycerol requires 16 mole
Cell mass
Lipid content
Lipid yield
Cell mass (g/L), Lipid content (%) Lipid yield x 100 (g/g)
50
40
30
20
10
0 20
30
40
50
Loading volume (mL) Fig. 3. Effects of the loading volume on lipid production. Synthetic crude glycerol (50 g L−1 glycerol, 5 g L−1 K2 SO4 and 5 g L−1 methanol) was used, and the culture was held at 30 ◦ C, 200 rpm, pH 5.5 for 120 h.
of glucose, and the lipid yield is 0.31 g g−1 [3,4]. Similarly, the theoretical lipid yield is 0.30 g g−1 glycerol because about 32 mole of glycerol is used to produce 1 mole of trioleoylglycerol. While high lipid yields of over 0.20 g g−1 glucose have been documented in many reports [18,19,25], comparable results have been only achieved in a few reports for glycerol [22]. A number of oleaginous strains have been tested for the conversion of glycerol into lipids, but lipid yields were usually below 0.15 g g−1 glycerol [3,16,26–29]. Lipid yield of 0.19 g g−1 crude glycerol was reported using rapeseed hydrolysates as nitrogen source instead of yeast extract [15]. In this study, the yield was improved to 0.22 g g−1 glycerol in the absence of exogenous nitrogen source, suggesting that the crude glycerol was good feedstock for microbial lipid production. 3.4. The effects of potential impurities on lipid production Currently the biodiesel industry is mainly operated with inorganic acid/base catalysts in the presence of excess methanol and lipids used for biodiesel production, especially waste oils, contain various amounts of phospholipids, phosphate are generated in the process. Depending on product separation processes, the raw glycerol stream contained 60–90 wt% glycerol and 5–6 wt% methanol [10–12,16]. Therefore, salts such as sulfates and phosphates, and methanol are major impurities in crude glycerol. Therefore, many experiments have been performed to demonstrate how these impurities could play roles in biological utilization of crude glycerol, and findings have been reviewed elsewhere [30]. Here, experiments were designed to include various amounts of K2 SO4 , K2 HPO4 or methanol in the media for lipid production by R. toruloides Y4 using the two-stage process. Compared with the control experiment in the absence of these impurities, the addition of K2 SO4 up to 8 g L−1 clearly increased cell mass, lipid content and lipid yield (Table 2). Similar phenomena have been described recently [16]. The presence of K2 SO4 provided sulphur element which is essential for provision of acyl-S-CoA and S-containing amino acids. Besides, potassium ion is an activator for many enzymes. However, when K2 HPO4 was added to an initial concentration of 0.5 g L−1 or 1.0 g L−1 , slightly better results were obtained; but it was slightly worse in the presence of 2.0 g L−1 K2 HPO4 (Table 2). It should be noted that initial and final media pH values were 8.5 and 6.2, 8.8 and 6.8, and 8.9 and 7.1, respectively, for the cultures with initial K2 HPO4 concentrations of 0.5, 1.0 g and 2.0 g L−1 . Experimental results indicated that cell growth and lipid production by R. toruloides Y4 were seriously inhibited in buffered YEPD media at pH above 7.0 (data not shown). Thus, an increased media pH in the presence of 2.0 g L−1 K2 HPO4 led to slightly inhibiting the lipid production. As shown in Table 2, cell mass and lipid yield were a little lower in the presence of methanol up to 8.0 g L−1 . These results indicated that methanol slightly inhibited lipid production, which
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Table 2 The effects of potential impurities on lipid production.a K2 SO4 (g L−1 )
K2 HPO4 (g L−1 )
Methanol (g L−1 )
Total cell mass (g L−1 )
Control 2 4 8 – – – – – –
– – – – 0.5 1 2 – – –
– – – – – – – 2 4 8
17.7 18.9 19.5 20.9 20.1 19.6 18.0 17.0 16.7 16.8
a
± ± ± ± ± ± ± ± ± ±
Lipid (g L−1 )
1.3 0.4 0.1 0.8 1.3 1.4 1.3 0.5 0.4 0.5
6.2 6.7 6.9 7.6 8.6 6.6 5.6 5.7 5.7 5.8
± ± ± ± ± ± ± ± ± ±
Lipid content (%)
0.2 0.7 0.3 0.2 0.7 0.3 0.2 0.7 0.3 0.2
34.8 35.7 35.7 36.5 33.9 31.9 31.3 33.6 34.1 34.5
± ± ± ± ± ± ± ± ± ±
Glycerol used (g L−1 )
Lipid yield (g 100 g−1 )
2.2 2.6 1.4 1.2 1.9 3.0 3.0 1.5 0.8 2.7
19.3 21.7 21.4 21.1 22.2 20.0 18.5 22.8 22.6 21.5
± ± ± ± ± ± ± ± ± ±
0.6 1.7 1.9 0.9 1.6 1.3 1.0 0.6 0.2 1.1
30.6 31.8 32.4 34.5 30.3 28.4 26.4 24.3 23.5 24.7
± ± ± ± ± ± ± ± ± ±
DCW yield (g 100 g−1 )
0.5 2.7 0.2 0.8 2.6 1.3 3.0 1.0 1.3 1.1
32.7 33.6 37.3 38.5 42.4 44.0 41.4 39.8 41.5 39.8
± ± ± ± ± ± ± ± ± ±
0.6 3.8 0.6 1.7 1.7 0.6 1.2 2.8 2.5 2.3
Pure glycerol (50 g L−1 ) was used and the culture conditions were 30 ◦ C, 200 rpm, 120 h, pH 5.5.
