Article pubs.acs.org/EF
Enhanced Lipid Accumulation by Chlorella vulgaris in a Two-Stage Fed-Batch Culture with Glycerol Yuan Sun,† Jing Liu,‡ Tonghui Xie,† Xiaolan Xiong,† Wenbin Liu,† Bin Liang,† and Yongkui Zhang*,† †
Department of Pharmaceutical & Biological Engineering, School of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610065, P. R. China ‡ Chengdu Environmental Monitoring Center, Chengdu, Sichuan 610072, P. R. China S Supporting Information *
ABSTRACT: A large amount of crude glycerol is generated during biodiesel production, but it has little commercial value. In this study, glycerol was used to cultivate Chlorella vulgaris to recycle its surplus, and its effects on microalgal growth and lipid accumulation were investigated. Lipid production in glycerol batch culture was higher than in glucose batch culture. However, the cell growth was slow, and a lag phase was more obvious as glycerol concentration increased from 1 to 30 g L−1. A two-stage fed-batch culture was developed to achieve high cell density and lipid production in C. vulgaris. In this culture, high microalgal cell concentration was first obtained by glucose, and lipid accumulation was enhanced by later additions of crude glycerol. Lipid production and lipid content reached 1663.02 mg L−1 and 36.39%, respectively. Lipids produced by C. vulgaris were good feedstock for biodiesel production.
1. INTRODUCTION The search for renewable diesel fuels has been driven by global concerns because of greenhouse gas emissions and emergency diesel fuels.1 Biodiesels are a promising alternative energy resource, and they can reduce 57−86% greenhouse gas emissions. Currently, they are mainly produced from oil crops (soybeans, corns, jatropha, etc.), animal fat, and waste cooking oil. However, biodiesels from these sources are far from satisfying the demand for transportation fuels.2,3 Microalgae are a reliable source of biodiesel that can to meet global demand, due to their environmental friendliness, fast growth, high lipid content, and low requirement for cultivated land.4−6 Furthermore, biodiesel produced from microalgae is highly biodegradable and nontoxic.7 Recently, a large amount of crude glycerol is generated as the main byproduct of the fast-growing biodiesel industry. In general, each pound of biodiesel is accompanied by 10% (W/ W) crude glycerol.8 To purify the crude glycerol into higher quality products for use in the health and cosmetics industries is too costly.9 Therefore, sustainable processes that make use of this organic raw material are needed. There are several approaches (such as fermentation and thermochemical processing) to the conversion of crude glycerol into valueadded products, such as 1,3-propanediol, pigment, acetol, and propylene glycol.10−13 Crude glycerol was converted into microalgal lipids by fermentation with C. vulgaris in this study. Microalgae have the ability to grow autotrophically, heterotrophically, or mixotrophically. Heterotrophic cultivation is attractive, because it is carefully controlled and does not depend on sunlight or air. Heterotrophic microalgae can easily achieve high cell density, growth rate, and lipid content.14 Glucose is the major carbon source for heterotrophic cultivation, but the high cost of glucose is a major hurdle to microalgal biodiesel production.15 Compared with glucose, crude glycerol is much cheaper.9 The application of crude © 2014 American Chemical Society
glycerol to microalgal fermentation is potentially an important contribution to biodiesel production.16,17 The culture mode affects microalgal growth and lipid accumulation. In batch cultures, where glycerol was the sole carbon source for microalgae, the rate of substrate consumption was low, and substrate inhibition was observed.16 Mixed carbon sources of glucose and glycerol enhance microalgal growth and lipid accumulation.16 However, the individual effects of each carbon source on growth and lipid accumulation needed clarification. As a novel culture mode, two-stage fed-batch culture is an efficient way to enhance cell growth and lipid accumulation. Microalgal cells are first cultivated in a batch culture with glucose as the sole carbon source to reach high cell density; the cells are then stimulated to accumulate lipids by the addition of glycerol. Thus, substrate inhibition is reduced, and high cell density and lipid production are easily obtained. In this study, the individual effects of glycerol and glucose on the growth and lipid production of the microalgae Chlorella vulgaris were explored. A two-stage fed-batch cultivation process was developed. The growth, lipid production, and fatty acids of C. vulgaris in the two-stage fed-batch culture were investigated.
