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Microbial conversion of biodiesel byproduct glycerol to triacylglycerols by oleaginous yeast Rhodosporidium toruloides and the individual effect of some impurities on lipid production
Biochemical Engineering Journal 65 (2012) 30–36
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Regular Article
Microbial conversion of biodiesel byproduct glycerol to triacylglycerols by oleaginous yeast Rhodosporidium toruloides and the individual effect of some impurities on lipid production Jingyang Xu, Xuebing Zhao, Wencong Wang, Wei Du, Dehua Liu ∗ Institute of Applied Chemistry, Department of Chemical Engineering, Tsinghua University, Beijing 10084, PR China
a r t i c l e
i n f o
Article history: Received 4 October 2011 Received in revised form 26 March 2012 Accepted 4 April 2012 Available online 11 April 2012 Keywords: Batch processing Bioconversion Glycerol Yeast Microbial lipid Biodiesel
a b s t r a c t With the development of biodiesel industry, the byproduct glycerol will become a considerable resource as feedstock for production of many other chemicals. In present work, microbial conversion of crude glycerol to triacylglycerols (microbial lipid) was proposed and investigated using the oleaginous yeast Rhodosporidium toruloides (R. toruloides) by one-stage batch fermentation. Compared with glucose and refined glycerol, the crude glycerol could obtain significantly higher biomass concentration and lipid yield. The highest biomass concentration of R. toruloides obtained from crude glycerol in a 5 L fermenter reached 26.7 g/L with an intracellular lipid content of 70%. Further study was performed to investigate the individual effect of five representative compounds which were present in crude glycerol as impurities. It was found that within the general concentration ranges, only methanol displayed somewhat inhibitive effect, while others showed positive influence on lipid production. These results indicated that crude glycerol could be directly converted to triacylglycerols by R. toruloides without purification. Contrarily, certain amount of salt and soap could promote the accumulation of biomass and lipid. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Biodiesel industry has become important due to the shortage of fossil oil and increasing awareness of environmental issue. For its sustainable development, however, it is primarily necessary to insure that triacylglycerol (TAG) can be produced continually without competing with food industry. Lipids derived from microorganisms, known as microbial lipid, can be fast synthesized and accumulated intracellularly, with fatty acids compositions resembling these of vegetable oils. Since the production of microbial lipid has many advantages over that of vegetable oils such as short life cycle and no need of agricultural land, it has attracted much interest in recent decades, and considered as a potential non-food feedstock for biodiesel production. Importantly, however, during microbial conversion, abundance of carbon source that can be effectively utilized by microorganisms is required. Traditionally this conversion process is based on starch glucose. Nevertheless high expense of glucose feedstock would significantly limit the industrialization of microbial lipid production. For this reason, renewable and affordable carbon source in abundance is favored.
∗ Corresponding author. Tel.: +86 10 62772130; fax: +86 10 62772130. E-mail address: [email protected] (D. Liu). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.04.003
Crude glycerol is a main byproduct of biodiesel production. As the production of biodiesel grows, it will be generated in a considerable quantity, and its utilization will become an urgent topic. Although refined glycerol could be a valuable product, the purification process is always costly and generally out of the range of economic feasibility for small to medium-sized plants [1]. In some European countries, crude glycerol is even simply treated as industrial wastewater [2]. Nowadays, trials are underway and researchers have already successfully converted crude glycerol not only to useful chemicals such as 1,3-propanediol [3], phytase [4], citric acid [3], but also single cell oil, which can further be transformed to biodiesel [3]. In present work, Rhodosporidium toruloides (R. toruloides) was employed as a promising candidate for prospective industrial application. It can accumulate intracellular lipid up to 60% of its cellular dry weight [5], which is mainly triacylglycerol, resembling that of oils from food crops in terms of fatty acid composition, and has been successfully converted to biodiesel [6]. Besides, R. toruloides has already been proved to be capable to grow on a broad range of substrates including but not limited to: sugars like glucose and xylose, lignocellulosic hydrolysate, and excess sludge hydrolysate [5,7]. Furthermore, it can simultaneously produce carotenoids as by-products, which may have some potential values (data not published yet). However, no related data on the conversion of glycerol to microbial lipid by this robust yeast are found in the
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2. Materials and methods
was 40 ◦ C. The drift pipe temperature was 70 ◦ C, and the nitrogen pressure was 320 kPa. The ash content of crude glycerol was determined by burning the glycerol sample in muffle furnace until a constant weight was reached. The yeast biomass concentration was expressed as the dry weight concentration of cell biomass (CB , g/L). After the fermentation broth was centrifuged, the wet cells were washed with deioned water and dried at 105 ◦ C to a constant weight. The dry weight concentration of cell biomass was then calculated according to following equation:
2.1. Microorganism and chemicals
CB (g/L) =
literatures. Besides, for effective utilization of biodiesel byproduct, detailed knowledge is required to comprehensively evaluate its fermentability and also the effects of impurities. Therefore, the objective of present work is to find out whether the crude glycerol can be directly used as carbon source for microbial lipid production, and how the impurities present in crude glycerol affect the growth, lipid accumulation and as well as the lipid fatty acids composition of R. toruloides.
