4166

Energy & Fuels 2009, 23, 4166–4173

Alga-Based Biodiesel Production and Optimization Using Sugar
Cane as the Feedstock
Yun Cheng, Yue Lu, Chunfang Gao, and Qingyu Wu*
Department of Biological Science and Biotechnology, Tsinghua UniVersity,
Beijing 100084, People’s Republic of China
ReceiVed April 30, 2009. ReVised Manuscript ReceiVed June 4, 2009

The alga Chlorella protothecoides is known to produce oil suitable for biodiesel preparation by heterotrophic
cultivation in media containing glucose as a carbon source. In this study, sugar cane juice was used as alternative
carbon supply for oil production. As a result, the highest oil content of 53.0% by cell dry weight was achieved.
Fermentation in a 5 L bioreactor showed that algae using sugar cane juice hydrolysate (SCH) grow faster than
that using glucose. Conversion ratios of sugar/biomass and sugar/oil using SCH were 15.2 and 8.8% higher
than that using glucose, respectively. Biodiesel prepared from algal oil by transesterification is mainly composed
of 9-octadecenoic acid methyl ester, 9,12-octadecadienoic acid methyl ester, and hexadecenoic acid methyl
ester. Our results suggest that sugar cane is a good feedstock for biodiesel production. Response surface
methodology upon exploring the effect of C/N and concentration of yeast extraction (YE) on the yield of
biomass and oil was performed. The optimal production with the highest output-cost coefficient of 0.061 (
0.004 was achieved when C/N was 26.9 and YE was 0.60 g L-1.

1. Introduction
The recent decade witnessed a surge of interest in algal oil
as a promising supplemental oil source for biodiesel production.
Many specific species of algae can use a carbon source for oil
accumulation under special conditions.1 Not all algal oils are
suitable for producing biodiesel; however, satisfactory oils occur
commonly. Usually triglyceride and fatty acids are the dominating components of algal oil, from which biodiesel can be
prepared by transesterification, yielding monoalkyl esters of fatty
acids and alcohols.2
Algae can grow with CO2 as the carbon source and sunlight
as the energy supply. However, both biomass productivity and
oil content of photoautotrophic cultures are extremely low. The
photoautotrophic technology mainly suffers from a limited
supply of light and lower energy conversion efficiencies.3 In
comparison to photoautotrophic cultivation, heterotrophic fermentation allows algae to accumulate a much higher proportion
of oil within less time and the scale-up is much easier. Thus, it
offers a potential pathway to produce oil for diesel production
in large scale.4 Various strains of algae, including Chlorella
protothecoides,5 Crypthecodinium cohnii,6 and Porphyridium
propureum,7 have been studied to achieve both high biomass
and oil or fatty acids productivity in heterotrophic cultures. In
our previous studies, we have reported a strategy of pilot-scale
* To whom correspondence should be addressed. Telephone/Fax: +8610-62781825. E-mail: qingyu@mail.tsinghua.edu.cn.
(1) Li, Y.; Horsman, M.; Wu, N.; Lan, C. Q.; Dubois-Calero, N.
Biotechnol. Prog. 2008, 24, 815–820.
(2) Meng, X.; Yang, J.; Xu, X.; Zhang, L.; Nie, Q.; Xian, M. Renewable
Energy 2009, 34, 1–5.
(3) Grobbelaar, J. U. J. Appl. Phycol. 2000, 12, 201–206.
(4) Eriksen, N. T. Biotechnol. Lett. 2008, 30, 1525–1536.
(5) Xiong, W.; Li, X. F.; Xiang, J. Y.; Wu, Q. Y. Appl. Microbiol.
Biotechnol. 2008, 78, 29–36.
(6) Barclay, W. R.; Meager, K. M.; Abril, J. R. J. Appl. Phycol. 1994,
6, 123–129.
(7) Wen, Z. Y.; Chen, F. Biotechnol. AdV. 2003, 21 (4), 273–294.

fermentation of heterotrophic microalgae with high biomass
productivity and oil yield. The oil content of microalgae has
reached up to 50% of dry cell weight.8
To further improve the yield of algal oil and minimize the
cost of fermentation, optimizing the medium to achieve a
maximum performance of oil productivity seems to be of critical
importance, because medium composition can significantly
affect the yield and cost of products. The key factors associated
with oil yield are nitrogen content, C/N (ratio of carbon source/
nitrogen source), and carbon source.1,9 Currently, the cost of
producing biodiesel from algal oil is much higher than that of
diesel derived from petroleum, and the cost of carbon source
represents 50% of the cost of medium for algal cultivation.8
Therefore, economic considerations need to explore cheap and
easily available alternate feedstock.
Sugar cane is a highly efficient C4 crop and can store about
1% of the radiation on biomass per year.10 It is estimated that
750 L of raw sugar cane juice and 250 kg of wet bagasse can
be harvested from 1 ton of sugar cane stalks. The juice contains
18.5% sucrose and 1.7% other fermentable sugars, such as
glucose and fructose, indicating a yield of about 150 kg of
sugars/ton of stalks. Recently, sugar cane has received increasing
attention for its application in ethanol production. The most wellknown example is The Brazilian National Alcohol Program
´ lcool). A total of 50% of sugar cane
(Programa Nacional do A
production in Brazil is used to satisfy alcohol consumption.11
However, sugar cane has never been reported for biodiesel
production.
