Efficient conversion of biomass into lipids by using the simultaneous saccharification and enhanced lipid production process
© Gong et al.; licensee BioMed Central Ltd. 2013
Received: 4 December 2012
Accepted: 28 February 2013
Published: 5 March 2013
Microbial lipid production by using lignocellulosic biomass as the feedstock holds a great promise for biodiesel production and biorefinery. This usually involves hydrolysis of biomass into sugar-rich hydrolysates, which are then used by oleaginous microorganisms as the carbon and energy sources to produce lipids. However, the costs of microbial lipids remain prohibitively high for commercialization. More efficient and integrated processes are pivotal for better techno-economics of microbial lipid technology.
Here we describe the simultaneous saccharification and enhanced lipid production (SSELP) process that is highly advantageous in terms of converting cellulosic materials into lipids, as it integrates cellulose biomass hydrolysis and lipid biosynthesis. Specifically, Cryptococcus curvatus cells prepared in a nutrient-rich medium were inoculated at high dosage for lipid production in biomass suspension in the presence of hydrolytic enzymes without auxiliary nutrients. When cellulose was loaded at 32.3 g/L, cellulose conversion, cell mass, lipid content and lipid coefficient reached 98.5%, 12.4 g/L, 59.9% and 204 mg/g, respectively. Lipid yields of the SSELP process were higher than those obtained by using the conventional process where cellulose was hydrolyzed separately. When ionic liquid pretreated corn stover was used, both cellulose and hemicellulose were consumed simultaneously. No xylose was accumulated over time, indicating that glucose effect was circumvented. The lipid yield reached 112 mg/g regenerated corn stover. This process could be performed without sterilization because of the absence of auxiliary nutrients for bacterial contamination.
The SSELP process facilitates direct conversion of both cellulose and hemicellulose of lignocellulosic materials into microbial lipids. It greatly reduces time and capital costs while improves lipid coefficient. Optimization of the SSELP process at different levels should further improve the efficiency of microbial lipid technology, which in turn, promote the biotechnological production of fatty acid-derived products from lignocellulosic biomass.
KeywordsMicrobial lipids Cryptococcus curvatus Biodiesel Corn stover Simultaneous saccharification and enhanced lipid production
Cell dry weight
Filter paper unit
Saturated fatty acid
Separated hydrolysis and lipid production
Separated hydrolysis and enhanced lipid production
Simultaneous saccharification and lipid production
Simultaneous saccharification and enhanced lipid production
Yeast peptone dextrose
Biodiesel is an important renewable fuel that is usually produced from vegetable oils or animal fats by transesterification with methanol/ethanol . However, traditional oil-rich crops and plants are limited by land availability, and are in constant debate due to the food versus fuel issues. Thus, feedstock supply is the major obstacle for large-scale application of biodiesel. Alternative feedstocks for biodiesel have been pursued intensively in recent years. Oleaginous yeasts produce neutral lipids consisting of long-chain fatty acids comparable to those of conventional vegetable oils. Thus, microbial lipids have been suggested as a potential feedstock for biodiesel production [2, 3]. However, the costs of microbial lipids remain prohibitively high for commercialization. To reduce the costs, it is important to explore sustainable raw materials such as lignocellulosic biomass and to develop integrated processes.
Carbohydrates derived from lignocellulosic materials have been used to culture oleaginous yeasts for microbial lipids [4–8]. In those cases, biomass was pretreated, hydrolyzed and the corresponding hydrolysates were used. There were also a few reports that integrated cellulose hydrolysis and lipid biosynthesis in a single bioreactor, where both cellulolytic enzymes and auxiliary nutrients were present. In one recent example of integration of lipid production and enzymatic hydrolysis of corn stover, lipid titre reached 3.23 g/L when the yeast Trichosporon cutaneum was cultivated in 50-L stirred-tank bioreactor . In another example, lipid production by Microsphaeropsis sp. was carried out using steam-exploded wheat straw under solid-state culture conditions . We recently reported the two-staged culture mode for lipid production, where cells were loaded in glucose solution without auxiliary nutrients [11, 12]. Because cell density was high and cell propagation was inhibited by nutrient deficiency, lipid productivity and yield were significantly enhanced.