Table 3 The fatty acid compositional profiles of lipid samples produced by R. toruloides Y4. Entry
Key culture parametersa
Lipid content (%)
1 2 3 4 5 6 7 8 9 10 11 12 13b
– 8 g L−1 , K2 SO4 0.5 g L−1 , K2 HPO4 2 g L−1 , methanol pH 3.7, in MES pH 5.5, in MES pH 6.0, in MES pH 6.5, in MES Initial pH 5.5 Initial pH 7.0 Initial pH 9.0 Initial pH 11.0 Crude glycerol
35.0 35.9 33.9 33.6 39.3 41.0 40.8 41.2 39.4 38.5 37.6 37.0 48.9
± ± ± ± ± ± ± ± ± ± ± ± ±
1.2 1.7 1.9 1.6 1.5 1.9 1.7 1.8 0.5 1.9 1.7 1.4 0.6
C14:0 1.4 1.5 1.7 1.2 1.4 1.7 1.7 1.7 1.2 1.3 1.4 1.3 1.5
± ± ± ± ± ± ± ± ± ± ± ± ±
C16:0 0.2 0.2 0.2 0.0 0.2 0.2 0.3 0.1 0.1 0.2 0.2 0.2 0.1
27.8 27.4 29.1 23.8 28.5 34.1 36.5 36.9 25.8 26.0 27.7 27.1 33.2
± ± ± ± ± ± ± ± ± ± ± ± ±
C16:1 1.2 3.7 2.6 2.0 5.8 4.6 2.0 2.2 4.6 4.1 4.9 5.5 2.1
0.6 0.9 0.9 0.8 0.7 1.0 1.3 1.1 0.8 0.8 0.8 0.8 3.0
± ± ± ± ± ± ± ± ± ± ± ± ±
C18:0 0.1 0.3 0.3 0.4 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.2 1.0
21.8 15.9 17.2 19.3 18.2 15.0 14.1 14.1 16.9 15.7 16.7 15.3 20.1
± ± ± ± ± ± ± ± ± ± ± ± ±
C18:1 0.3 0.6 2.1 1.6 2.6 0.1 1.0 0.8 1.8 2.2 2.5 1.3 1.1
43.8 46.8 45.9 46.9 46.1 43.5 45.6 45.9 48.5 47.6 46.3 45.8 28.6
± ± ± ± ± ± ± ± ± ± ± ± ±
C18:2 1.5 3.2 2.5 1.1 5.8 2.2 1.7 5.6 2.8 1.1 2.8 0.5 1.7
2.9 5.3 3.4 6.4 3.2 3.1 4.6 4.1 5.5 5.6 2.0 5.8 13.5
± ± ± ± ± ± ± ± ± ± ± ± ±
C18:3 0.0 0.6 2.2 3.7 0.1 1.2 1.7 2.3 2.8 2.6 1.1 3.6 1.0
1.2 1.9 1.5 1.1 1.8 1.4 1.2 1.2 1.4 1.3 1.6 1.3 0.5
± ± ± ± ± ± ± ± ± ± ± ± ±
0.8 0.5 0.6 0.7 0.8 0.1 1.0 0.3 0.1 0.0 0.4 0.3 0.1
a Lipid production media for Entries 1–4 were made with pure glycerol (50 g L−1 ); for Entries 5–12, with synthetic crude glycerol (50 g L−1 glycerol, 5 g L−1 K2 SO4 and 5 g L−1 methanol). b Crude glycerol (50 g L−1 ) was home-made from the base-catalyzed biodiesel production process.
might be due to the alteration of the fluidity of cell membranes by methanol, as has been reported elsewhere [16,31]. Similar results have been found for other oleaginous species [29], such as microalgae Schizochytrium limacinum [32] and oleaginous mold Pythium irregulare [33].