The major objective of this study was to evaluate the
feasibility of producing biodiesel from algal oil with sugar cane
(8) Li, X. F.; Xu, H.; Wu, Q. Y. Biotechnol. Bioeng. 2007, 98, 764–
771.
(9) Yamaberi, K.; Takagi, M.; Yoshida, T. J. Mar. Biotechnol. 1998, 6,
44–48.
(10) Lunn, J. E.; Furbank, R. T. New Phytol. 1999, 143, 221–237.
(11) Moreira, J. R.; Goldemberg, J. Energy Policy 1999, 27, 229–245.

10.1021/ef9003818 CCC: $40.75  2009 American Chemical Society
Published on Web 06/26/2009

X. 1-10 g L-1) was studies and considered as the most suitable (1-4 g L-1) nitrogen source to provide high biomass and oil yield. Technol. among three inorganic nitrogen sources (urea.4. The methods of preparation of medium. Kosaric. 0. However. Biotechnol. 841–846. 1 mL L-1 A5 trace mineral solution. 2. J. cells were collected when cultures reached the stationary growth phase. The oil content (%). sucrose and sugar cane juice were enzymatically hydrolyzed.01 mg L-1 vitamin B1..7. The generated models were also subjected to analysis of variation (ANOVA) using Design Expert 7.5 In this work. and 21 was set as -1. Samples were taken at regular intervals to determine the cell density (g L-1) and reducing sugar concentration (g L-1). Heterotrophic fermentation was completed in a 5 L bioreactor (Minifors. Through diluting the SH or SCH prepared in section 2. which greatly contributed to biomass productivity. Upon exploring the effect of C/N and YE concentration on the production of biomass and oil.5 Heterotrophic cells in exponential phase were used to inoculate fresh media. A two-factor CCD. Lett. C/N when referred to cultivations with SCH is considered as an apparent C/N ratio. glucose was used as a control to evaluate the cultivation kinetics with that of SCH.13 cell density was monitored. The cell density and consumption of carbon source were determined and recorded at regular intervals. The quadratic model for predicting the optimal point was expressed as the equation 2. containing about 600-700 g L-1 sucrose). MN) to estimate the responses to the variables. Minneapolis. J. Sugarcane Canesugar 2003. 637–642.3. was employed for the optimization of oil production. 0. Bioresour. Preparation of Sucrose Hydrolysate (SH) and SCH.1. The oil content was analyzed by time-domain nuclear magnetic resonance (TDNMR) on a Bruker Minispec MQ20 NMR analyzer (Bruker. Media and Cultivation in a Shake Flask. China. (12) Li.3 (Static Made Easy.12 MPa for 30 min. Bottmingen. Y. Alga-based biodiesel was prepared by transesterification of algal oil from bioreactor cultures. 2.5 Therefore. which represents the ratio of added carbon source/added YE.. b1 and b2 are linear effects. and six axial points (R ) 1). C.8.7 g L-1 KH2PO4.3. KOH solution (0. A concentrated solution of YE (100 g L-1) and glucose or SCH (400-500 g L-1) were fed to keep the concentration of YE and carbon source at certain levels. All media and the cultivation apparatus were sterilized with steam at 112 °C and 0. leading to a total of 11 runs (described below. 2. H. 3 mg L-1 FeSO4 · 7H2O. Q. C/N at 9. C/N and YE concentration were chosen as independent variables. Concentrated sugar cane juice was commercially available (ZK007. B. Therefore. Lu. a range of C/N covering 9-21 was investigated for further optimization.1. the content of sucrose in SC can be evaluated. and +1. 15. The components of basal medium for alga growth were as follows: 0. it is unknown whether nitrogenous compounds in SC can be used by alga.3 g L-1 MgSO4 · 7H2O. Curves of cell growth and sugar consumption were recorded by software Origin 7. C/N at 10 was used for oil production. 97. The total oil in algal cells was extracted by Soxhlet extraction. According to the central composite design (CCD). Cell Collection. Nitrogenous compounds may be present in sugar cane juice (SC). 23. Monitor of Cell Growth. 2009 4167 media at an equivalent initial cell density. The sugar cane juice or 500 g L-1 sucrose solution was supplemented with fructofuranosidase (Valisase Ivertase ANL. According to our previous work. To obtain a carbon source suitable for microalgal growth. 2006. and 1. conversion ratio. YE was selected as the nitrogen supplement in all cultures. TX). Microorganisms. and culture apparatus were described before. respectively. C.3. Northampton.. Then.0 (OriginLab Corporation.5 M) was fed to keep pH at 6. 