The oleaginous microorganism used in the experiments was R. toruloides AS2.1389, which was obtained from China General Microbiological Culture Collection Center (CGMCC). The yeast was maintained at 4 ◦ C on yeast extract peptone dextrose (YEPD) slopes containing: 20 g/L glucose, 10 g/L peptone, 10 g/L yeast extract, and 20 g/L agar. For preparing inocula, the organism was pre-cultured overnight in the medium containing 20 g/L glucose (or 15 g/L glycerol), 10 g/L peptone and 10 g/L yeast extract at 30 ◦ C and 200 rpm in an air-bath shaker. The chemicals and reagents in all the experiments were analytically pure and purchased locally. The crude glycerol samples were provided by Hunan Rivers Bioengineering Co., Ltd., Hunan, China. 2.2. Fermentation process Batch fermentation experiments with crude glycerol as carbon substrate were conducted in either flasks or 5-L fermenters. In flask fermentation, the experiments were conducted in 500 mL flasks with 100 mL liquid medium containing 50 g/L glycerol (for crude glycerol, the addition amount was decided by its exact glycerol concentration which was tested by HPLC), 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. The pH value of the liquid medium was adjusted to 6.0. Followed by sterilization at 115 ◦ C for 15 min, 10 mL inocula were added to the medium, and the culture was conducted at 30 ◦ C in an air-bath shaker at 200 rpm for 5–6 days. Before inoculation, the biomass concentration of the inocula was carefully controlled at 0.5–0.6 g/L. Each culture was performed by duplicate test. For culture in 5-L fermenters, the experiments were conducted with 4 L culture volume. The initial glycerol concentration was 60 g/L and other nutrients concentrations were the same as those in flask culture. pH was controlled at 6.0 by feeding 40 wt% NaOH. Dissolved oxygen was controlled at 20–30% saturation by automatic adjustment of stirring speed with 2 vvm aeration. For comparison, glucose was also used as carbon source at the same initial concentration. 2.3. Analytical methods The quantitative analysis of methanol, glucose, and glycerol was performed with a Shimadzu10AVP HPLC system (Shimadzu Corp., Japan) equipped with a RID-10A 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. The estimation of glyceride was performed with a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) with an ELSD-LT II low temperature-evaporative light scattering detector. Before analysis, 2 L sample and 1 mL acetone were mixed thoroughly, and 20 L of the aforementioned mixture was injected. A C18 column (5 m, 250 mm × 4.6 mm) (Dikma Technology, PLATISIL ODS, China) and a gradient elution program by acetonitrile and dichloromethane at 1.5 mL/min was employed, respectively. The column temperature
dry weight of cell biomass (g) volume of fermentation broth (L)
Total crude intracellular lipid was extracted by acid-heating procedure with mixture of chloroform and methanol as extractant [8]. Crude lipid content was expressed as gram lipid per gram of dry biomass. For determination of fatty acid compositions, the crude lipid was first converted to fatty acid methyl ester (FAME) with excess methanol in tert-butanol system under the catalysis of excess loading of immobilized lipase (NS 435 and NS 40044, produced by Novozymes) [9]. The obtained FAME was then 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 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. For describing the efficiency of the microbial lipid production, several response variables, namely, lipid concentration (CL ), lipid yield (YL ), and lipid content (CnL ) are defined as follows, respectively: CL (g/L) =
weight of lipid (g) volume of fermentation broth (L)
YL (g/100 g carbon substrate) =
weight of lipid (g) × 100 weight of carbon substrate consumed (g)
CnL (%) =
weight of lipid (g) × 100% weight of cell biomass (g)
3. Results and discussion 3.1. Lipid production by R. toruloides in flask fermentation Generally, in the biodiesel industry, about 0.1 t of crude glycerol is produced for every 1 t of biodiesel. Some soap and alcohol (usually methanol) together with small amount of fat soluble substance are also present in the glycerol phase depending on the feedstock and production processes. Two types of crude glycerol samples used in present work were analyzed, and their main components are shown in Table 1. Glycerol A contained 85% glycerol, a small amount of glyceride and fatty acid methyl ester (FAME). Notably, after burning the sample to a constant weight, the content of ash residue reached 6.5%, most of which was soluble in water. The ash generally consisted of sodium salts from the catalyst (e.g. sodium hydroxide) of transesterification reaction. Therefore, ash content can be considered as the total percentage of salt and soap components in the crude glycerol sample. Glycerol B was originated from lipase-catalyzed transesterification of palm oil. It contained 32.97% glycerol, a small amount of glyceride and fatty acid methyl ester, and a little ash. However, no
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Table 1 Main components of crude glycerol samples used in present work. Samplea
Glycerol (%)
Methanol (%)
Biodiesel and glyceride (%)
Ash content (%)
A B
85.19 ± 2.16 32.97 ± 0.96
– 14.89 ± 1.10
0.09 ± 0.00 1.81 ± 0.00
6.52 ± 0.16 0.11 ± 0.00
a Crude glycerol A was from an alkaline-catalyzed biodiesel production process; crude glycerol B was from an enzyme-catalyzed biodiesel production process. Both two samples had a brown color with a pH around 6–7 and the rest component was water.
distillation pretreatment was performed, and the methanol concentration was as high as 14.89%. To evaluate whether R. toruloides can grow on crude glycerol, we first examined the growth of this yeast on 50 g/L crude glycerol. For comparison, glucose and refined glycerol were also used as carbon source for culture. The experimental data after 160 h incubation are summarized in Table 2. It can be known that R. toruloides could well utilize refined glycerol for lipid accumulation. Furthermore, R. toruloides grew better in crude glycerol than in refined glycerol. For instance, the biomass concentration obtained by crude glycerol A was 50% higher than that obtained by refined glycerol. Corresponding lipid concentration and yield were also higher. For the scenario of crude glycerol B, similar results were obtained. It was also interesting to find that the yeast even grew better on crude glycerol than on glucose, which shows that crude glycerol from biodiesel production process is a promising feedstock for microbial lipid production. However, the experimental results should be further verified by larger scale cultivations. 3.2. Lipid production by R. toruloides in 5-L bioreactors To further increase the cell growth and production of lipids, batch fermentation was carried out in a 5-L fermentor equipped with pH control and aeration systems. The time courses of cell biomass concentration, substrate consumption and lipid concentration are shown in Fig. 1. As shown in the figure, the growth of R. toruloides on glucose substrate was fast at first, but gradually speeded down as the fermentation proceeded. The final biomass concentration was 16.7 g/L with lipid concentration of 11 g/L. On refined glycerol substrate, glycerol consumption was obviously slower than that of glucose; the lipid production, however, was comparable. On crude glycerol substrates, the kinetics of biomass growth, glycerol consumption and lipid production showed some difference depending on the characteristics of the crude glycerol. On crude glycerol B, the growth and glycerol consumption slowed down after the 150 h, and the final biomass reached 18 g/L with
lipid concentration of 13.4 g/L. On crude glycerol A, the biomass concentration, glycerol consumption, and lipid production were impressively fast at an almost stable rate. The final biomass and lipid concentrations reached 26.7 g/L and 18.5 g/L, respectively, with lipid yield of as high as 17.5 g/100 g consumed glycerol. Compared with refined glycerol substrate, crude glycerol A could obtain 42.0% higher biomass concentration and 68.2% higher lipid concentration. This performance indicated a remarkable advantage when using crude glycerol as carbon source. Compared with the some reported data from the literatures shown in Table 3, crude glycerol seems to be effectively utilized by this yeast, and led to similar or higher biomass and lipid productivity, which demonstrates that crude glycerol from biodiesel production could be a promising feedstock for microbial lipid production. According to the literatures, palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) constitute the majority of fatty acids in microbial triacylglycerols (TAGs) [10,11]. We thus further determined the fatty acid compositions of R. toruloides lipids derived from different carbon sources as shown in Table 4. It was found that the predominant fatty acids were palmitic acid, stearic acid, oleic acid, and linoleic acid; and palmitic acid and oleic acid accounted for about 70% of the total fatty acid composition. There was no significant difference observed for the lipids obtained from refined glycerol and crude glycerol substrates. However, the oleic acid and linolenic acid contents of the lipid obtained from glucose were a little higher than that obtained from glycerol, correspondingly palmitic acid and stearic acid contents were somewhat lower. Above experimental results showed that crude glycerol could be well utilized by R. toruloides with obvious advantages over refined glycerol and even glucose. Scott et al. [12] also demonstrated such advantages when they used crude glycerol from fish oil production as carbon source to produce oil rich in DHA by a thraustochytrid. Since the crude glycerol from biodiesel plant unavoidably contains some impurities such as salts, methanol and soap, which might exert influences on the yeast growth and lipid accumulation, we thus further performed experiments to investigate the individual effects of these impurities on the fermentation process. 3.3. Individual effects of some impurities on lipid production by R. toruloides Some compounds contained in crude glycerol were added into the refined glycerol medium to study the individual effects of the impurities on microbial lipid production. Taking into account that the concentrations of those substances in real crude glycerol composition, we selected the compounds and their concentration ranges as follows: sodium oleate (0–2 g/L), methyl oleate (0–2 g/L), glyceryl monooleate (0–2 g/L), sodium chloride (0–16 g/L), and methanol (0–16 g/L). In most cases, the real concentrations of the impurities do not exceed the upper limit as set.