2. MATERIALS AND METHODS 2.1. Algae Strain, Medium, and Culture Conditions. C. vulgaris was isolated from freshwater as reported previously.6 Microalgal cells for inoculum were incubated with soil extract (SE) medium supplemented with 10 g L−1 glucose in a shake-flask.6 The culture medium was autoclaved at 115 °C for 30 min, inoculated with 10% Received: January 6, 2014 Revised: April 23, 2014 Published: April 28, 2014 3172
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(V/V) microalgal culture and cultivated at 28 °C and 180 rpm in the dark. Crude glycerol was kindly provided by Prof. Houfang Lu of Sichuan University. It was obtained from the alkali-catalyzed transesterification of jatropha curcas oil with methanol, which is a common reaction for synthesizing biodiesel. The crude glycerol contained glycerol, soap, methanol and ester at 66.28%, 4.10%, 10.59% and 18.42% concentrations, respectively (W/W). Almost all of the methanol (99.89%) evaporated when the crude glycerol was autoclaved. 2.2. Batch Culture. In the batch culture, microalgal cells were incubated in 250 mL shake-flasks containing 100 mL SE medium supplemented with glycerol or glucose at the indicated concentrations. The effect of glycerol or glucose as the sole carbon source at 10 g L−1 on the growth and lipid production of C. vulgaris was investigated. The effects of different concentrations (1, 5, 10, 15, and 30 g L−1) of glycerol were also examined. 2.3. Two-Stage Fed-Batch Culture. In the two-stage fed-batch culture, microalgal cells were first cultivated in shake-flasks with SE medium supplemented with 10 g L−1 glucose, a fed-batch process was then carried out with successive additions of crude glycerol (containing 1 g L−1 glycerol) every 10 h. Two-stage culture was used as the control. Lab-scale cultivation was performed in a 5-L stirred tank bioreactor (Baoxing, China). Microalgal cells were inoculated into 3 L medium and cultivated by two-stage fed-batch culture. The batch culture stage was carried out for 44 h with 10 g L−1 glucose, followed by a fed-batch culture stage with additions of crude glycerol (containing 1 g L−1 glycerol) every 4 h. Dissolved oxygen (DO) concentration was maintained at above 20% air saturation by regulating agitation speed and airflow. The aeration rate and the agitation speed were initially set at 0.8 vvm and 200 rpm. The temperature was controlled at 28 ± 0.2 °C. 2.4. Analytical Methods. 2.4.1. Cell Growth. Cell growth was estimated by the absorbance at 680 nm by a spectrophotometer (Mapada, China). The result was correlated with microalgal cell concentration:
y = 2.6838x − 0.0394(R2 = 0.9992)
y = 0.0210x + 0.0119(R2 = 0.9990)
(6)
−1
where y is glycerol concentration (g L ) and x is OD412. 2.4.4. Statistical Analysis. Each experiment was carried out in triplicate. The average values were reported, and the errors were estimated by the standard deviation of replicates. Results were analyzed by ANOVA in Origin 8.0 software.
3. RESULTS AND DISCUSSION 3.1. Comparison of the Effects of Glycerol and Glucose on the Growth and Lipid Production of C. vulgaris. To study the potential use of glycerol as the sole carbon source for microalgal lipid production, the cell concentrations, lipid production and substrate consumption were studied. As shown in Figure 1A, C. vulgaris grew well
(1)
−1
6
where y is cell concentration (10 mL ) and x is OD680. When microalgal cells reached stationary phase, biomass production was obtained by the gravimetric method.18 Biomass yield was calculated by the following equation:
YB/S = B /S
(2) −1
where YB/S is biomass yield (mg g ), B is biomass production (mg L−1) and S is consumed substrate (g L−1). 2.4.2. Lipid Extraction and Analysis. Lipid generation was monitored daily by the rapid sulfo-phospho-vanillin (SPV) method.19 After fermentation, C. vulgaris intracellular lipids were extracted from dry algae powder by the chloroform−methanol method.18 The extracted lipids were converted to methyl esters and analyzed by gas chromatography and mass spectrometry (GC/MS-QP2010, Shimadzu, Japan) to evaluate the biodiesel quality.6,18 Lipid yield and lipid content were calculated according to YL/S = L /S
Figure 1. Comparison of glycerol and glucose as carbon sources.