crude glycerol into lipid with high yield using the two-stage lipid production process. Our data provided an attractive route to integrate biodiesel production with microbial lipid technology for better economical viability. Acknowledgements
3.5. Fatty acid compositional profile of lipid products Lipid samples were transmethylated and the resulting fatty acid methyl esters (FAMEs) were analyzed by GC. Results listed in Table 3 showed the distributions of the following fatty acids, oleic acid (C18:1), palmitic acid (C16:0), stearic acid (C18:0), linoleic acid (C18:2), myristic acid (C14:0), linolenic acid (C18:3) and palmitoleic acid (C16:1). It should be noted that when home-made crude glycerol was used, there were more palmitoleic acid and linolenic acid, but less oleic acid. Nonetheless, those with 16 and 18 carbon atoms took over 95% of the total fatty acids. This result was in line with previous observations that similar fatty acid compositions were found for lipids produced from refined glycerol and crude glycerol [3,16,30]. Such fatty acid compositional profiles were similar to those of vegetable oils, indicating that microbial lipids produced from crude glycerol are of great potential as biodiesel feedstock. Except for the sample with home-made crude glycerol, the contents of linoleic acid were below the specified limit of 12% (w/w) according to the European Standard EN 14214. 4. Conclusions Direct utilization of biodiesel-derived glycerol is imperative in terms of resource economy and environmental friendliness for large-scale biodiesel production. We demonstrated that the oleaginous yeast R. toruloides Y4 had great capacity to convert
This work was financially supported by the National Basic Research and Development Program of China (2011CB707405), the National Natural Scientific Foundation of China (21325627), and the Knowledge Innovation Program of Chinese Academy of Sciences (KGZD-EW-304-2). References [1] A. Röttig, L. Wenning, D. Bröker, A. Steinbüchel, Fatty acid alkyl esters: perspectives for production of alternative biofuels, Appl. Microbiol. Biotechnol. 85 (2010) 1713–1733. [2] B.D. Wahlen, M.R. Morgan, A.T. McCurdy, R.M. Willis, M.D. Morgan, D.J. Dye, et al., Biodiesel from microalgae, yeast, and bacteria: engine performance and exhaust emissions, Energ. Fuels 27 (2013) 220–228. [3] S. Papanikolaou, G. Aggelis, Lipids of oleaginous yeasts. Part I: Biochemistry of single cell oil production, Eur. J. Lipid Sci. Technol. 113 (2011) 1031–1051. [4] C. Ratledge, J.P. Wynn, The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms, Adv. Appl. Microbiol. 51 (2002) 1–51. [5] B. Liu, Z.K. Zhao, Biodiesel production by direct methanolysis of oleaginous microbial biomass, J. Chem. Technol. Biotechnol. 82 (2007) 775–780. [6] X. Meng, J. Yang, X. Xu, L. Zhang, Q. Nie, M. Xian, Biodiesel production from oleaginous microorganisms, Renew. Energ. 34 (2009) 1–5. [7] X. Chen, Z. Li, X. Zhang, F. Hu, D.D. Ryu, J. Bao, Screening of oleaginous yeast strains tolerant to lignocellulose degradation compounds, Appl. Biochem. Biotechnol. 159 (2009) 591–604. [8] Z. Gong, H. Shen, Q. Wang, X. Yang, H. Xie, Z.K. Zhao, Efficient conversion of biomass into lipids by using the simultaneous saccharification and enhanced lipid production process, Biotechnol. Biofuels 6 (2013) 36. [9] F. Yang, M.A. Hanna, R. Sun, Value-added uses for crude glycerol – a byproduct of biodiesel production, Biotechnol. Biofuels 5 (2012) 13.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Recycling biodiesel-derived glycerol by the oleaginous yeast Rhodosporidium toruloides Y4 through the two-stage lipid production process Xiaobing Yang a,b,c , Guojie Jin b , Zhiwei Gong b , Hongwei Shen b , Fengwu Bai a,∗∗ , Zongbao Kent Zhao b,∗ a
Dalian University of Technology, Dalian 116024, PR China Dalian National Laboratory for Clean Energy, Dalian Institute of Chemical Physics, CAS, Dalian 116023, PR China c University of Chinese Academy of Sciences, Beijing 100049, PR China b
a r t i c l e
i n f o
Article history: Received 5 May 2014 Received in revised form 26 July 2014 Accepted 28 July 2014 Available online 5 August 2014 Keywords: Bioconversion Biodiesel Glycerol Microbial growth Optimization Oleaginous yeast
a b s t r a c t Direct utilization of crude glycerol, a major byproduct in biodiesel industry, becomes imperative, because its production has outpaced the demand recently. We demonstrated that the oleaginous yeast Rhodosporidium toruloides Y4 had a great capacity to convert glycerol into lipids with high yield using the two-stage production process. Significantly higher cell mass and lipid yield were observed when the media were made with synthetic crude glycerol than pure glycerol. The process achieved a lipid yield of 0.22 g g−1 glycerol, which was comparable with the lipid yield using glucose as the substrate. Lipid samples showed similar fatty acid compositional profiles to those of vegetable oils, suggesting that such microbial lipids were potential feedstock for biodiesel production. Our data provided an attractive route to integrate biodiesel production with microbial lipid technology for better resource efficiency and economical viability. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Concerns about the costs and supply of fossil resources and environmental deterioration have been driving our society to explore alternative transportation fuels. Biodiesel is a proven biofuel that can be dropped directly in the established infrastructure and blended legally with petroleum diesel in any percentages [1]. Our current technology for biodiesel production is depending on vegetable oils and animal fats, however, these resources are limited. Microbial lipids prepared from oleaginous yeasts, bacteria, and microalgae have been employed to produce biodiesel of comparable properties with those made from typical plant oils [2]. Besides, yeast lipids can be utilized as substitutes of high-added value lipids (e.g. cocoa-butter of other exotic fats equivalents) [3,4]. Microbial lipid technology has a number of advantages, such as shorter life cycle and without competing with agriculture for land use. Thus,
∗ Corresponding author. Tel.: +86 411 84379211; fax: +86 411 84379211. ∗∗ Corresponding author. Tel.: +86 411 84379066; fax: +86 411 84706329. E-mail addresses: [email protected] (F. Bai), [email protected], [email protected] (Z.K. Zhao). http://dx.doi.org/10.1016/j.bej.2014.07.015 1369-703X/© 2014 Elsevier B.V. All rights reserved.