Fig. 1. Cultivation of R. toruloides in a 5 L fermenter with crude glycerol as carbon substrate. (A) Biomass concentration and (B) lipid concentration.
3.3.1. Effects of soap Oleic acid is one of the main fatty acid compositions of biodiesel feedstock oil [6,13]. In an alkali (for example, sodium hydroxide) catalyzed transesterification reaction, sodium oleate is produced
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Table 2 Lipid production by R. toruloides in flask fermentation for 160 h. Carbon source
CB (g/L)
Glucose Refined glycerol Crude glycerol A Crude glycerol B
14.4 12.8 19.2 20.1
± ± ± ±
CL (g/L) 0.1 0.6 0.4 1.0
7.2 5.6 9.2 8.6
± ± ± ±
YL (g/100 g carbon source)
0.1 0.0 0.8 0.9
13.4 13.9 14.9 14.4
± ± ± ±
CnL (wt %)
0.2 0.6 1.5 1.8
50.0 43.4 47.7 42.9
± ± ± ±
0.4 1.0 2.0 2.0
Table 3 Comparison of lipid production by R. toruloides with those of some other microorganisms. Strain
Carbon source
Biomass concentration (g/L)
Lipid content (%)
Lipid concentration (g/L)
Cultivation
Reference
Cunninghamella echinulata Thraustochytrium sp. Rhodosporidium toruloides Mortierella isabellina Cryptococcus curvatus Yarrowia lipolytica Rhodosporidium toruloides
Glucose Glucose Glucose Crude glycerol Crude glycerol Crude glycerol Glucose Pure glycerol Crude glycerol A Crude glycerol B
15 23.9 18.2 8.5 32.9 8.1 16.7 18.8 26.7 18.0
46 30.1 76.1 51 52.9 43 66.9 58.7 69.5 74.1
6.9 7.2 13.9 4.4 18 3.5 11.2 11.0 18.5 13.4
Batch in flask Batch in flask Batch in flask Batch in fermenter Fed-batch in fermenter Continuous in fermenter Batch in fermenter Batch in fermenter Batch in fermenter Batch in fermenter
[27] [12] [28] [3] [2] [26] This work This work This work This work
Table 4 Fatty acid composition of R. toruloides lipid obtained from different carbon sources. C14 Glucose Glycerol Crude glycerol A Crude glycerol B
1.5 1.6 1.6 1.3
C16 ± ± ± ±
0.2 0.2 0.0 0.3
26.1 28.7 29.1 29.2
C16:1 ± ± ± ±
0.8 1.1 0.6 0.0
0.0 0.2 1.0 1.0
± ± ± ±
C18:0 0.0 0.0 0.1 0.1
13.0 15.3 17.8 13.9
± ± ± ±
C18:1 1.5 1.0 1.0 0.7
46.4 41.5 38.1 41.4
± ± ± ±
C18:2 1.8 0.6 1.5 0.3
9.2 10.1 9.7 10.4
± ± ± ±
C18:3 0.5 0.2 0.0 0.4
3.8 2.6 2.6 2.9
± ± ± ±
0.4 0.1 0.1 0.0
and remains in the reaction system. As shown in Fig. 2, positive effect was observed when sodium oleate was added, and all of the response variables were increased with sodium oleate concentration. For control experiments, the biomass, lipid concentrations and lipid yield were 12.8 g/L, 5.6 g/L and 13.9%, respectively. However, the addition of 0.5–2 g/L sodium oleate resulted in increases of biomass concentration by 25–41%, lipid concentration by 34–59% and lipid yield by 13–43%, respectively. The effect of sodium oleate, as a representative of soap, deserves further discussion. Soap plays a role as surfactant, which may provide some influences in fermentation process. It has been reported by Pyle et al. [14] that soap showed a negative effect on Schizochytrium limacinum for DHA production, and the inhibition was pronounced at higher levels. They suggested that this phenomenon should relate to the function of soap as a surfactant, and
the inhibition is caused by the complex interaction between the cell wall (membrane) and the surfactants. In our experiment, it was found that with the addition of 2 g/L sodium oleate, the medium could be emulsified to a great extent. Thus it can be assumed that sodium oleate should function as a surfactant in the culture medium. However, no significantly negative effect on cell growth and lipid production was observed when sodium oleate concentration was increased to 4 g/L. Contrarily, the presence of sodium oleate in culture medium led to an increase of biomass concentration with associated increase of lipid concentration and yield, which is in accordance with some researches that a proper concentration of surfactants benefits the microbial fermentation process [15–17]. It is very likely that some surfactants can interfere with the permeability of the cell membrane, and enhance the nutritional input from the surroundings to the cells [18].
Fig. 2. The effect of sodium oleate on cell growth, lipid production and lipid yield of R. toruloides cultivated with refined glycerol as carbon source for 7 days.
3.3.2. Effects of glycerides and fatty acid methyl esters After transesterification reaction, biodiesel is separated out as product through pH adjustment and settlement or centrifugation. However, unavoidably there are still some glycerides and fatty acid methyl esters (biodiesel) remaining in the aqueous glycerol phase. Since oleic acid is always the main fatty acid component in most of biodiesel feedstocks and monoglyceride is more or less present in the solution, in this work we selected methyl oleate and glyceryl monooleate as the representatives to investigate their effects on cell growth and lipid accumulation. The results are shown in Figs. 3 and 4. It can be known that both two esters showed somewhat positive influence on cell growth and lipid production. For methyl oleate, the biomass was increased by 9–12%, and lipid production was increased by 7–22%. For glyceryl monooleate, the biomass was increased by 8–13%, and lipid production was increased by 10–16%, respectively. Some yeasts have specific metabolic pathways to utilize hydrophobic substrate such as alkanes, fatty acids and oils. For example, a widely studied yeast Yarrowia lipilytica can utilize oils
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Fig. 3. The effect of methyl oleate on cell growth, lipid production and lipid yield of R. toruloides cultivated with refined glycerol as carbon source for 7 days.
Fig. 5. The effect of sodium chloride on cell growth, lipid production and lipid yield of R. toruloides cultivated with refined glycerol as carbon source for 7 days.
like glycerides by first hydrolyzing the lipid into free fatty acids, and then taking up by the cell as carbon source [19,20]. On the other hand, hydrophobic compounds are known to partition into cell membranes, interfering with the bilayer structure, and modifying its fluidity [16]. Considering that glycerides and fatty acid methyl esters have similar structures to that of sodium oleate while differ in the hydrophilic group, it is likely that such chemicals play a role as a “week” surfactant (compared with soap), and may also have some interactions with cell membranes as well as the culture mediums. However, since the effect of hydrophobic compounds on R. toruloides is not clear yet, corresponding mechanisms should be further investigated.