when glycerol was the sole carbon source. However, a pronounced lag phase was observed. Glycerol was consumed slowly in the first 48 h, which was consistent with the slow growth and lipid production of C. vulgaris. Microalgal cells require an acclimation period to develop a special transport system that is necessary for glycerol uptake.21 From 48 to 96 h, lipid production rapidly increased (Figure 1B), although cell concentration remained low. After 288 h, a second rise in lipid production corresponded with increased glycerol consumption. A different phenomenon was observed with glucose. A total of 80.83% glucose was consumed, and cell concentration and lipid production increased sharply in 96 h. At 144 h, almost all the glucose was consumed, which terminated the cell growth. Thus, glucose could not maintain high cell density for a period of time. After 360 h, lipid production declined and was even lower than in the glycerol culture. Lipids might be employed to maintain cell growth when substrate was absent.
(3)
lipid content = L /B
(4) −1
−1
where YL/S is lipid yield (mg g ) and L is lipid production (mg L ). 2.4.3. Substrate Concentration Determination. Glucose concentration was determined by 3,5-dinitrosalicylic acid (DNS) method6 and correlated with the absorbance at 540 nm:
y = 0.6257x + 0.0035(R2 = 0.9991)
(5)
−1
where y is glucose concentration (g L ) and x is OD540 Glycerol concentration was determined by the colorimetric periodate oxidation method.20 It was quantified by the absorbance at 412 nm with the following equation: 3173
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Table 1. Biomass and Lipid Production of C. vulgaris Using Different Carbon Resources and Culture Modesa culture mode batch
b
two-stagec two-stage fed-batch (A)d two-stage fed-batch (B)e
carbon substrate
biomass production (g L−1)
glycerol glucose glycerol and glucose glycerol and glucose crude glycerol and glucose glycerol and glucose crude glycerol and glucose
± ± ± ± ± ± ±
1.32 1.65 2.36 2.39 2.82 3.94 4.57
f
0.27 0.11f 0.08 0.14 0.09 0.03 0.19
lipid content (%)
YB/S (mg g−1)
YL/S (mg g−1)
± ± ± ± ± ± ±
205.45 ± 8.41 172.05 ± 6.56 159.03 ± 8.12 121.11 ± 5.41 142.02 ± 2.10 197 ± 4.09 231.21 ± 2.87
55.31 32.48 38.69 36.98 44.15 59.14 84.14
26.90 18.88 24.37 30.56 31.04 30.01 36.39
0.21 0.36 0.81 0.67 0.34 0.20 0.17
± ± ± ± ± ± ±
3.03 2.90 1.47 2.43 3.11 1.11 2.03
Data are reported as means ± standard deviation (n = 3). bIn a batch culture, C. vulgaris was cultivated in SE medium supplemented with 10 g L−1 glucose in shake-flasks. cIn a two-stage culture, a batch culture with 10 g L−1 glucose was followed by a single addition of 10 g L−1 glycerol in shakeflasks. dIn a two-stage fed-batch culture (A), a batch culture with 10 g L−1 glucose was followed by a fed-batch culture with successive additions of 1 g L−1 glycerol every 10 h in shake-flasks. eIn a two-stage fed-batch culture (B), a batch culture with 10 g L−1 glucose was followed by a fed-batch culture with successive additions of 1 g L−1 glycerol every 4 h in bioreactors. fp = 0.12 a
When glycerol or glucose was as the sole carbon source, no significant difference (p > 0.05) in biomass production was observed (Table 1). However, lipid content and lipid yield in the glycerol culture were substantially higher than in the glucose culture. Glycerol enters the cells by simple diffusion without any extra energy, and it is not a source of carbons for biosynthesis.21,22 During glycerol assimilation, the pentose phosphate pathway (PPP) is inhibited.22 More glycerol would be used in the Embden−Meyerhof pathway (EMP), and then sacrifice to generate lipids. Moreover, glycerol is a substrate for triacylglycerol (TAG) synthesis. For glucose uptake, C. vulgaris possess an inducible active hexose/H+ system, and it invests one molecule of ATP per molecule of sugar transported.21 In completely dark conditions, glucose is mainly heterotrophically metabolized via PPP, as EMP fades, or even fully silent.21,23 In addition, glucose is not a substrate of TAG. Therefore, glycerol was more conducive for lipid accumulation than glucose in microalgal fermentation. 