A number of oleaginous yeasts have been applied for lipid production . Cryptococcus curvatus is an excellent lipid producer. It can use various cheap substrates such as biomass hydrolysates and has a good tolerance to major biomass-derived inhibitors [5, 7, 14]. In this paper, we describe the simultaneous saccharification and enhanced lipid production (SSELP) process that integrates the biomass hydrolysis step and an enhanced lipid accumulation step, to effectively convert lignocellulosic materials into lipids. Specifically, cells prepared in a nutrient-rich medium were inoculated at high dosage for lipid production in biomass suspension in the presence of hydrolytic enzymes without auxiliary nutrients. When corn stover regenerated from an ionic liquid-based pretreatment procedure was used, both cellulose and hemicellulose were consumed simultaneously. No xylose accumulation was observed, indicating that glucose effect was circumvented. The SSELP process offers high lipid coefficient, greatly reduces time and costs and appears promising for lipid production from lignocellulosic biomass.
Results and discussion
Effect of initial pH, enzyme dosages and temperature on the SSELP process
Features of different culture modes used for lipid production
Summary of experimental parameters tested for the SSELP process
Effect of initial pH
Effect of enzymes
Effect of temperature
Agitation speed (rpm)
Cellulase (FPU/g) & cellobiose (CBU/g)
15 & 30
15 &30, 15 & 0,
7.5 & 15, 7.5 & 0
7.5 & 15
25, 30, 34,
It was found that dosages of cellulase and cellobiase per gram cellulose at 15 FPU and 30 CBU, respectively, led to the highest lipid titre, cellulose conversion and residual glucose concentration (Figure 1B), which suggested that cell viability was the major constraint in the system. The lowest lipid titre and cellulose conversion were found when cellulase alone was used at 7.5 FPU/g cellulose. Lipid titre and cellulose conversion improved significantly when cellobiase was added. This was likely due to cellobiase-mediated clearance of cellobiose, which could inhibit cellulose hydrolysis . Lipid titre increased little but cellulose conversion improved significantly when cellulase dosage was doubled. Since the costs of enzymes remain one of the major hurdles to the development of an economically viable cellulosic biofuel industry , a lower enzyme loading is more appealing. Thus, dosages of cellulase and cellobiase per gram cellulose at 7.5 FPU and 15 CBU, respectively, were chosen for further optimization.
Experiments were also done at different temperatures for 48 h with initial pH 5.2 (Table 2), and results are shown in Figure 1C. Lipid titre and cellulose conversion increased when the culture temperature increased from 25°C to 37°C, and residual glucose were all below 0.5 g/L. Lipid titre and cellulose conversion reached 7.2 g/L and 92.3%, respectively, at 37°C. However, when the experiment was done at 40°C, both lipid titre and cellulose conversion were significantly dropped, and residual glucose was 10.0 g/L, indicating that lipid biosynthesis was inhibited but cellulose hydrolysis was efficient at 40°C.
A major challenge for the traditional simultaneous saccharification and fermentation process is that saccharification and fermentation have different temperature optima [18, 19]. Oleaginous yeasts often have optimal growth temperature around 30°C whereas enzymatic saccharification is optimal at about 50°C. Because C. curvatus is a mesophilic yeast, cell growth in YPD medium was significantly inhibited at 37°C (data not shown). However, C. curvatus cells were found highly active for lipid production at 37°C when the SSELP process was employed, as lipid titre reached the highest. This was likely due to the fact that the absence of auxiliary nutrients inhibited cell propagation and enabled lipid biosynthesis at a higher temperature. The shift of optimal temperature to 37°C was advantageous because enzymatic cellulose hydrolysis was faster.
Time course of lipid production with different strategies
Results shown in Figure 2 and Figure 3 were obtained with almost identical amounts of glucose equivalent carbon sources and initial inoculum sizes. Within 60 h, 30.8 g/L glucose was consumed in Figure 2, while 30.9 g/L cellulose was consumed in Figure 3. It was clear lipid titre data in Figure 3 were always higher than those in Figure 2 at given time points. For example, at 48 h, the SSELP process and the two-staged culture gave a lipid titre of 6.9 g/L and 6.5 g/L, respectively. The fact that glucose in the SSELP process was maintained at lower concentrations might avoid substrate inhibition. Moreover, the culture at 37°C should inhibit cell propagation but improve lipid biosynthesis than 30°C. By integration of cellulose hydrolysis and the two-staged lipid production into a single reactor, the SSELP process reduced the total time and capital costs, and improved efficiency for cellulose hydrolysis and lipid production. Similar results have been demonstrated for ethanol fermentation when lignocellulosic materials were used [20, 21].