As shown in Fig. 5, the addition of sodium chloride led to obvious increase of biomass concentration, lipid concentration and yield. It indicated that the concentration of salt in crude glycerol was not high enough to cause an osmotic pressure. Contrarily, with the addition of 4–16 g/L sodium chloride, the biomass concentration, lipid concentration and lipid yield were increased by 12–40%, 20–48%, and 9–20%, respectively. This result was in accordance with the reported phenomena that salt can lead to a higher intracellular lipid content and biomass concentration if added in optimized amount [23–25]. It is probably that a low concentration of salt contributes to the physiological state in favor of lipid synthesis, in which circumstance cells can adapt to the salts with an increased production of carbonhydrates and lipids as the osmoprotectants.
3.3.3. Effect of inorganic salt In alkali-catalyzed production of biodiesel, alkali like sodium hydroxide is used as the catalyst. After transesterfication, pH adjustment, and separation process, part of the sodium remains in glycerol phase in the form of salts. It has been reported that a suitable salt content provides a positive effect on oleaginous microorganisms for cell growth and lipid production. Many microalgae even can utilize salt streams, and produce lipids under high salt concentrations [21,22]. However, excessive amount of salt may cause osmotic pressure. For this reason, sodium chloride was added to culture medium until its concentrations were 4 g/L, 8 g/L, and 16 g/L, respectively, to investigate its effect on cell growth and lipid production.
Fig. 4. The effect of glyceryl monooleate on cell growth, lipid production and lipid yield of R. toruloides cultivated with refined glycerol as carbon source for 7 days.
3.3.4. Effect of methanol Methanol is largely used for transesterification reaction; therefore the unreacted methanol remains in the aqueous phase. Depending on separation process, methanol concentration in the aqueous phase is varied. To cover the methanol concentration ranges of most crude glycerol, in this work, methanol was added to the culture medium until its concentration was 4 g/L, 8 g/L, and 16 g/L, respectively. In our experiments, methanol was found to be somewhat inhibitive to R. toruloides. As shown in Fig. 6, the biomass, lipid concentrations and lipid yield was decreased by about 0–5%, 6–24%, and 6–23%, respectively, when methanol was added. Whereas, it also can be observed that the inhibitory effect did not become even
Fig. 6. The effect of methanol on cell growth, lipid production and lipid yield of R. toruloides cultivated with refined glycerol as carbon source for 7 days.
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Table 5 Fatty acid composition of lipid obtained from R. toruloides growing on refined glycerol in the present of selected crude glycerol impurities. Impurities
C14
Control Sodium chloride Methanol Sodium oleate Methyl oleate Glyceryl momooleate
1.3 1.2 1.3 1.6 1.7 1.6
C16 ± ± ± ± ± ±
0.1 0.0 0.0 0.2 .0.2 0.1
26.3 25.9 25.4 27.2 25.2 23.5
C16:1 ± ± ± ± ± ±
1.4 0.1 0.2 0.7 0.2 0.1
2.7 0.9 2.3 2.1 3.2 3.5
± ± ± ± ± ±
C18:0 0.0 0.0 0.2 0.2 0.1 0.1
11.7 12.1 12.6 12.1 10.8 11.0
± ± ± ± ± ±
C18:1 0.6 0.0 0.0 0.1 0.5 0.5
severer when the methanol concentration was increased to as high as 16 g/L. This observation accords with some literatures that methanol can lead to inhibitory effects on cell growth and lipid synthesis of some oleaginous microorganisms at high concentrations. For instance, Pyle et al. [14] reported that when methanol concentration is above 10 g/L in the medium, several important objective variables including biomass productivity, growth yield, DHA content, DHA yield and DHA productivity go down. Liang et al. also found that methanol could harm the cell growth of S. limacinum, but the inhibitory effect was not significant during the fed-batch fermentation process with a methanol concentration no more than 8 g/L [26]. However, the exact mechanism of methanol to oleaginous microorganisms is not clear yet. It has been hypothesized that methanol might be uptake into the cell and even utilized [2]. In this sense, it is probably that the methanol metabolism could exert some influence on the cell growth and lipid synthesis. Anyway, the experimental results indicate that although methanol can pose somewhat inhibitory effect on R. toruloides, a small amount of methanol remaining in the crude glycerol does not show severe impact on the fermentation process. Additionally, methanol is easy to be separated from crude glycerol through evaporation process, and should be recovered for recycling. Therefore, the effect of methanol actually can be neglected.
3.3.5. Effects of impurities on fatty acid compositional profiles of the yeast lipid To further investigate whether the impurities could affect the lipid synthesis pathways, the fatty acid composition of microbial lipid was analyzed as shown in Table 5. It can be known that the major fatty acid composition of lipids was C16:0 (palmitic acid), C18:0 (stearic acid), C18:1 (oleic acid) and C18:2 (linoleic acid), which accounted for about 26%, 12%, 45% and 11% of the total fatty acids, respectively. The addition of the compounds did not result in significant change of fatty acid compositions. This result was in accordance with the aformentioned conclusion that refined glycerol and crude glycerol led to similar fatty acid compositions. Additionally, it also implied that the impurities of crude glycerol did not affect the fatty acid biosynthetic pathways.
4. Conclusion This study has shown that oleaginous yeast R. touloides had great potential for converting biodiesel byproduct glycerol to valuable lipids, although some impurities such as soap, glycerides, fatty acid methyl esters, inorganic salt and methanol were present in crude glycerol. It was found that some of those impurities could even increase biomass concentration and lipid accumulation, and the impurities did not affect the fatty acid compositions of the obtained lipid. The results demonstrated that biodiesel byproduct glycerol could be directly used as carbon source for microbial lipid production and even more that it could induce higher biomass concentration and lipid yield than glucose.
45.1 46.3 45.4 44.1 43.1 47.1
± ± ± ± ± ±
C18:2 2.4 0.0 0.6 0.3 0.3 0.8
10.8 11.7 11.3 11.3 13.4 11.0
± ± ± ± ± ±
C18:3 0.5 0.0 0.0 0.5 0.5 0.5
2.1 2.0 1.8 1.6 2.6 2.3
± ± ± ± ± ±
Saturated FAs 0.2 0.0 0.0 0.3 0.2 0.1
39.3 39.2 39.4 40.9 37.7 36.1
± ± ± ± ± ±
2.5 0.1 0.2 1.0 0.2 0.3
Unsaturated FAs 60.7 ± 3.0 60.8 ± 0.1 60.6 ± 0.2 59.1 ± 1.0 62.3 ± 0.2 63.9 ± 0.2
Acknowledgments This work is supported by International Cooperation Project of the Ministry of Science and Technology of China (No. 2010DFB40170), and Tsinghua Research Funding (No. 20121080046), which are greatly appreciated by the authors.
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[22] N.M. Verma, S. Mehrotra, A. Shukla, B.N. Mishra, Prospective of biodiesel production utilizing microalgae as the cell factories: a comprehensive discussion, Afr. J. Biotechnol. 9 (2010) 1402–1411. [23] M. Takagi, Karseno, T. Yoshida, Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells, J. Biosci. Bioeng. 101 (2006) 223–226. [24] S. Verma, B.K. De, Lipid, protein and fatty acid profiles of Emericella sp. in different media, Eur. J. Lipid Sci. Technol. 112 (2010) 417–424. [25] A.R. Rao, C. Dayananda, R. Sarada, T.R. Shamala, G.A. Ravishankar, Effect of salinity on growth of green alga Botryococcus braunii and its constituents, Bioresour. Technol. 98 (2007) 560–564.