3.2. Effect of Initial Glycerol Concentration on Batch Fermentation. As shown in Figures 2 and 3, the initial glycerol level had different effects on C. vulgaris growth and lipid accumulation. A high glycerol concentration resulted in obvious lag phases. Moreover, the substrate utilization rate remarkably dropped with an increase in glycerol concentration (Figure 2 C). Approximately 80% of the glycerol was assimilated in 10 h when its concentration was 1 g L−1, while it took 240 h to assimilate 80% of the substrate when the glycerol concentration was 5 g L−1. The difference might have resulted from a long adaption period for growth in high glycerol environments.24 C. vulgaris cell concentration increased when the glycerol concentration increased from 1 to 30 g L−1 (Figure 2A). The highest biomass production (1.51 g L−1) was achieved when the culture was supplemented with 30 g L−1 glycerol (Figure 3), and a similar result was observed in the glucose culture (1.65 g L−1). However, biomass yield decreased when the glycerol concentration increased. Thus, high glycerol concentration did not benefit biomass accumulation by C. vulgaris. Depending on the microalgal species, substrate inhibition can be induced by an excess of organic nutrients.24 C. vulgaris growth is inhibited at a glycerol concentration of 15 g L−1.16 Lipid yield was negatively affected when glycerol exceeded 10 g L−1 (Figure 3). From 10 to 30 g L−1 glycerol, there was no significant difference in lipid content. These results revealed that glycerol was not easily converted into C. vulgaris lipids when the glycerol concentration was high.
Figure 2. Effects of initial glycerol concentration on C. vulgaris growth and lipid production.
3.3. Two-Stage Fed-Batch Culture. 3.3.1. Shake-Flask Cultivation. To promote lipid accumulation and avoid 3174
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Figure 3. C. vulgaris biomass and lipid production with different glycerol concentrations.
substrate inhibition, the two-stage fed-batch culture was developed. A glucose batch culture was first used to obtain high microalgal cell density, a fed-batch culture followed with additions of glycerol to boost lipid synthesis. As shown in Figure 4A, more growth of cells was observed after glycerol additions were added. While a lag phase was not observed in the two-stage fed-batch culture, it was obvious in the two-stage culture. Because substrate inhibition was minimized in the twostage fed-batch culture by continuous nutrient feeding, cells experienced log phase growth during almost the whole cultivation cycle.25 As a result, biomass production was approximately 1.4-fold that obtained in batch culture (Table 1). Lipid production was noticeably enhanced in the two-stage fed-batch culture (Figure 4 B). The maximum lipid production was 729.67 mg L−1, which was higher than the other two processes. This result strongly suggested that glycerol allowed excellent lipid accumulation in the C. vulgaris two-stage fedbatch culture. Lipid content reached 30.56% (Table 1), which was higher than that in the two-stage and batch cultures and was 1.2-fold that observed in a Cryptococcus curvatus single fedbatch culture with glycerol.8 Furthermore, lipid yield was increased by 13.85% compared with batch culture. The conversion of substrate into lipids was 36.98 mg g−1, which was equivalent to the two-stage culture. Microalgal lipid accumulation was stimulated in the second stages of twostage fed-batch and two-stage cultures, and more substrate was utilized for TAG synthesis to accumulate lipids. With regard to lipid productivity, glycerol was more efficiently converted into lipids by two-stage fed-batch culture. Compared with pure glycerol, crude glycerol was more beneficial to biomass production and lipid accumulation in C. vulgaris (Figure 5). Cells in cultures with crude glycerol grew better than in cultures with pure glycerol, achieving biomass production of 2.82 g L−1 and biomass yield of 142.02 mg g−1 (Table 1). These results may have been caused by other nutrients present in the crude glycerol. The small amount (