0. Liu. The most common carbon source maintaining heterotrophic growth was glucose.. and SCH was added separately into basal medium with 3 g L-1 YE to prepare media containing 20 g L-1 different carbon source.5 g L-1 was set as -1. Vol. Algal cells were harvested and weighed after lyophilization. Heterotrophic cultivation was carried out in 500 mL flasks at 28 ( 1 °C with continuous shaking (220 rpm). and b12 is the interaction term. To study the feasibility of SCH as the carbon source for biodiesel production. 3539 (in Chinese). In our previous observation. The manipulation was previously reported in details in a previous publication. Equation 1 was calculated with software Design Expert 7. Table 2). SC.6. The conditions of inoculation and heterotrophic cultivation of 11 total runs were as described in section 2.4.. Maintenance. INFORS AG CH-4103.Alga-Based Biodiesel Production Using Sugar Cane as the feedstock. 0. and +1. Reducing Sugar Consumption.14 2.2. Because the triglycerides usually accumulate to a maximum amount and remain constant during the stationary phase in nitrogen-depleted culture. Wu. the components and amount is limited and vary between sugar cane species. MA). 0. 2.12 Besides. Switzerland) containing 3 L of basal medium. The concentration of reducing sugar was analyzed by the DNS method. protothecoides in exponential phase was inoculated into where Y is the predicted response. Temperature was controlled at 28 ( 1 °C. b0 is the intercept. Cells were collected by centrifugation and lyophilized to a constant weight. L. Different amounts of SCH and YE were used to set different C/N and added into basal medium. To investigate the cell growth and oil accumulation in algal cells using sugar cane as the feedstock. The dissolved oxygen concentration was controlled between 20 and 50% air saturation by airflow and agitation speed. X. The concentration of reducing sugar (hydrolysate) was monitored at regular intervals by the dinitrosalicyclic acid (DNS) method to determine the complete digestion. We have achieved the optimized medium composition for algal oil production and have significantly improved the oil yield and efficiency of carbon source conversion.8. Materials and Methods Energy & Fuels.3. protothecoides was originally provided by Culture Collection of Algae at University of Texas (Austin. with three central points. 15 000 Valley Summer units g-1) and hydrolyzed according to the instructions provided by the manufacturer. 1989.5. G. two factorial points. SC media were prepared by diluting the concentrated SC and adjusting the amount of sucrose to 20 g L-1. N. A and B are independent variables that represent C/N and YE concentration.1. sucrose. 4. potassium nitrate. preparation of inoculum. an equivalent amount of glucose. D. and output-cost coefficient (described below) were used as responses. The variables and corresponding responses were analyzed on the basis of experimental results to evaluate the significance of quadratic models. Our results suggest that sugar cane juice hydrolysate (SCH) was an available and effective carbon source for algal fermentation and oil production. 2 (9). The YE concentration at 0.3 g L-1 K2HPO4. and Biodiesel Preparation. 2. SH. yeast extract (YE. Sweden). 2. in which glucose or sucrose powder was weighted and directly added into media. The quantity of amino acids decreases during storage. Experimental Design and Optimization Using Response Surface Methodology (RSM). On the basis of the concentration of reducing sugar in SCH. (13) Turcotte.3 ( 0. Selection of Nitrogen and Carbon Sources. the concentration of reducing sugar in SH or SCH media was measured by the DNS method and adjusted to 20 g L-1. b11 and b22 are squared effects. (14) Miao. 0.1.1. Ltd. Y ) b0 + b1A + b2B + b12AB + b11A2 + b22B2 (1) . Oil Extraction. and ammonium nitrate) and two organic nitrogen sources (glycine and yeast extract). Cell growth was monitored by optical density measurements at 540 nm using a UV/vis spectrophotometer (Pharmacia Biotech Ultrospec 2000.. This may provide a positive pathway for industrial application of sugar cane in biodiesel production. Bai Guo Yuan Juice and Food Co. 2. and Inoculum. Fermentation Conditions. and Oil Content.