[26] Y. Liang, N. Sarkany, Y. Cui, J.W. Blackburn, Batch stage study of lipid production from crude glycerol derived from yellow grease or animal fats through microalgal fermentation, Bioresour. Technol. 101 (2010) 6745–6750. [27] S. Fakas, S. Papanikolaou, A. Batsos, M. Galiotou-Panayotou, A. Mallouchos, G. Aggelis, Evaluating renewable carbon sources as substrates for single cell oil production by Cunninghamella echinulata and Mortierella isabellina, Biomass Bioenergy 33 (2009) 573–580. [28] Y. Li, B. Liu, Z. Zhao, F. Bai, Optimization of culture conditions for lipid production by Rhodosporidium toruloides, Chin. J. Biotechnol. 22 (2006) 650–656.
Contents lists available at SciVerse ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular Article
Microbial conversion of biodiesel byproduct glycerol to triacylglycerols by oleaginous yeast Rhodosporidium toruloides and the individual effect of some impurities on lipid production Jingyang Xu, Xuebing Zhao, Wencong Wang, Wei Du, Dehua Liu ∗ Institute of Applied Chemistry, Department of Chemical Engineering, Tsinghua University, Beijing 10084, PR China
a r t i c l e
i n f o
Article history: Received 4 October 2011 Received in revised form 26 March 2012 Accepted 4 April 2012 Available online 11 April 2012 Keywords: Batch processing Bioconversion Glycerol Yeast Microbial lipid Biodiesel
a b s t r a c t With the development of biodiesel industry, the byproduct glycerol will become a considerable resource as feedstock for production of many other chemicals. In present work, microbial conversion of crude glycerol to triacylglycerols (microbial lipid) was proposed and investigated using the oleaginous yeast Rhodosporidium toruloides (R. toruloides) by one-stage batch fermentation. Compared with glucose and refined glycerol, the crude glycerol could obtain significantly higher biomass concentration and lipid yield. The highest biomass concentration of R. toruloides obtained from crude glycerol in a 5 L fermenter reached 26.7 g/L with an intracellular lipid content of 70%. Further study was performed to investigate the individual effect of five representative compounds which were present in crude glycerol as impurities. It was found that within the general concentration ranges, only methanol displayed somewhat inhibitive effect, while others showed positive influence on lipid production. These results indicated that crude glycerol could be directly converted to triacylglycerols by R. toruloides without purification. Contrarily, certain amount of salt and soap could promote the accumulation of biomass and lipid. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Biodiesel industry has become important due to the shortage of fossil oil and increasing awareness of environmental issue. For its sustainable development, however, it is primarily necessary to insure that triacylglycerol (TAG) can be produced continually without competing with food industry. Lipids derived from microorganisms, known as microbial lipid, can be fast synthesized and accumulated intracellularly, with fatty acids compositions resembling these of vegetable oils. Since the production of microbial lipid has many advantages over that of vegetable oils such as short life cycle and no need of agricultural land, it has attracted much interest in recent decades, and considered as a potential non-food feedstock for biodiesel production. Importantly, however, during microbial conversion, abundance of carbon source that can be effectively utilized by microorganisms is required. Traditionally this conversion process is based on starch glucose. Nevertheless high expense of glucose feedstock would significantly limit the industrialization of microbial lipid production. For this reason, renewable and affordable carbon source in abundance is favored.
∗ Corresponding author. Tel.: +86 10 62772130; fax: +86 10 62772130. E-mail address: [email protected] (D. Liu). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.04.003
Crude glycerol is a main byproduct of biodiesel production. As the production of biodiesel grows, it will be generated in a considerable quantity, and its utilization will become an urgent topic. Although refined glycerol could be a valuable product, the purification process is always costly and generally out of the range of economic feasibility for small to medium-sized plants [1]. In some European countries, crude glycerol is even simply treated as industrial wastewater [2]. Nowadays, trials are underway and researchers have already successfully converted crude glycerol not only to useful chemicals such as 1,3-propanediol [3], phytase [4], citric acid [3], but also single cell oil, which can further be transformed to biodiesel [3]. In present work, Rhodosporidium toruloides (R. toruloides) was employed as a promising candidate for prospective industrial application. It can accumulate intracellular lipid up to 60% of its cellular dry weight [5], which is mainly triacylglycerol, resembling that of oils from food crops in terms of fatty acid composition, and has been successfully converted to biodiesel [6]. Besides, R. toruloides has already been proved to be capable to grow on a broad range of substrates including but not limited to: sugars like glucose and xylose, lignocellulosic hydrolysate, and excess sludge hydrolysate [5,7]. Furthermore, it can simultaneously produce carotenoids as by-products, which may have some potential values (data not published yet). However, no related data on the conversion of glycerol to microbial lipid by this robust yeast are found in the
J. Xu et al. / Biochemical Engineering Journal 65 (2012) 30–36
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2. Materials and methods
was 40 ◦ C. The drift pipe temperature was 70 ◦ C, and the nitrogen pressure was 320 kPa. The ash content of crude glycerol was determined by burning the glycerol sample in muffle furnace until a constant weight was reached. The yeast biomass concentration was expressed as the dry weight concentration of cell biomass (CB , g/L). After the fermentation broth was centrifuged, the wet cells were washed with deioned water and dried at 105 ◦ C to a constant weight. The dry weight concentration of cell biomass was then calculated according to following equation:
2.1. Microorganism and chemicals
CB (g/L) =
literatures. Besides, for effective utilization of biodiesel byproduct, detailed knowledge is required to comprehensively evaluate its fermentability and also the effects of impurities. Therefore, the objective of present work is to find out whether the crude glycerol can be directly used as carbon source for microbial lipid production, and how the impurities present in crude glycerol affect the growth, lipid accumulation and as well as the lipid fatty acids composition of R. toruloides.