microbial lipids are considered as potential feedstock for biodiesel [5,6]. It remains challenging to produce microbial lipids at large scale because costs for feedstock and product recovery are high. Attempts to use lignocellulosic biomass have been reported by a number of groups, but the overall efficiency has been low [7,8]. Crude glycerol is a major byproduct in biodiesel and oleochemical industries, and its production has outpaced the demand recently [9]. Because of the presence of impurities, such as salts, methanol, organic acids and heavy metals, it is difficult to find a direct utilization of crude glycerol in chemical industry. Although refined glycerol is valuable, the purification process is costly. In some European countries, crude glycerol is even simply treated as industrial wastewater. Glycerol has been explored as carbon source for the fermentative production of value-added products such as 1,3propanediol [10,11], citric acid [10], biopolymers [12] and succinic acid [13]. Microbial lipid production by fed-batch culture of oleaginous species on glycerol has also been known, but lipid yields usually remained lower than those with glucose as the carbon source [14–17]. We have recently developed the two-stage lipid production process [18,19]. In the first stage, cells are cultivated in a nutrientrich medium for propagation; and in the second stage, cells are
X. Yang et al. / Biochemical Engineering Journal 91 (2014) 86–91
A
87
Media with C–, N– and P–sources
Pre-cultures
Lipid-rich cells
B
Cells
Media with C–source only
Fig. 1. Microbial lipid production based on conventional (path A) and two-stage (path B) process.
resuspended in a medium contained carbon source without auxiliary nutrients for lipid production (Fig. 1, Path B). Because of the absence of auxiliary nutrients such as nitrogen and phosphorous sources, biosynthesis of protein and nucleic acids ceased, and cell propagation was inhibited. However, the conversion of carbon source into lipid was more efficient, leading to higher lipid yield and productivity [19]. We have further demonstrated the usefulness of this two-stage lipid production process for direct conversion of cellulose and lignocellulosic materials into lipids [8]. In this study, we applied the two-stage process for the conversion of crude glycerol into lipid by the oleaginous yeast Rhodosporidium toruloides Y4. This yeast strain is a robust lipid producer capable of accumulating lipids up to 76% of cell dry weight [19] and tolerating major inhibitors found in biomass hydrolysates [20]. Our data provided an attractive route to integrate biodiesel production with microbial lipid technology for better resource utilization efficiency and economical viability. 2. Materials and methods 2.1. Strain, reagents and media When R. toruloides AS 2.1389 was cultivated in cornstalk hydrolysates, a more robust strain R. toruloides Y4 was isolated [19]. It was maintained at 4 ◦ C on YPD agar slant containing (g L−1 ) glucose, 20; yeast extract, 10; peptone, 10 and agar powder 15. Compound 2-(N-morpholino)ethanesulfonic acid (MES) was purchased from Sigma. All other chemicals were of analytical grade and bought locally. The edible soy bean oil was from China Oil & Foodstuffs Corporation (China). Because the composition of crude glycerol varies and usually contains 60–90% glycerol, 5–6% salts and 4–6% methanol [10–12,16], the synthetic crude glycerol stock solution in this study was made by dissolving glycerol, methanol and K2 SO4 in distilled water to final concentrations of 60 wt%, 6 wt% and 6 wt%, respectively. The preculture medium contained (g L−1 ) glucose or glycerol, 20; yeast extract 10 and peptone 10, pH 6.0. To make lipid production media using pure glycerol, appropriate amounts of glycerol, K2 SO4 , K2 HPO4 and methanol were dissolved in distilled water or 50 mM MES buffer. To make lipid production media using synthetic crude glycerol, the stock solution was diluted to an appropriate glycerol concentration with distilled water or 50 mM MES. Initial pH was adjusted with 2 M NaOH when necessary. No sterilization was done for all lipid production media. Crude glycerol samples were also prepared in the lab. Briefly, a mixture of vegetable oil and 5% KOH methanol solution (v/v, 5/1) was treated at 65 ◦ C for 3 h. Then the reaction mixture was placed in a funnel overnight to ensure the separation of biodiesel from crude glycerol into two layers. The lower layer was evaporated, neutralized with H2 SO4 , and dilution with distilled water. This home-made crude glycerol solution contained: glycerol 50 g L−1 , K2 SO4 5 g L−1 , methanol 5 g L−1 , and others 6 g L−1 . 2.2. Lipid production All cultures were performed with 250-mL Erlenmeyer flasks on a rotary shaker. R. toruloides Y4 cells were incubated in the
preculture medium at 200 rpm, 30 ◦ C for 48 h, collected by centrifugation at 8000 × g for 5 min. The seed cells had a lipid content of 10%. Cells corresponding an initial concentration of 7.0 g L−1 were inoculated into lipid production media and incubated at 200 rpm, 30 ◦ C for 120 h, while the time intervals were 24 h for pH analysis. The control experiments were performed with 50 g L−1 synthetic crude glycerol under identical culture conditions unless otherwise specified. Cells were harvested by centrifugation at 8000 × g for 5 min, washed twice with distilled water (for experiments performed with crude glycerol, the cells were washed with ethanol once before water washes to remove the vegetable oil), and dried at 105 ◦ C overnight to a constant weight to obtain dry cell weight (DCW).