The highest oil content of 53. This suggests that sucrose and sugar cane juice could not be used directly by C. the range of this design has been effective enough to provide information for analysis.15 2. media without carbon source supplementation (basal media with YE) and glucose media were used as a comparison to evaluate the effects of SCH media on heterotrophic cultivation of C. Vol.7 ( 1. algal cells grow fast. and conversion ratio obtained in SCH media reached comparable levels to those of glucose media (Table 1). Data of 11 runs show that a higher YE concentration leads to a higher biomass yield (run 3 in Table 2) and a higher biomass productivity (Figure 2a) but lower conversion ratio of sugar/biomass (Figure 2b). both SH and SCH are available for algal use. Table 1 showed that the biomass. the cultivation condition in a shake flask restrains the air and light supply. when hydrolyzed sucrose (i. experiments at higher C/N or higher YE concentrations are not necessary. On the other hand. (a) Cell density (-b-) and reducing sugar (-O-) in the medium with 20 g L-1 glucose and cell density ( · · · b · · · ) and reducing sugar ( · · · O · · · ) in the medium without glucose (basal medium).22 ( 0. Both high and low YE concentrations had a negative influence on oil content (Figure 3a). and the GC was manipulated with a flow rate of 10 mL min-1. Wu. Figure 1.64 ( 0.23 ( 0.2.5. Results 3.28 ( 0.1. DW)c glucose SH SCH 1. Xiong.5%. such as glucose. protothecoides.05 1. 23. The YE concentration was shown to significantly affect the oil production. protothecoides can also grow with CO2 and light as energy. Although maximum and minimum points did not shown up on the response curves in Figure 2. 3.4 12. C. Q. algal cells grow fast and the glucose is consumed. protothecoides. Cell Growth and Oil Accumulation in C. 437–440.20 44.. etc. dualstage quadrupoles GC apparatus (Thermo. Table 1. and the conversion ratios of sugar/biomass and sugar/oil were not higher than that in our previous publications.5 3.07 1. It is known that C. including sucrose as well as fructose and glucose. DF ) 0. protothecoides Using Sugar Cane as the Feedstock. n ) 3]a carbon source biomass (20 g L-1) yield (g. In Figure 1a. SCH) is added. The lack of enzyme-digesting sucrose may account for this phenomenon. Y. Rheinstetten. However.1 ( 2.e. (b) Cell density (-b-) and reducing sugar (-O-) in the medium with 20 g L-1 sucrose hydrolysate (SH) and cell density ( · · · b · · · ) and reducing sugar ( · · · O · · · ) in the medium with 20 g L-1 sucrose without hydrolysis.65 ( 0.9. Comparison of different carbon sources for microalgal heterotrophic cultivation. there is still considerable room for improvement on them after optimization of C/N and YE concentration (see section below). However.07 conversion ratio (%)b consumed sugar (g) oil content (%) 4.8 However. 2009 Cheng et al.4 ( 2. (c) Cell density (-b-) and reducing sugar (-O-) in the medium with 20 g L-1 sugar cane juice hydrolysate (SCH) and cell density ( · · · b · · · ) and reducing sugar ( · · · O · · · ) in the medium with 20 g L-1 sugar cane juice without hydrolysis. g of oil/g of consumed sugar). L. Methods 2008.5 33. algal cells hardly grow with nonhydrolyzed sucrose or nonhydrolyzed SC (panels b and c of Figure 1). oil content. C. g of biomass/g of consumed sugar) and conversion ratio of sugar/oil (%. algal cells do not exhibit a significant growth in a shake flask. To provide the carbon source available to algal assimilation. We also noticed that the oil content (15) Gao. Considering this. because the conversion ratio of the carbon source will be extremely low (Figure 2b).8 28. MA). protothecoides before hydrolysis treatment.95 MHz. C.1 g L-1) leads to a high conversion ratio of sugar/biomass of 66. Zhang. Composition Analysis of Biodiesel. a low YE concentration (0. SH) or hydrolyzed SC (i.. Oppositely.3 biomass/ sugar oil/ sugar 30. As far as we know. C/N showed no significant influence on the cell growth and conversion ratio. Whereas. protothecoides was shown to use hexose.e.0% was observed in run 4 when C/N was set at a high level (Table 2).. However. The composition of biodiesel produced from extracted algal oil was analyzed on gas chromatograph (GC)-linked mass spectrometry (GC-MS).15 43.. 75. F. Yuan. Effects of YE Concentration and C/N on Biomass Production and Oil Accumulation. cultivation in medium without an organic carbon source has difficulty in maintaining algal growth (Figure 1a). This result is coincidence with previous publications.8 14. it is necessary to investigate the effects of YE concentration and C/N on biomass production and oil accumulation when using SCH as the feedstock for alga-based biodiesel production..6 14. b Conversion ratio of sugar/biomass (%.25 mm inner diameter.1 4. which indicated that a high C/N ratio . sugar cane juice was processed by hydrolysis. Microbiol.43 ( 0. Sugar cane juice is rich in carbohydrate.4168 Energy & Fuels. at a resonance frequency of 19.6 a The final volume of media in flasks was 200 mL. W. The biomass yield includes the weight of oil. Accordingly..18 46. Low efficiency of use would not be considered in industrial application. W. Because (1) nitrogen source and concentration are important for heterotrophic growth in algal cells and (2) SCH is an efficient carbon source for heterotrophic growth of C. J. c The initial inoculum was subtracted from the final biomass yield. thus not enough to support algal growth under heterotrophic conditions. equipped with a Varian VF-5 ms column (30 m × 0. fructose. Germany) with a 35 mm absolute probe. Y. When glucose is supplemented. Waltham. Similar to the condition in the medium without a carbon source supply (Figure 1a). We observed that the reducing sugar in nonhydrolyzed sugar cane juice was very low (Figure 1c). 3. Effects of Different Carbon Sources on Heterotrophic Cultivation [Data Are Given as Means ( Standard Deviation (SD).25 µm). galactose.