The oleaginous microorganism used in the experiments was R. toruloides AS2.1389, which was obtained from China General Microbiological Culture Collection Center (CGMCC). The yeast was maintained at 4 ◦ C on yeast extract peptone dextrose (YEPD) slopes containing: 20 g/L glucose, 10 g/L peptone, 10 g/L yeast extract, and 20 g/L agar. For preparing inocula, the organism was pre-cultured overnight in the medium containing 20 g/L glucose (or 15 g/L glycerol), 10 g/L peptone and 10 g/L yeast extract at 30 ◦ C and 200 rpm in an air-bath shaker. The chemicals and reagents in all the experiments were analytically pure and purchased locally. The crude glycerol samples were provided by Hunan Rivers Bioengineering Co., Ltd., Hunan, China. 2.2. Fermentation process Batch fermentation experiments with crude glycerol as carbon substrate were conducted in either flasks or 5-L fermenters. In flask fermentation, the experiments were conducted in 500 mL flasks with 100 mL liquid medium containing 50 g/L glycerol (for crude glycerol, the addition amount was decided by its exact glycerol concentration which was tested by HPLC), 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. The pH value of the liquid medium was adjusted to 6.0. Followed by sterilization at 115 ◦ C for 15 min, 10 mL inocula were added to the medium, and the culture was conducted at 30 ◦ C in an air-bath shaker at 200 rpm for 5–6 days. Before inoculation, the biomass concentration of the inocula was carefully controlled at 0.5–0.6 g/L. Each culture was performed by duplicate test. For culture in 5-L fermenters, the experiments were conducted with 4 L culture volume. The initial glycerol concentration was 60 g/L and other nutrients concentrations were the same as those in flask culture. pH was controlled at 6.0 by feeding 40 wt% NaOH. Dissolved oxygen was controlled at 20–30% saturation by automatic adjustment of stirring speed with 2 vvm aeration. For comparison, glucose was also used as carbon source at the same initial concentration. 2.3. Analytical methods The quantitative analysis of methanol, glucose, and glycerol was performed with a Shimadzu10AVP HPLC system (Shimadzu Corp., Japan) equipped with a RID-10A 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. The estimation of glyceride was performed with a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) with an ELSD-LT II low temperature-evaporative light scattering detector. Before analysis, 2 L sample and 1 mL acetone were mixed thoroughly, and 20 L of the aforementioned mixture was injected. A C18 column (5 m, 250 mm × 4.6 mm) (Dikma Technology, PLATISIL ODS, China) and a gradient elution program by acetonitrile and dichloromethane at 1.5 mL/min was employed, respectively. The column temperature
dry weight of cell biomass (g) volume of fermentation broth (L)
Total crude intracellular lipid was extracted by acid-heating procedure with mixture of chloroform and methanol as extractant [8]. Crude lipid content was expressed as gram lipid per gram of dry biomass. For determination of fatty acid compositions, the crude lipid was first converted to fatty acid methyl ester (FAME) with excess methanol in tert-butanol system under the catalysis of excess loading of immobilized lipase (NS 435 and NS 40044, produced by Novozymes) [9]. The obtained FAME was then 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 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. For describing the efficiency of the microbial lipid production, several response variables, namely, lipid concentration (CL ), lipid yield (YL ), and lipid content (CnL ) are defined as follows, respectively: CL (g/L) =
weight of lipid (g) volume of fermentation broth (L)
YL (g/100 g carbon substrate) =
weight of lipid (g) × 100 weight of carbon substrate consumed (g)
CnL (%) =
weight of lipid (g) × 100% weight of cell biomass (g)
3. Results and discussion 3.1. Lipid production by R. toruloides in flask fermentation Generally, in the biodiesel industry, about 0.1 t of crude glycerol is produced for every 1 t of biodiesel. Some soap and alcohol (usually methanol) together with small amount of fat soluble substance are also present in the glycerol phase depending on the feedstock and production processes. Two types of crude glycerol samples used in present work were analyzed, and their main components are shown in Table 1. Glycerol A contained 85% glycerol, a small amount of glyceride and fatty acid methyl ester (FAME). Notably, after burning the sample to a constant weight, the content of ash residue reached 6.5%, most of which was soluble in water. The ash generally consisted of sodium salts from the catalyst (e.g. sodium hydroxide) of transesterification reaction. Therefore, ash content can be considered as the total percentage of salt and soap components in the crude glycerol sample. Glycerol B was originated from lipase-catalyzed transesterification of palm oil. It contained 32.97% glycerol, a small amount of glyceride and fatty acid methyl ester, and a little ash. However, no
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Table 1 Main components of crude glycerol samples used in present work. Samplea
Glycerol (%)
Methanol (%)
Biodiesel and glyceride (%)
Ash content (%)
A B
85.19 ± 2.16 32.97 ± 0.96
– 14.89 ± 1.10
0.09 ± 0.00 1.81 ± 0.00
6.52 ± 0.16 0.11 ± 0.00
a Crude glycerol A was from an alkaline-catalyzed biodiesel production process; crude glycerol B was from an enzyme-catalyzed biodiesel production process. Both two samples had a brown color with a pH around 6–7 and the rest component was water.
distillation pretreatment was performed, and the methanol concentration was as high as 14.89%. To evaluate whether R. toruloides can grow on crude glycerol, we first examined the growth of this yeast on 50 g/L crude glycerol. For comparison, glucose and refined glycerol were also used as carbon source for culture. The experimental data after 160 h incubation are summarized in Table 2. It can be known that R. toruloides could well utilize refined glycerol for lipid accumulation. Furthermore, R. toruloides grew better in crude glycerol than in refined glycerol. For instance, the biomass concentration obtained by crude glycerol A was 50% higher than that obtained by refined glycerol. Corresponding lipid concentration and yield were also higher. For the scenario of crude glycerol B, similar results were obtained. It was also interesting to find that the yeast even grew better on crude glycerol than on glucose, which shows that crude glycerol from biodiesel production process is a promising feedstock for microbial lipid production. However, the experimental results should be further verified by larger scale cultivations. 3.2. Lipid production by R. toruloides in 5-L bioreactors To further increase the cell growth and production of lipids, batch fermentation was carried out in a 5-L fermentor equipped with pH control and aeration systems. The time courses of cell biomass concentration, substrate consumption and lipid concentration are shown in Fig. 1. As shown in the figure, the growth of R. toruloides on glucose substrate was fast at first, but gradually speeded down as the fermentation proceeded. The final biomass concentration was 16.7 g/L with lipid concentration of 11 g/L. On refined glycerol substrate, glycerol consumption was obviously slower than that of glucose; the lipid production, however, was comparable. On crude glycerol substrates, the kinetics of biomass growth, glycerol consumption and lipid production showed some difference depending on the characteristics of the crude glycerol. On crude glycerol B, the growth and glycerol consumption slowed down after the 150 h, and the final biomass reached 18 g/L with
lipid concentration of 13.4 g/L. On crude glycerol A, the biomass concentration, glycerol consumption, and lipid production were impressively fast at an almost stable rate. The final biomass and lipid concentrations reached 26.7 g/L and 18.5 g/L, respectively, with lipid yield of as high as 17.5 g/100 g consumed glycerol. Compared with refined glycerol substrate, crude glycerol A could obtain 42.0% higher biomass concentration and 68.2% higher lipid concentration. This performance indicated a remarkable advantage when using crude glycerol as carbon source. Compared with the some reported data from the literatures shown in Table 3, crude glycerol seems to be effectively utilized by this yeast, and led to similar or higher biomass and lipid productivity, which demonstrates that crude glycerol from biodiesel production could be a promising feedstock for microbial lipid production. According to the literatures, palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) constitute the majority of fatty acids in microbial triacylglycerols (TAGs) [10,11]. We thus further determined the fatty acid compositions of R. toruloides lipids derived from different carbon sources as shown in Table 4. It was found that the predominant fatty acids were palmitic acid, stearic acid, oleic acid, and linoleic acid; and palmitic acid and oleic acid accounted for about 70% of the total fatty acid composition. There was no significant difference observed for the lipids obtained from refined glycerol and crude glycerol substrates. However, the oleic acid and linolenic acid contents of the lipid obtained from glucose were a little higher than that obtained from glycerol, correspondingly palmitic acid and stearic acid contents were somewhat lower. Above experimental results showed that crude glycerol could be well utilized by R. toruloides with obvious advantages over refined glycerol and even glucose. Scott et al. [12] also demonstrated such advantages when they used crude glycerol from fish oil production as carbon source to produce oil rich in DHA by a thraustochytrid. Since the crude glycerol from biodiesel plant unavoidably contains some impurities such as salts, methanol and soap, which might exert influences on the yeast growth and lipid accumulation, we thus further performed experiments to investigate the individual effects of these impurities on the fermentation process. 3.3. Individual effects of some impurities on lipid production by R. toruloides Some compounds contained in crude glycerol were added into the refined glycerol medium to study the individual effects of the impurities on microbial lipid production. Taking into account that the concentrations of those substances in real crude glycerol composition, we selected the compounds and their concentration ranges as follows: sodium oleate (0–2 g/L), methyl oleate (0–2 g/L), glyceryl monooleate (0–2 g/L), sodium chloride (0–16 g/L), and methanol (0–16 g/L). In most cases, the real concentrations of the impurities do not exceed the upper limit as set.