2.3. Analysis methods Glycerol was quantified by ion chromatography (IC) on the Dionex ICS2500 system (Dionex, America) at 30 ◦ C with a flow rate at 1 mL/min using an analytical column CarboPac PA10 4 × 250 mm and the guard column CarboPac PA10 4 × 50 mm by isocratic elution with a mobile phase consisting of 18 mM NaOH. Under such conditions, the retention time for glycerol was 1.63 min. Methanol was determined using the alcohol oxidase method [21]. Cell density was determined at 600 nm with a JASCO V-530 UV-visible spectrophotometer (JASCO, Japan). Cellular lipid was extracted by a mixture of chloroform and methanol according to the reported procedure [19]. Briefly, 1.0 g of dry cell was treated with 4 M HCl at 78 ◦ C for 90 min and extracted twice with chloroform and methanol (1/1, v/v), the obtained organic layer were combined and dried using anhydrous Na2 SO4 , then the solvent was removed under vacuum conditions. The lipids were heated to a constant weight at 105 ◦ C. Lipid content was calculated as g lipid per g DCW. Lipid yield was defined as g lipid per g glycerol consumed. The yield of DCW was calculated as per g DCW per g glycerol consumed. Five milliliter of crude glycerol was sampled to determine residual lipids using the chloroform/methanol (1/1, v/v) extraction. To calculate DCW and lipid yields, the amounts of cell mass (7.0 g L−1 ) and lipid (0.7 g L−1 ) of the inocula cells were subtracted, respectively, from the total cell mass and lipid data of each experiment. Experimental data were based on four independent cultures, and were shown as mean ± SD. Lipid samples were transmethylated according to the published procedure with minor modification [19]. Briefly, wet cell pellets from 5 ml of culture were treated with 0.5 ml 5% KOH methanol solution at 65 ◦ C for 50 min, followed by the addition of 0.2 ml BF3 diethyl etherate and 0.5 ml methanol, refluxed for 10 min, cooled, diluted with distilled water and extracted with petroleum ether (bp 60–90 ◦ C). The organic layer was washed twice with distilled water and subjected to fatty acid compositional analysis. Fatty acids were profiled with a 7890F gas chromatography instrument (Techcomp Scientific Instrument Co. Ltd., Shanghai, China) equipped with a cross-linked capillary FFAP column (30 m × 0.32 mm × 0.4 mm) and flame ionization detector. Operating conditions: N2 carrier as 40 ml/min, injection port temperature 250 ◦ C, oven temperature 190 ◦ C and the detector temperature was 280 ◦ C. Fatty acids were identified by comparison of their retention times with those of
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standards (Sigma, USA) and quantified based on their respective peak areas and normalized.
A 12
3. Results and discussion
Control
7
9
11
9
3.2. The effect of pH on lipid production Culture pH has major effects on cell growth and product accumulation in biological processes. In addition, acid (H2 SO4 ) and base (KOH or NaOH) were utilized in the process of biodiesel production. If R. toruloides Y4 can tolerate a wide range of initial pH, there will be less chemicals consumption to control pH when the crude glycerol is employed. Thus, two sets of experiments were done to test the effect of pH on lipid production by R. toruloides Y4. Firstly, cells were inoculated in lipid production media of different initial pH values. Media pH dropped rapidly to about 3.3 within 24 h for all experiments (Fig. 2A). These results suggested that R. toruloides Y4 might initially produce some organic acids, such as oxalic acid, that brought culture pH to lower values. While the production of organic acids from glycerol has been demonstrated previously [22], oxalic acid concentration in this study was below 0.5 g L−1 , and thus only a minus amount of glycerol was converted into acids. The lipid content was slightly affected by higher initial pH while the cell mass and lipid yield were similar (Fig. 2B). Interestingly, other oleaginous yeasts have also been demonstrated to acidify the culture broth and be able to tolerate high initial pH values [24]. When the media were buffered with 50 mM MES to hold constant pH at a range of 3.7–6.5, there was no significant difference (Fig. 2C). Specifically, higher
6
3
0 0
20
40
60
80
100
120
Time (h)
B
Cell mass
Lipid content
Lipid yield
Cell mass(g/L), Lipid content (%) Lipid yield x100 (g/g)
40
30
20
10
0 Control
I7.0
I9.0
I11.0
pH
C
Cell mass
Lipid content
Lipid yield
40 Cell mass (g/L), Lipid content (%) Lipid yield x100 (g/g)
Two sets of experiments were done where lipid production media were made with pure glycerol or synthetic crude glycerol at different initial glycerol concentrations, and results are shown in Table 1. It was clear that cell mass and lipid titer were the highest when initial glycerol concentration was 100 g L−1 . When initial glycerol concentration increased further, cell mass and lipid titer decreased (Table 1). This was likely due to the inhibition of cell growth by an increased osmotic pressure [10,14]. Except for experiments with initial glycerol at 20 g L−1 , lipid contents were within the range of 34.2–42.5% for all other experiments. Because the inoculated cells had residual nitrogen sources, those experiments with 20 g L−1 initial glycerol might have lower carbon-to-nitrogen ratio than others with higher initial glycerol concentrations, and thus leading to low lipid contents. Lipid yields were very close to 0.20 g g−1 glycerol in the experiments with 50 g L−1 of initial glycerol, and were higher than 0.21 g g−1 glycerol for those where initial glycerol concentrations of the media were 100 g L−1 or 150 g L−1 (Table 1). The maximum net lipid yield of 0.22 g g−1 glycerol, comparable with lipid yields using glucose as the substrate [22,23], was achieved for the experiments using 150 g L−1 pure glycerol or 50 g L−1 crude glycerol (Table 1). The fact that high lipid yields were achieved across a wide concentration range of initial glycerol indicated that consumed glycerol was efficiently used for lipid biosynthesis. It was interesting to note that synthetic crude glycerol, at 50 g L−1 , gave better results than pure glycerol in terms of cell mass, lipid and lipid content. It should be noted that, for the experiment with synthetic crude glycerol at 50 g L−1 , the medium also contained 5.0 g L−1 methanol and 5.0 g L−1 K2 SO4 . Therefore, it seemed that the presence of low concentrations of methanol or K2 SO4 favored lipid production (vide infra). However, when glycerol concentrations were higher than 100 g L−1 , higher concentrations of methanol and K2 SO4 might inhibit lipid production due to toxicity or high osmotic pressure. Similar phenomena have been observed for lipid production recently [16].