75 3.976 output-cost coefficient f oil content (%) consumed sugar (g) cultivation time (day)d biomass/sugar oil/ sugar experimental estimated 42.200 1. Figure 3.0 19.0552 0.0371 0.87 0.08 5.67 5.47 2.8 1.0520 0. DW) is defined here as the daily biomass production (g) when cells are collected at stationary phase.2 53.0348 0.1 0. (a) Biomass productivity (g day-1. 53% of oil content in dry algal cells is satisfied in biodiesel production. Figure 2.875 0.4 09.5 2.13 Figure 3a indicated that the maximum oil content was obtained when C/N was 19. It means that increasing the C/N ratio further over 19.0 22.3 49.75 2.8 42.7 08.2 1.0329 0.9 31. in the medium has been considered to be essential for oil overproduction.0500 0. 8.1 55.0547 0.58 2.5 12.2 40. and cells were collected at stationary phase.25 3.228 0. 2009 4169 Table 2.5 0.5 20. and +1 (1. and 10 are central points.38 0.5 15.7 38. Response surface plot representing the effects of C/N and YE concentration on (a) oil content and (b) conversion ratio of sugar/oil.5 39.0547 0.5 36. We also noticed that the highest conversion ratio .261 1. d The cell density was monitored.8 0.00 2.2 0.0335 0.1 46.1 12.5 16. b A represents C/N: -1 (9).40 3.4 34.25 3.75 4.173 1.5).0 49.5 0.5 0.72 2.8 0.42 3. DW) 1 2 3 4 5 6 7 8 9 10 11 9 9 21 21 9 21 15 15 15 15 15 0.0518 0. 0 (0.435 1. Experimental Design and Estimated Data for Response Surface Analysis central composite design conversion ratio (%) runa Ab Bc SCH (g L-1) biomass yield (g.2 43.8 1.5 49.0 42.0 20. However.8).66 0.6 0.0 1.1).35 5.1 16.0392 0. and +1 (21).00 4.0399 0.0458 0.0518 0. nitrogen starvation usually triggers the oil accumulation and contributes to a greater extent of triglyceride content during the stationary phase of growth.0 61. Response surface plot representing the effects of C/N and YE concentration on (a) biomass production and (b) conversion ratio of sugar/biomass.0446 0.80 3.8 did not cause a proportionally increasing oil content.45 2.3 19. c B represents the concentration of YE (g L-1): -1 (0.0518 0.4 16.0339 0.0462 0.7 44. The final volume of media in flasks is 260 mL.0 17.0308 0.8 0.3 44.75 46.0365 0.4 07.16 g L-1.6 50. 23.8 13.1 0.233 2.0519 0.184 1.279 1.8 66.3 49.Alga-Based Biodiesel Production Using Sugar Cane Energy & Fuels.453 0.0 12.16 In addition.5 7.91 3. Vol.8 and YE was 1.0372 a Runs 7. 0 (15).1 1.

9 and YE concentration was 0.706 × 10-5 3.0076 0.18 0. the quadratic model eq 2 was generated and suggested as the best fitting model Y ) 0.878 × 10-6 7.693 × 10-4 1. 3. This means that the combined effects of C/N and YE concentration contribute significantly to the variation in oil production. Accordingly.0076 showed that the model was statistically significant at a 5% level of significance (Table 3).279 × 10-5 8. Media prepared according to the optimum C/N and YE concentration for the highest output-cost coefficient may optimize the algal oil production by heterotrophic cultivation with sugar cane as the feedstock.4%) in run 4. As the result.012 × 10-7 8.5% in run 2 (Table 2).012 × 10-7 8. To evaluate the fitness of the model eq 2 in practical application. Thus. the commercialization of alga-based biodiesel depends upon its oil yield.05. Therefore.193 × 10-4 3. The conversion ratio of sugar/oil (%) is an indicator of how efficient the algae convert sugars to oil.4.0009 4. and consumed sugar. and cultivation time in 11 runs listed in Table 2 were also conducted and analyzed through Table 3. carbon consumption. consumed feedstock.333 × 10-5 6.1723 RSM. the value of .654 × 10-4B 4. When R is set as 0. Poor efficiency of carbon source conversion is therefore not desirable.1537 0.439 × 10-4 3.1723 and larger than R.7% in run 8. It showed that a high or low YE concentration had negative influences on oil content (Figure 3a) and the conversion ratio of sugar/oil (Figure 3b).8196 0.0. However. when the nitrogen source was set constant. the value of 0.0439 0. 2009 Cheng et al. had an inhibition on both cell growth and conversion efficiency.146 × 10-4A2 . which demonstrates a model representing the actual interaction of tested parameters. The main cost for oil production from algae comes from the cultivation time and carbon source (sugar) consumed. Response surface plot representing the effects of C/N and YE concentration on the output-cost coefficient f.058 49. we set an output-cost coefficient f that is in a direct ratio with the oil yield (g) and an inverse ratio with both time (days) and consumed sugar (g) to evaluate and optimize the alga-based biodiesel production: f ) oil yield/time × sugar.161 × 10-5 1.5 For this reason. a proper combination of the YE concentration and C/N leading to both a satisfying oil yield and efficiency of carbon source use should be considered.439 × 10-6 12. Justification of the Quadratic Model Generated by RSM. Higher C/N seems to slightly increase the efficiency of sugar conversion. The cost of carbon source contributes to the main cost of medium and product.069 7. all data related to oil yield. The carbon source at high concentrations. the p value is 0.2.3.806 × 10-5 5.39 2.052 . and production time.03 0. Using data from Table 2 related to oil yield. coefficients with smaller p values vary more significantly.8033 0. alga can be considered as a cell factory that processes the carbon source into oil. such as 60 g of glucose L-1. 