Fig. 1. Cultivation of R. toruloides in a 5 L fermenter with crude glycerol as carbon substrate. (A) Biomass concentration and (B) lipid concentration.
3.3.1. Effects of soap Oleic acid is one of the main fatty acid compositions of biodiesel feedstock oil [6,13]. In an alkali (for example, sodium hydroxide) catalyzed transesterification reaction, sodium oleate is produced
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Table 2 Lipid production by R. toruloides in flask fermentation for 160 h. Carbon source
CB (g/L)
Glucose Refined glycerol Crude glycerol A Crude glycerol B
14.4 12.8 19.2 20.1
± ± ± ±
CL (g/L) 0.1 0.6 0.4 1.0
7.2 5.6 9.2 8.6
± ± ± ±
YL (g/100 g carbon source)
0.1 0.0 0.8 0.9
13.4 13.9 14.9 14.4
± ± ± ±
CnL (wt %)
0.2 0.6 1.5 1.8
50.0 43.4 47.7 42.9
± ± ± ±
0.4 1.0 2.0 2.0
Table 3 Comparison of lipid production by R. toruloides with those of some other microorganisms. Strain
Carbon source
Biomass concentration (g/L)
Lipid content (%)
Lipid concentration (g/L)
Cultivation
Reference
Cunninghamella echinulata Thraustochytrium sp. Rhodosporidium toruloides Mortierella isabellina Cryptococcus curvatus Yarrowia lipolytica Rhodosporidium toruloides
Glucose Glucose Glucose Crude glycerol Crude glycerol Crude glycerol Glucose Pure glycerol Crude glycerol A Crude glycerol B
15 23.9 18.2 8.5 32.9 8.1 16.7 18.8 26.7 18.0
46 30.1 76.1 51 52.9 43 66.9 58.7 69.5 74.1
6.9 7.2 13.9 4.4 18 3.5 11.2 11.0 18.5 13.4
Batch in flask Batch in flask Batch in flask Batch in fermenter Fed-batch in fermenter Continuous in fermenter Batch in fermenter Batch in fermenter Batch in fermenter Batch in fermenter
[27] [12] [28] [3] [2] [26] This work This work This work This work
Table 4 Fatty acid composition of R. toruloides lipid obtained from different carbon sources. C14 Glucose Glycerol Crude glycerol A Crude glycerol B
1.5 1.6 1.6 1.3
C16 ± ± ± ±
0.2 0.2 0.0 0.3
26.1 28.7 29.1 29.2
C16:1 ± ± ± ±
0.8 1.1 0.6 0.0
0.0 0.2 1.0 1.0
± ± ± ±
C18:0 0.0 0.0 0.1 0.1
13.0 15.3 17.8 13.9
± ± ± ±
C18:1 1.5 1.0 1.0 0.7
46.4 41.5 38.1 41.4
± ± ± ±
C18:2 1.8 0.6 1.5 0.3
9.2 10.1 9.7 10.4
± ± ± ±
C18:3 0.5 0.2 0.0 0.4
3.8 2.6 2.6 2.9
± ± ± ±
0.4 0.1 0.1 0.0
and remains in the reaction system. As shown in Fig. 2, positive effect was observed when sodium oleate was added, and all of the response variables were increased with sodium oleate concentration. For control experiments, the biomass, lipid concentrations and lipid yield were 12.8 g/L, 5.6 g/L and 13.9%, respectively. However, the addition of 0.5–2 g/L sodium oleate resulted in increases of biomass concentration by 25–41%, lipid concentration by 34–59% and lipid yield by 13–43%, respectively. The effect of sodium oleate, as a representative of soap, deserves further discussion. Soap plays a role as surfactant, which may provide some influences in fermentation process. It has been reported by Pyle et al. [14] that soap showed a negative effect on Schizochytrium limacinum for DHA production, and the inhibition was pronounced at higher levels. They suggested that this phenomenon should relate to the function of soap as a surfactant, and
the inhibition is caused by the complex interaction between the cell wall (membrane) and the surfactants. In our experiment, it was found that with the addition of 2 g/L sodium oleate, the medium could be emulsified to a great extent. Thus it can be assumed that sodium oleate should function as a surfactant in the culture medium. However, no significantly negative effect on cell growth and lipid production was observed when sodium oleate concentration was increased to 4 g/L. Contrarily, the presence of sodium oleate in culture medium led to an increase of biomass concentration with associated increase of lipid concentration and yield, which is in accordance with some researches that a proper concentration of surfactants benefits the microbial fermentation process [15–17]. It is very likely that some surfactants can interfere with the permeability of the cell membrane, and enhance the nutritional input from the surroundings to the cells [18].
Fig. 2. The effect of sodium oleate on cell growth, lipid production and lipid yield of R. toruloides cultivated with refined glycerol as carbon source for 7 days.
3.3.2. Effects of glycerides and fatty acid methyl esters After transesterification reaction, biodiesel is separated out as product through pH adjustment and settlement or centrifugation. However, unavoidably there are still some glycerides and fatty acid methyl esters (biodiesel) remaining in the aqueous glycerol phase. Since oleic acid is always the main fatty acid component in most of biodiesel feedstocks and monoglyceride is more or less present in the solution, in this work we selected methyl oleate and glyceryl monooleate as the representatives to investigate their effects on cell growth and lipid accumulation. The results are shown in Figs. 3 and 4. It can be known that both two esters showed somewhat positive influence on cell growth and lipid production. For methyl oleate, the biomass was increased by 9–12%, and lipid production was increased by 7–22%. For glyceryl monooleate, the biomass was increased by 8–13%, and lipid production was increased by 10–16%, respectively. Some yeasts have specific metabolic pathways to utilize hydrophobic substrate such as alkanes, fatty acids and oils. For example, a widely studied yeast Yarrowia lipilytica can utilize oils
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Fig. 3. The effect of methyl oleate on cell growth, lipid production and lipid yield of R. toruloides cultivated with refined glycerol as carbon source for 7 days.
Fig. 5. The effect of sodium chloride on cell growth, lipid production and lipid yield of R. toruloides cultivated with refined glycerol as carbon source for 7 days.
like glycerides by first hydrolyzing the lipid into free fatty acids, and then taking up by the cell as carbon source [19,20]. On the other hand, hydrophobic compounds are known to partition into cell membranes, interfering with the bilayer structure, and modifying its fluidity [16]. Considering that glycerides and fatty acid methyl esters have similar structures to that of sodium oleate while differ in the hydrophilic group, it is likely that such chemicals play a role as a “week” surfactant (compared with soap), and may also have some interactions with cell membranes as well as the culture mediums. However, since the effect of hydrophobic compounds on R. toruloides is not clear yet, corresponding mechanisms should be further investigated.