Initial pH
3.1. The effect of initial glycerol concentration on lipid production
30
20
10
0 Control
M3.7
M5.5 pH
M6.0
M6.5
Fig. 2. Effects of pH on lipid production by R. toruloides Y4. Synthetic crude glycerol (50 g L−1 glycerol, 5 g L−1 K2 SO4 and 5 g L−1 methanol) was used, and the cultures were held at 30 ◦ C, 200 rpm for 120 h. (A) The evolution of media pH over time. (B) Results of lipid production at different initial pH values. (C) Results of lipid production where media pH was maintained with 50 mM MES.
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Table 1 The effect of initial glycerol concentration on lipid production by the two-stage process.a Glycerol (g L−1 )
Glycerol quality
Total cell mass (g L−1 )
20
Pure Crude Pure Crude Crudeb Pure Crude Pure Crude Pure Crude
11.7 11.7 17.7 19.4 24.9 21.1 20.3 18.5 16.6 16.3 13.3
50
100 150 200 a b
± ± ± ± ± ± ± ± ± ± ±
0.5 1.4 1.3 1.4 0.5 0.2 2.4 0.5 2.2 0.1 1.4
Lipid (g L−1 ) 2.3 2.5 6.2 6.9 12.2 8.5 8.6 7.6 6.6 6.8 5.6
± ± ± ± ± ± ± ± ± ± ±
0.4 0.3 0.2 0.6 0.2 1.1 0.7 0.6 0.3 0.3 0.2
Lipid content (%) 19.7 21.6 34.8 35.4 48.9 40.3 42.5 41.1 40.4 39.6 41.5
± ± ± ± ± ± ± ± ± ± ±
3.3 0.3 2.2 0.7 0.6 4.7 2.3 3.8 3.0 4.9 4.0
Lipid yield (g 100 g−1 ) 16.3 14.8 19.3 19.7 22.0 21.9 21.8 22.2 21.8 18.4 18.5
± ± ± ± ± ± ± ± ± ± ±
1.8 1.4 0.6 1.1 0.1 0.5 1.6 0.5 1.5 2.1 0.3
Glycerol used (g L−1 ) 12.9 18.3 30.6 36.8 50.0 38.0 42.2 35.7 29.0 35.9 27.3
± ± ± ± ± ± ± ± ± ± ±
2.3 1.5 0.5 2.1 0.0 2.5 2.6 0.7 0.5 1.3 2.2
DCW yield (g 100 g−1 ) 36.4 26.1 32.7 33.6 35.8 37.3 31.6 32.9 33.1 26.1 23.0
± ± ± ± ± ± ± ± ± ± ±
4.0 8.5 0.6 2.3 1.1 5.5 6.1 1.3 1.7 1.8 4.2
Pure glycerol was used, and the culture conditions were 30 ◦ C, 200 rpm, 120 h, pH 5.5. Crude glycerol was home-made from the base-catalyzed biodiesel production process.
initial pH resulted in slightly lower cell mass and lipid yield but similar lipid content. The highest cell mass and lipid content of 22.2 g L−1 and 40%, respectively, were achieved at pH 6.0. The highest lipid yield of 0.21 g g−1 , however, was observed at pH 3.7. These results demonstrated that R. toruloides Y4 could maintain good capacity for the conversion of glycerol into lipid at a relatively broad range of initial pH. 3.3. The effect of loading volume on lipid production Since lipid synthesis is an aerobic process, dissolved oxygen should be a crucial factor. To evaluate the effect of aeration on lipid production, cultures were done in 250-mL flasks with different media volumes, and results are shown in Fig. 3. It was obvious that cell mass, lipid content and lipid yield were decreasing when the media volume increased from 20 mL to 50 mL. The highest cell mass, lipid content and net lipid yield were 24.3 g L−1 , 47.3% and 0.22 g g−1 , respectively, obtained at the loading volume of 20 mL. Compared with results of the loading volume of 50 mL, significantly higher cell mass (17.4% increase) and lipid content (24.4% increase) were obtained. The net lipid yield was 0.22 g g−1 if lipids from the seed cells, which contained 8–10% lipid, were subtracted. These results suggested that oxygen supply was limiting, and that oxygen-transfer should be intensified to further improve lipid productivity. Detailed biochemical analysis of lipid biosynthesis indicated that the production of 1 mole of trioleoylglycerol requires 16 mole
Cell mass
Lipid content
Lipid yield
Cell mass (g/L), Lipid content (%) Lipid yield x 100 (g/g)
50
40
30
20
10
0 20
30
40
50
Loading volume (mL) Fig. 3. Effects of the loading volume on lipid production. Synthetic crude glycerol (50 g L−1 glycerol, 5 g L−1 K2 SO4 and 5 g L−1 methanol) was used, and the culture was held at 30 ◦ C, 200 rpm, pH 5.5 for 120 h.