3. The optimum values of C/N and YE concentration were calculated by solving eq 2 through Design Expert 7.693 × 10-4 5.4170 Energy & Fuels.709 × 10-7 5. which was almost similar to that (20. ANOVA for the Response Surface Quadratic Model source model A B AB A2 B2 residual lack of fit pure error correlation total sum of squares DF 7.564 × 10-3AB + 5.015B2 (2) Please see eq 1 to understand eq 2. RSM Analysis and the Optimal Production Based on Feedstock and Cultivation Time. of sugar/biomass was 66.279 × 10-5 8. we suggest that the high conversion ratio of sugar/oil is more important than the high sugar/ biomass in alga-based biodiesel production. therefore. In the other words. The optimal production with the highest output-cost coefficient is suggested to be obtained when C/N was 26. To optimize the oil production.3.1. cultivation time.338 × 10-3A + 3. No significant “lack of fit” is desired and obtained in this design.773 × 10-4 5 1 1 1 1 1 5 3 2 10 mean square F value p value 1. The p value is the probability that the variation between conditions may have occurred by chance. which mainly refer to labors and consumption of electric power.60 g L-1. Figure 4.333 × 10-5 6. The highest conversion ratio of sugar/oil was 20. Vol.709 × 10-7 5. In the process of oil production.96 0. higher C/N often leads to a higher concentration of carbon source.118 × 10-5 6. As a result. 23. ANOVA was conducted to fit the model using the experimental data. a response surface plot indicating the effect of C/N and YE concentration on the output-cost coefficient was achieved (Figure 4).82 0.

62 18. 12.5 40. The production pathway from sugar cane to biodiesel by microalgae fermentation has some advantages in comparison to fuel-ethanol production by yeast fermentation.7 35. respectively (Table 5).2 and 8.Alga-Based Biodiesel Production Using Sugar Cane Energy & Fuels. 0. Therefore.9 17. The present work focusing on reducing the cost of feedstock and optimizing oil yield to some extent helps to reduce the cost in algal oil production. c n ) 3.05.864 kg L-1.75% (w/w). Effects of Different Carbon Sources on Cell Growth and Oil Production of Fed-Batch Fermentation in a 5 L Bioreactor (the Harvesting Volume Is 2. The results demonstrate no significant differences between estimated and experimental values.0 296.98 ( 1. The carbon source and YE were batch-fed whenever the carbon source was exhausted. This result further confirms the feasibility of using sugar cane as the feedstock for biodiesel production through heterotrophic algal fermentation. and hexadecenoic acid methyl ester. Discussion Figure 5. The conversion ratios of sugar/biomass and sugar/oil with SCH were higher than those with glucose by 15. protothecoides provided higher biomass productivity and oil yield with SCH than with glucose (Figure 5). the price of industrial glucose was about 330-360 USD/ton and the price of sugar cane stalks is about 40 USD/ton (October 2008). 2009 4171 Table 4. Thus. This work. instead of cultivation in a shake flask.56 a C/N ) 26.061 ( 0. illustrates the feasibility of sugar cane juice served as a fermentable carbon source for oil production in C.3 51.6. less than that of glucose. In comparison to yeast fermentation for ethanol production. For example. Algal heterotrophic fermentation in a bioreactor. The main fatty acid methyl esters detected in biodiesel from both SCH and glucose carbon source include 9-octadecenoic acid methyl ester.8%.01 1.71 0.22 ( 1. we are clearly conscious that this is just a beginning for biodiesel production using microalgae to meet the commercial needs.8 54.5 L) conversion ratio (%) carbon source consumed sugar (g) biomass yield (g) oil yield (g) biomass/ sugar oil/ sugar glucose SCH 304. respectively: (b) biomass yield and (O) concentration of reducing sugar.12-octadecadienoic acid methyl ester. Feasibility of SCH as the Carbon Source for Oil Production. In alga-based biodiesel production by heterotrophic fermentation.009 showed that B2 was the most significant term among others (Table 3).15 56. Heterotrophic fermentation in a 5 L bioreactor using (a) glucose and (b) SCH as the feedstock. Within 7 days of cultivation. DF ) 2.90 18. Because algal cells are easily separated from medium and oil is easily extracted by solvent.40 0. Three independent experiments in optimized media were conducted to verify the predictions of the model. Estimated and Experimental Values of Responses under Optimized Conditionsa responses estimated experimentalb t valuec oil content (%) oil/sugar (%) biomass/sugar (%) output-cost coefficient f time (h) 43.56 and 53.1.34% (w/w). High-quality biodiesel from a glucose carbon source has previously been characterized by a heating value of 41 MJ kg-1. respectively.303. a lower price of carbon source means less cost of oil production and better economic feasibility.12 46.60 g L-1.95 0.8 121. it is interesting to note that algal cells grow faster and provide a higher yield of biomass and oil with SCH than that with glucose.48% (w/w). protothecoides. for the first time.5.9. R ) 0.42 3. 