Lipid production using regenerated corn stover as the feedstock
Results of lipid production by C. curvatus on corn stover regenerated from the EmimOAc–NMP system
Enzyme loading (/g corn stover)
Culture time (h)
Cell mass (g/L)
Lipid content (%)
Lipid coefficient (mg/g corn stover)
Cellulase: 4.0 FPU
6.0 ± 0.2
Cellobiase: 8.0 CBU
Xylanase: 5.0 mg
Cellulase: 10 FPU
16.5 ± 0.4
7.2 ± 0.2
43.4 ± 1.0
Cellobiase: 20 CBU
Xylanase: 10 mg
Fatty acid compositional profiles
Fatty acid compositions of lipids from C. curvatus cultivated on cellulose according to the SSELP process
Culture temp. (ºC)
Myristic acid (%)
Palmitic acid (%)
Palmitoleic acid (%)
Stearic acid (%)
Oleic acid (%)
Linoleic acid (%)
We developed the SSELP process for efficient conversion of lignocellulosic materials into microbial lipids with high lipid coefficient. Because cells are inoculated in the auxiliary nutrient-free suspension of lignocellulosic materials containing hydrolytic enzymes, it can be performed without sterilization. It ensures efficient utilization of both cellulose and hemicellulose simultaneously, because glucose concentration remains low in the culture and glucose repression is circumvented. The SSELP process greatly reduces time and costs and appears promising for the production of fatty acid-derived products from lignocellulosic biomass.
Reagents and strain
Ionic liquid 1-ethyl-3-methylimidazolium acetate (EmimOAc) was supplied by Lanzhou Greenchem ILs, LIPC, CAS (Lanzhou, China) and used without further purification. Cellulose (sigmacell cellulose Type101, moisture content 10% (wt/wt)), cellulase and cellobiase were purchased from Sigma. The activities of cellulase and cellobiase were determined as 161.0 FPU/mL and 674.7 CBU/mL, respectively [31, 32]. Xylanase was obtained from Imperial Jade Bio-Technology Co., Ltd. (Yinchuan, China), and the activity was 5000 kU/g. One unit of xylanase (U) was defined as the amount of enzyme which produces 1.0 μg of xylose from 1% xylan solution at pH 5.0, 50°C within 1 min. N-Methylpyrrolidone (NMP) and other reagents were analytical grade and purchased from local company.
The yeast C. curvatus ATCC 20509 was obtained from the American Type Culture Collection, and maintained at 4°C every two weeks on yeast peptone dextrose (YPD) agar slant (yeast extract 10 g/L, peptone 10 g/L, glucose 20 g/L, agar 15 g/L, pH 6.0).
Pretreatment of corn stover with the EmimOAc–NMP solution
Corn stover harvested from countryside of Changchun, China, was milled to a particle size of 1–2 mm. The milled materials were washed to remove the field dirt, stones and metals, dried at 105°C until the weight was constant, and stored in desiccate before use. Analysis of the corn stover sample according to the procedures of the National Renewable Energy Laboratory revealed a composition (dry weight basis) of 37.9% glucan, 20.1% xylan, 2.3% arabinan, and 20.8% lignin.
Corn stover was pretreated by the EmimOAc–NMP solution . Briefly, to a solution of EmimOAc (60 g) and NMP (140 g) preheated in an oil bath at 140°C was added corn stover sample (20 g). The suspension was held at the same temperature with stirring for 1 h, until a viscous black yellow solution formed. The solution was cooled down to 50°C, and methanol (500 mL) was added with vigorously stirring. The precipitates were filtrated, washed with methanol (500 mL) and water (2 × 500 mL), and freeze-dried to obtain regenerated corn stover samples.
Enzymatic hydrolysis of cellulose and regenerated corn stover
Cellulose hydrolysates were made by enzymatic hydrolysis of 3.6% (w/v) sigmacell cellulose in 0.3 M phosphate buffer (pH 5.2) at 37°C, 200 rpm. Cellulase and cellobiase per gram cellulose were loaded at 7.5 FPU and 15 CBU, respectively. Corn stover hydrolysates were made by enzymatic hydrolysis of 5.0% (w/v) regenerated corn stover in 50 mM phosphate buffer (pH 4.8) at 50°C, 200 rpm. Cellulase, cellobiase and xylanase per gram dry materials were loaded at 10 FPU, 20 CBU and 10 mg, respectively.