of glucose, and the lipid yield is 0.31 g g−1 [3,4]. Similarly, the theoretical lipid yield is 0.30 g g−1 glycerol because about 32 mole of glycerol is used to produce 1 mole of trioleoylglycerol. While high lipid yields of over 0.20 g g−1 glucose have been documented in many reports [18,19,25], comparable results have been only achieved in a few reports for glycerol [22]. A number of oleaginous strains have been tested for the conversion of glycerol into lipids, but lipid yields were usually below 0.15 g g−1 glycerol [3,16,26–29]. Lipid yield of 0.19 g g−1 crude glycerol was reported using rapeseed hydrolysates as nitrogen source instead of yeast extract [15]. In this study, the yield was improved to 0.22 g g−1 glycerol in the absence of exogenous nitrogen source, suggesting that the crude glycerol was good feedstock for microbial lipid production. 3.4. The effects of potential impurities on lipid production Currently the biodiesel industry is mainly operated with inorganic acid/base catalysts in the presence of excess methanol and lipids used for biodiesel production, especially waste oils, contain various amounts of phospholipids, phosphate are generated in the process. Depending on product separation processes, the raw glycerol stream contained 60–90 wt% glycerol and 5–6 wt% methanol [10–12,16]. Therefore, salts such as sulfates and phosphates, and methanol are major impurities in crude glycerol. Therefore, many experiments have been performed to demonstrate how these impurities could play roles in biological utilization of crude glycerol, and findings have been reviewed elsewhere [30]. Here, experiments were designed to include various amounts of K2 SO4 , K2 HPO4 or methanol in the media for lipid production by R. toruloides Y4 using the two-stage process. Compared with the control experiment in the absence of these impurities, the addition of K2 SO4 up to 8 g L−1 clearly increased cell mass, lipid content and lipid yield (Table 2). Similar phenomena have been described recently [16]. The presence of K2 SO4 provided sulphur element which is essential for provision of acyl-S-CoA and S-containing amino acids. Besides, potassium ion is an activator for many enzymes. However, when K2 HPO4 was added to an initial concentration of 0.5 g L−1 or 1.0 g L−1 , slightly better results were obtained; but it was slightly worse in the presence of 2.0 g L−1 K2 HPO4 (Table 2). It should be noted that initial and final media pH values were 8.5 and 6.2, 8.8 and 6.8, and 8.9 and 7.1, respectively, for the cultures with initial K2 HPO4 concentrations of 0.5, 1.0 g and 2.0 g L−1 . Experimental results indicated that cell growth and lipid production by R. toruloides Y4 were seriously inhibited in buffered YEPD media at pH above 7.0 (data not shown). Thus, an increased media pH in the presence of 2.0 g L−1 K2 HPO4 led to slightly inhibiting the lipid production. As shown in Table 2, cell mass and lipid yield were a little lower in the presence of methanol up to 8.0 g L−1 . These results indicated that methanol slightly inhibited lipid production, which
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X. Yang et al. / Biochemical Engineering Journal 91 (2014) 86–91
Table 2 The effects of potential impurities on lipid production.a K2 SO4 (g L−1 )
K2 HPO4 (g L−1 )
Methanol (g L−1 )
Total cell mass (g L−1 )
Control 2 4 8 – – – – – –
– – – – 0.5 1 2 – – –
– – – – – – – 2 4 8
17.7 18.9 19.5 20.9 20.1 19.6 18.0 17.0 16.7 16.8
a
± ± ± ± ± ± ± ± ± ±
Lipid (g L−1 )
1.3 0.4 0.1 0.8 1.3 1.4 1.3 0.5 0.4 0.5
6.2 6.7 6.9 7.6 8.6 6.6 5.6 5.7 5.7 5.8
± ± ± ± ± ± ± ± ± ±
Lipid content (%)
0.2 0.7 0.3 0.2 0.7 0.3 0.2 0.7 0.3 0.2
34.8 35.7 35.7 36.5 33.9 31.9 31.3 33.6 34.1 34.5
± ± ± ± ± ± ± ± ± ±
Glycerol used (g L−1 )
Lipid yield (g 100 g−1 )
2.2 2.6 1.4 1.2 1.9 3.0 3.0 1.5 0.8 2.7
19.3 21.7 21.4 21.1 22.2 20.0 18.5 22.8 22.6 21.5
± ± ± ± ± ± ± ± ± ±
0.6 1.7 1.9 0.9 1.6 1.3 1.0 0.6 0.2 1.1
30.6 31.8 32.4 34.5 30.3 28.4 26.4 24.3 23.5 24.7
± ± ± ± ± ± ± ± ± ±
DCW yield (g 100 g−1 )
0.5 2.7 0.2 0.8 2.6 1.3 3.0 1.0 1.3 1.1
32.7 33.6 37.3 38.5 42.4 44.0 41.4 39.8 41.5 39.8
± ± ± ± ± ± ± ± ± ±
0.6 3.8 0.6 1.7 1.7 0.6 1.2 2.8 2.5 2.3
Pure glycerol (50 g L−1 ) was used and the culture conditions were 30 ◦ C, 200 rpm, 120 h, pH 5.5.