9. b Table 5. It is reasonable that there are other unknown nutritions in SCH to benefit cell growth and oil accumulation. Other minor methyl esters were also detected (data not shown). Although the advantages mentioned above. 4. There are bottlenecks in this pathway. It means that sugar cane is not only a good feedstock for fuel-ethanol production but also for biodiesel production. 3. RSM combining an effective design can be applied to the optimization of medium composition for algal oil production.1 g L-1.49 and 11.33 and 19. Arrows indicated the points of feeding. 23.051 72 47. Composition of Biodiesel Produced from Algal Oil by Transesterification. According to the observation in this study. The estimated and experimental responses were compared and subjected to a two-tailed t test (Table 4). microalgae accumulate oils as intracel- . 4. algal fermentations in a 5 L bioreactor were conducted with 3 g L-1 YE and 30 g L-1 glucose or SCH as the starting conditions. Data were shown as means ( SD. their corresponding contents from SCH and glucose carbon source were 52. it is interesting to note that C. This result illustrates that the main components of biodiesel from sugar cane are similar to that of glucose. YE ) 0.91 ( 0. the heating value of alga-based biodiesel is much higher than that of yeast-based fuel-ethanol.4 107. a density of 0.5 3. SCH ) C/N × YE ) 16.004 75 3. and a viscosity of 5. it takes a lot of heating power to prepare ethanol by distillation from yeast fermentation medium. 10.0 18.14 We expect that biodiesel produced from SCH will show a similar performance to that from glucose. Two-tailed t ) 4. is commonly used in practical production.2 × 10-4 Pa s (at 40 °C). indicating that the price of fermentable sugars from sugar cane juice is about 260 USD/ ton. Both conversion ratios of sugar/biomass and sugar/oil with SCH were higher than that with glucose. On the basis of the market in China. Heterotrophic Fermentation of Algal Cells in a 5 L Bioreactor. Vol.

100–112. M. Through comparison. genetic manipulation performed on critical enzymes for lipid synthesis may also be possible to improve oil yield. G. N. C19H36O2 (64. Within the range of the design... (18) Hu. Vol. enough ATP and reductant for CO2 fixation can hardly be supplied. A. a quadratic model has been successfully generated from 11 runs and used to give optimum parameters for the output-cost coefficient in oil production. The moment cell density reaches the maximum value (stationary phase). 1989. The dominating fatty acid methyl ester is C19H34O2 (52. Larger quantities of ethanol could be produced with relatively small amounts of microbial cells for ethanol fermentation. When photoautotrophic algae were grown in a nitrogen-deprived outdoor system. it is important to notice that light deficiency is common in photosynthetic cultivation. Ghirardi. G. As a result. M. etc.. Biondi. Tredici.82 g L-1 day-1 (Table 2. and cell division. N. 1999. Bioeng. 868– 871. 2009 lular products. Neochloris oleoabundans was reported to give a remarkable lipid productivity of 0. reasonable improvements can be introduced to the pathway of alga-based biodiesel production. The oil/carbon source conversion is dependent upon the biomass/sugar conversion. it is obvious that heterotrophic culture has advantages in both high biomass and oil productivity. Jarvis. Microbiol. 2009.20 In industrial application. 4. M. 66. Second.7% conversation ratio of sugar/oil (run 8 in Table 2) were achieved at different C/N and YE concentrations separately. For example. with biomass productivity at 1.4172 Energy & Fuels. J. A. 2008. Even parts of cultures can be pumped out from the bioreactor. L. the highest lipid productivity in run 3 is 0. At present. the highest cell density in flasks was shown at 14-15 g L-1.. Genetic manipulation offering algal species with the ability of digesting sucrose is welcome in future studies. 23. Ann. cells were cultivated in a closed system (shake flask) and air supply and agitation speed were restrained. 54 (4). H. a proper combination of the YE concentration and C/N leading to both a satisfying oil yield and efficiency of carbon source use should be optimized. First. allowing further growth. Thomas... Sommerfeld. M... Karanth. pH. chlorophyll content.. Mohd. the oil cell content typically ranges from 30 to 60%. the main oil sources for biodiesel production are from oil crops. oneat-a-time.. A strategy of phototrophic/heterotrophic tandem cultivation can be conducted and may exhibit a benefit to both environmental and economic aspects.5. J..4%) in rapeseed (16) Sattur. 201–208. 102 (1). Accordingly. G. G. J.004) was achieved when C/N was 26. partial factorial. pretreatment of SC is inevitable and requires time and labor. from which the condition contributing to the highest efficiency of “less input-more output” can be judged. the cell density will be greatly improved.. Ind. and the other is heterotrophic fermentation using glucose as the carbon supply. (17) Rodolfi.4. the optimal oil production with the highest output-cost coefficient (0. The air. P. On the other hand. and semicontinuous flow feed have been widely applied in high-density fermentation. 