General procedure for the SSELP process
C. curvatus cells were cultivated in YPD liquid medium (yeast extract 10 g/L, peptone 10 g/L, glucose 20 g/L, pH 6.0) at 30°C, 200 rpm for 24 h. Cells were collected by centrifugation, washed with sterilized water, and then used as inocula for all two-stagedd culture processes. One unit optical absorbance at 600 nm (OD600 nm) for such inocula equaled to 0.36 g/L of cell dry weight (CDW). About 0.27 g of CDW equivalent inocula were transferred into a suspension of hydrolytic enzymes and 2.0 g of cellulose in 50 mL of 0.3 M phosphate buffer (pH 5.2), and other experimental details are summarized in Table 2.
Time course of lipid production with different culture processes
A two-stagedd lipid production process was carried out at 30°C, 200 rpm. About 0.27 g of CDW equivalent inocula were resuspended in 50 mL of 0.05 M phosphate buffer (pH 5.5) contained 2.08 g glucose · H2O and 1% (V/V) a trace element solution . The composition of the trace element solution contained (g/L): CaCl2 .2H2O 4.0, FeSO4 .7H2O 0.55, citric acid.H2O 0.52, ZnSO4 .7H2O 0.10, MnSO4 .H2O 0.076 and 100 uL of 18 M H2SO4.
For the SSELP process, 0.27 g of CDW equivalent inocula were transferred into a suspension contained 2.0 g of cellulose, 15 FPU cellulase and 30 CBU cellobiase in 50 mL of 0.3 M KH2PO4-Na2HPO4 buffer (pH 5.2), and the suspension was held at 37°C, 200 rpm for lipid production.
Lipid production from regenerated corn stover
Experiments were first done with the SHELP process. To 50 mL of the corn stover hydrolysates contained 31.9 g/L of glucose and xylose was inoculated with 0.27 g of CDW equivalent inocula, and the culture was held at 30°C, 200 rpm for lipid production.
For the SSELP process, 0.27 g of CDW equivalent inocula were transferred into a suspension contained 2.5 g of regenerated corn stover, 10 FPU cellulase, 20 CBU cellobiase and 12.5 mg xylanase in 50 mL of 0.3 M KH2PO4-Na2HPO4 buffer (pH 5.2), and the suspension was held at 37°C, 200 rpm for lipid production.
Glucose was determined using an SBA-50B glucose analyzer (Shandong Academy of Sciences, Jinan, China). Sugar mixtures were analyzed by ion chromatography (IC) on the Dionex ICS2500 system with a CarboPac PA10 analytical column (Dionex Co.) and an ED50 electrochemical detector (Dionex Co.). The column was washed with isocratic elution of NaOH at a speed of 1 mL/min at 30°C. The concentration of NaOH was 22 mM from 0 min to 20 min, retention time for glucose and xylose were 10.9 and 12.1 min, respectively. Cellulose concentration was determined as described . Residual cellulose was collected by repeated precipitation and washing with water to remove soluble carbohydrates. Precipitated sample was further washed using acetic acid-nitric acid reagent and water to remove non-cellulosic materials  and quantified by using the phenol-sulfuric acid method with glucose as the standard . Cellulose conversion was calculated based on the initial cellulose and residual cellulose.
Samples containing cellulose and yeast cells from 30 mL of culture broth were collected by centrifugation and washed twice with distilled water. Cell mass, expressed as CDW, was determined gravimetrically after drying the wet sample at 105°C overnight and deducting cellulose from the sample. Fat-free cell mass was calculated after subtraction of lipids from CDW.
Dried samples containing cellulose and yeast cells were digested with 4 M HCl at 78°C for 1 h before extraction with chloroform/methanol (1: 1, vol/vol). The extracts were washed with 0.1% NaCl, dried over anhydrous Na2SO4, evaporated in vacuo, and the residue was dried at 105°C for 24 h to give the total lipids . Lipid content was expressed as gram lipids per gram CDW. Lipid coefficient was expressed as gram lipids produced per gram cellulose.
The fatty acid compositional profiles of lipid samples were determined using a 7890F gas chromatography instrument after transmethylation according to a published procedure with minor modifications . Briefly, 70 mg of lipids were treated with 0.5 mL of 5% KOH solution in methanol at 65°C for 50 min, followed by the addition of 0.2 mL BF3 diethyletherate and 0.5 ml methanol. The mixture was refluxed for 10 min, cooled, and extracted with n-hexane. The organic layer was washed twice with distilled water, and used for fatty acid compositional analysis.
All data in this study were the averages of three independent experiments.
This work was financially supported by the National Basic Research and Development Program of China (2011CB707405), the Knowledge Innovation Program of Chinese Academy of Sciences (KSCX2-EW-G-1-3), and the National Natural Scientific Foundation of China (31170060).
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