Table 3 The fatty acid compositional profiles of lipid samples produced by R. toruloides Y4. Entry
Key culture parametersa
Lipid content (%)
1 2 3 4 5 6 7 8 9 10 11 12 13b
– 8 g L−1 , K2 SO4 0.5 g L−1 , K2 HPO4 2 g L−1 , methanol pH 3.7, in MES pH 5.5, in MES pH 6.0, in MES pH 6.5, in MES Initial pH 5.5 Initial pH 7.0 Initial pH 9.0 Initial pH 11.0 Crude glycerol
35.0 35.9 33.9 33.6 39.3 41.0 40.8 41.2 39.4 38.5 37.6 37.0 48.9
± ± ± ± ± ± ± ± ± ± ± ± ±
1.2 1.7 1.9 1.6 1.5 1.9 1.7 1.8 0.5 1.9 1.7 1.4 0.6
C14:0 1.4 1.5 1.7 1.2 1.4 1.7 1.7 1.7 1.2 1.3 1.4 1.3 1.5
± ± ± ± ± ± ± ± ± ± ± ± ±
C16:0 0.2 0.2 0.2 0.0 0.2 0.2 0.3 0.1 0.1 0.2 0.2 0.2 0.1
27.8 27.4 29.1 23.8 28.5 34.1 36.5 36.9 25.8 26.0 27.7 27.1 33.2
± ± ± ± ± ± ± ± ± ± ± ± ±
C16:1 1.2 3.7 2.6 2.0 5.8 4.6 2.0 2.2 4.6 4.1 4.9 5.5 2.1
0.6 0.9 0.9 0.8 0.7 1.0 1.3 1.1 0.8 0.8 0.8 0.8 3.0
± ± ± ± ± ± ± ± ± ± ± ± ±
C18:0 0.1 0.3 0.3 0.4 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.2 1.0
21.8 15.9 17.2 19.3 18.2 15.0 14.1 14.1 16.9 15.7 16.7 15.3 20.1
± ± ± ± ± ± ± ± ± ± ± ± ±
C18:1 0.3 0.6 2.1 1.6 2.6 0.1 1.0 0.8 1.8 2.2 2.5 1.3 1.1
43.8 46.8 45.9 46.9 46.1 43.5 45.6 45.9 48.5 47.6 46.3 45.8 28.6
± ± ± ± ± ± ± ± ± ± ± ± ±
C18:2 1.5 3.2 2.5 1.1 5.8 2.2 1.7 5.6 2.8 1.1 2.8 0.5 1.7
2.9 5.3 3.4 6.4 3.2 3.1 4.6 4.1 5.5 5.6 2.0 5.8 13.5
± ± ± ± ± ± ± ± ± ± ± ± ±
C18:3 0.0 0.6 2.2 3.7 0.1 1.2 1.7 2.3 2.8 2.6 1.1 3.6 1.0
1.2 1.9 1.5 1.1 1.8 1.4 1.2 1.2 1.4 1.3 1.6 1.3 0.5
± ± ± ± ± ± ± ± ± ± ± ± ±
0.8 0.5 0.6 0.7 0.8 0.1 1.0 0.3 0.1 0.0 0.4 0.3 0.1
a Lipid production media for Entries 1–4 were made with pure glycerol (50 g L−1 ); for Entries 5–12, with synthetic crude glycerol (50 g L−1 glycerol, 5 g L−1 K2 SO4 and 5 g L−1 methanol). b Crude glycerol (50 g L−1 ) was home-made from the base-catalyzed biodiesel production process.
might be due to the alteration of the fluidity of cell membranes by methanol, as has been reported elsewhere [16,31]. Similar results have been found for other oleaginous species [29], such as microalgae Schizochytrium limacinum [32] and oleaginous mold Pythium irregulare [33].
crude glycerol into lipid with high yield using the two-stage lipid production process. Our data provided an attractive route to integrate biodiesel production with microbial lipid technology for better economical viability. Acknowledgements
3.5. Fatty acid compositional profile of lipid products Lipid samples were transmethylated and the resulting fatty acid methyl esters (FAMEs) were analyzed by GC. Results listed in Table 3 showed the distributions of the following fatty acids, oleic acid (C18:1), palmitic acid (C16:0), stearic acid (C18:0), linoleic acid (C18:2), myristic acid (C14:0), linolenic acid (C18:3) and palmitoleic acid (C16:1). It should be noted that when home-made crude glycerol was used, there were more palmitoleic acid and linolenic acid, but less oleic acid. Nonetheless, those with 16 and 18 carbon atoms took over 95% of the total fatty acids. This result was in line with previous observations that similar fatty acid compositions were found for lipids produced from refined glycerol and crude glycerol [3,16,30]. Such fatty acid compositional profiles were similar to those of vegetable oils, indicating that microbial lipids produced from crude glycerol are of great potential as biodiesel feedstock. Except for the sample with home-made crude glycerol, the contents of linoleic acid were below the specified limit of 12% (w/w) according to the European Standard EN 14214. 4. Conclusions Direct utilization of biodiesel-derived glycerol is imperative in terms of resource economy and environmental friendliness for large-scale biodiesel production. We demonstrated that the oleaginous yeast R. toruloides Y4 had great capacity to convert
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