2 (3).1 However. more efficient and easier approaches are necessary.133 g L-1 day-1 achieved in phototrophic cultivation. We observed that 53% of oil content (run 4 in Table 2). a high nitrogen supply was observed to lead to poor efficiency of sugar use (Figure 2b). Posewitz. Bioeng. whereas those accumulating high oil content show little growth ability. H. Z. such as castor. in-depth research on the mechanism of oil production in heterotrophic alga is desired. Because of the lack of corresponding enzyme.. Biotechnol.. Bonini. P. Seibert. P. etc.. As for this work. M. the highest cell density in the 5 L bioreactor has been achieved at 48 g L-1.30 g L-1 day-1 and lipid content over 60%). continuous flow.5% conversation ratio of sugar/ biomass (run 2 in Table 2) and 20. 4. it is not likely for alga to prefer synthesizing fatty acids. the algal species used in this study cannot assimilate sucrose directly. fast-growing photosynthetic algal cells tend to produce poor amounts of oil. 11–21. M. through SCH feeding and fermentation process controls. E. Under the conditions of the bioreactor. J. M. This may provide useful information for further research of biodiesel production involving more variables.67 g L-1 day-1 and lipid content at 49.2. Plant J. 89 (1). microalgal biomass production has been achieved by at least two main approaches: one is photoautotrophic cultivation by using solar energy and carbon dioxide. leading to a limitation of oil/carbon source conversion. Krouse. Classical methods for medium designing and optimization include biological mimicry. Q. Chini. and agitation can be automatically controlled. A. For example.204 g L-1 day-1 and 5 times larger than 0. For example.19 The results in this study consist with previous publications. a higher concentration of sugar does not lead to a higher efficiency of sugar use.40 g L-1 day-1 and lipid content at 34%).204 g L-1 day-1 was achieved (with biomass productivity Cheng et al. Feasibility Algal Oil as a Biodiesel Oil Source. (19) Macduff. Currently. biofuel via a heterotrophic pathway should be further studied and considered as a transitional strategy. In our previous observation. 2002. (21) Bibhu. the highest lipid productivity of 0. It indicates the critical importance of C/N and nitrogen concentration for alga growth and oil accumulation.3%) in soybean biodiesel. Biotechnol. 4. The dilemma is that the synthesis of fatty acid needs a large amount of ATP and reducing power. 456–475. allowing the oil vesicle to be transported to an extracellular medium. Photoautotrophic Cultivation versus Heterotrophic Cultivation.7 When light is restricted. in the future. Biotechnol. in a recent study.60 g L-1. Darzins. Bassi. From the aspect of energy saving and greenhouse effect relief.21 In this study. . RSM with a Proper Experimental Design Is an Efficient Tool in Optimization. which is able to generate a mathematical model that accurately describes a given process. (20) Kennedy.9 and the YE concentration was 0.18 Nitrogen starvation has been widely reported to trigger lipid accumulation but leads to a decrease in protein synthesis. The dynamics of algal cells in flasks is different from that in a bioreactor. considering the following reasons. rapeseed. 23. Using RSM.17 In another example.133 g L-1 day-1 (with biomass productivity at 0. Because of the conditions of the experiments.2%). The strategy of feed batch. RSM used in this work is one kind of such an approach. O. 4. soybean. Res. For this reason. Padovani. When the YE concentration is constant... cotton seed. As observed in this study.061 ( 0. Many works have been published about this strategy. Thus. Bot. sunflower. the photoautotrophic alternative seems to be more economical and preferred. N. because many experiments are involved.3.. Saleem. a higher carbon supply does not contribute to continuous growth and oil accumulation. However. Microbiol. Humphreys. microalga is genetically modified. 621–639. 34. This result is almost 3 times larger than 0. Its advantage is the reduced number of experimental runs required to generate sufficient information for a statistically acceptable result. Influences of C/N and YE Concentration on Algal Growth and Oil Production. Therefore. 30 strains of photoautotrophic algae were screened for biomass productivity and oil content. Some of them are laborious and time-consuming. at 0. full factorial. R. D. 2007. the heterotrophic option nevertheless should not be completely ignored.

2009 4173 closely accordant with that of crop oils.22 respectively. The results proved that microalgal oil can also be used as another potential oil source. 4 (2). 9. 23.Alga-Based Biodiesel Production Using Sugar Cane biodiesel. 111–133. Energy & Fuels. Vol. P. 2000. and C19H36O2 (80. National 863 Project 2007AA05Z400. EF9003818 . This research was supported by NSF Guangdong Joint Project U0633009. and hexadecenoic acid methyl ester (C17H34O2). The biodiesel prepared from algal oil in this work is mainly composed of 9-octadecenoic acid methyl ester (C19H36O2).4%) in olive biodiesel.12-octadecadienoic acid methyl ester (C19H34O2). and China MOST Overseas Project 20070574.. Renewable Sustainable Energy ReV. Acknowledgment. A. Ram. which are (22) Srivastava.