As shown in Fig. 5, the addition of sodium chloride led to obvious increase of biomass concentration, lipid concentration and yield. It indicated that the concentration of salt in crude glycerol was not high enough to cause an osmotic pressure. Contrarily, with the addition of 4–16 g/L sodium chloride, the biomass concentration, lipid concentration and lipid yield were increased by 12–40%, 20–48%, and 9–20%, respectively. This result was in accordance with the reported phenomena that salt can lead to a higher intracellular lipid content and biomass concentration if added in optimized amount [23–25]. It is probably that a low concentration of salt contributes to the physiological state in favor of lipid synthesis, in which circumstance cells can adapt to the salts with an increased production of carbonhydrates and lipids as the osmoprotectants.
3.3.3. Effect of inorganic salt In alkali-catalyzed production of biodiesel, alkali like sodium hydroxide is used as the catalyst. After transesterfication, pH adjustment, and separation process, part of the sodium remains in glycerol phase in the form of salts. It has been reported that a suitable salt content provides a positive effect on oleaginous microorganisms for cell growth and lipid production. Many microalgae even can utilize salt streams, and produce lipids under high salt concentrations [21,22]. However, excessive amount of salt may cause osmotic pressure. For this reason, sodium chloride was added to culture medium until its concentrations were 4 g/L, 8 g/L, and 16 g/L, respectively, to investigate its effect on cell growth and lipid production.
Fig. 4. The effect of glyceryl monooleate on cell growth, lipid production and lipid yield of R. toruloides cultivated with refined glycerol as carbon source for 7 days.
3.3.4. Effect of methanol Methanol is largely used for transesterification reaction; therefore the unreacted methanol remains in the aqueous phase. Depending on separation process, methanol concentration in the aqueous phase is varied. To cover the methanol concentration ranges of most crude glycerol, in this work, methanol was added to the culture medium until its concentration was 4 g/L, 8 g/L, and 16 g/L, respectively. In our experiments, methanol was found to be somewhat inhibitive to R. toruloides. As shown in Fig. 6, the biomass, lipid concentrations and lipid yield was decreased by about 0–5%, 6–24%, and 6–23%, respectively, when methanol was added. Whereas, it also can be observed that the inhibitory effect did not become even
Fig. 6. The effect of methanol on cell growth, lipid production and lipid yield of R. toruloides cultivated with refined glycerol as carbon source for 7 days.
J. Xu et al. / Biochemical Engineering Journal 65 (2012) 30–36
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Table 5 Fatty acid composition of lipid obtained from R. toruloides growing on refined glycerol in the present of selected crude glycerol impurities. Impurities
C14
Control Sodium chloride Methanol Sodium oleate Methyl oleate Glyceryl momooleate
1.3 1.2 1.3 1.6 1.7 1.6
C16 ± ± ± ± ± ±
0.1 0.0 0.0 0.2 .0.2 0.1
26.3 25.9 25.4 27.2 25.2 23.5
C16:1 ± ± ± ± ± ±
1.4 0.1 0.2 0.7 0.2 0.1
2.7 0.9 2.3 2.1 3.2 3.5
± ± ± ± ± ±
C18:0 0.0 0.0 0.2 0.2 0.1 0.1
11.7 12.1 12.6 12.1 10.8 11.0
± ± ± ± ± ±
C18:1 0.6 0.0 0.0 0.1 0.5 0.5
severer when the methanol concentration was increased to as high as 16 g/L. This observation accords with some literatures that methanol can lead to inhibitory effects on cell growth and lipid synthesis of some oleaginous microorganisms at high concentrations. For instance, Pyle et al. [14] reported that when methanol concentration is above 10 g/L in the medium, several important objective variables including biomass productivity, growth yield, DHA content, DHA yield and DHA productivity go down. Liang et al. also found that methanol could harm the cell growth of S. limacinum, but the inhibitory effect was not significant during the fed-batch fermentation process with a methanol concentration no more than 8 g/L [26]. However, the exact mechanism of methanol to oleaginous microorganisms is not clear yet. It has been hypothesized that methanol might be uptake into the cell and even utilized [2]. In this sense, it is probably that the methanol metabolism could exert some influence on the cell growth and lipid synthesis. Anyway, the experimental results indicate that although methanol can pose somewhat inhibitory effect on R. toruloides, a small amount of methanol remaining in the crude glycerol does not show severe impact on the fermentation process. Additionally, methanol is easy to be separated from crude glycerol through evaporation process, and should be recovered for recycling. Therefore, the effect of methanol actually can be neglected.
3.3.5. Effects of impurities on fatty acid compositional profiles of the yeast lipid To further investigate whether the impurities could affect the lipid synthesis pathways, the fatty acid composition of microbial lipid was analyzed as shown in Table 5. It can be known that the major fatty acid composition of lipids was C16:0 (palmitic acid), C18:0 (stearic acid), C18:1 (oleic acid) and C18:2 (linoleic acid), which accounted for about 26%, 12%, 45% and 11% of the total fatty acids, respectively. The addition of the compounds did not result in significant change of fatty acid compositions. This result was in accordance with the aformentioned conclusion that refined glycerol and crude glycerol led to similar fatty acid compositions. Additionally, it also implied that the impurities of crude glycerol did not affect the fatty acid biosynthetic pathways.
4. Conclusion This study has shown that oleaginous yeast R. touloides had great potential for converting biodiesel byproduct glycerol to valuable lipids, although some impurities such as soap, glycerides, fatty acid methyl esters, inorganic salt and methanol were present in crude glycerol. It was found that some of those impurities could even increase biomass concentration and lipid accumulation, and the impurities did not affect the fatty acid compositions of the obtained lipid. The results demonstrated that biodiesel byproduct glycerol could be directly used as carbon source for microbial lipid production and even more that it could induce higher biomass concentration and lipid yield than glucose.
45.1 46.3 45.4 44.1 43.1 47.1
± ± ± ± ± ±
C18:2 2.4 0.0 0.6 0.3 0.3 0.8
10.8 11.7 11.3 11.3 13.4 11.0
± ± ± ± ± ±
C18:3 0.5 0.0 0.0 0.5 0.5 0.5
2.1 2.0 1.8 1.6 2.6 2.3
± ± ± ± ± ±
Saturated FAs 0.2 0.0 0.0 0.3 0.2 0.1
39.3 39.2 39.4 40.9 37.7 36.1
± ± ± ± ± ±
2.5 0.1 0.2 1.0 0.2 0.3
Unsaturated FAs 60.7 ± 3.0 60.8 ± 0.1 60.6 ± 0.2 59.1 ± 1.0 62.3 ± 0.2 63.9 ± 0.2
Acknowledgments This work is supported by International Cooperation Project of the Ministry of Science and Technology of China (No. 2010DFB40170), and Tsinghua Research Funding (No. 20121080046), which are greatly appreciated by the authors.
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