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Photosynthesis-fermentation hybrid system to produce lipid feedstock for algal biofuel a
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Yue Lu , Junbiao Dai & Qingyu Wu
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School of Life Sciences , Tsinghua University , Beijing , 100084 , People's Republic of China Published online: 08 Oct 2013.
To cite this article: Yue Lu , Junbiao Dai & Qingyu Wu (2013) Photosynthesis-fermentation hybrid system to produce lipid feedstock for algal biofuel, Environmental Technology, 34:13-14, 1869-1876, DOI: 10.1080/09593330.2013.824011 To link to this article: http://dx.doi.org/10.1080/09593330.2013.824011
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Environmental Technology, 2013 Vol. 34, Nos. 13–14, 1869–1876, http://dx.doi.org/10.1080/09593330.2013.824011
Photosynthesis-fermentation hybrid system to produce lipid feedstock for algal biofuel Yue Lu, Junbiao Dai∗ and Qingyu Wu∗ School of Life Sciences, Tsinghua University, Beijing 100084, People’s Republic of China
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(Received 29 March 2013; final version received 1 July 2013 ) To avoid bacterial contamination due to medium replacement in the expanded application of a photosynthesis–fermentation model, an integrated photosynthesis-fermentation hybrid system was set up and evaluated for algal lipid production using Chlorella protothecoides. In this system, the CO2 -rich off-gas from the fermentation process was recycled to agitate medium in the photobioreactor, which could provide initial cells for the heterotrophic fermentation. The cell concentration reached 1.03 ± 0.07 g/L during photoautotrophic growth and then the concentrated green cells were switched to heterotrophic fermentation after removing over 99.5% of nitrogen in the medium by a nitrogen removal device. At the end of fermentation in the system, the cell concentration could reach as high as 100.51 ± 2.03 g/L, and 60.05 ± 1.38% lipid content was achieved simultaneously. The lipid yield (60.36 ± 2.63 g/L) in the hybrid system was over 700 times higher than that in a photobioreactor and exceeded that by fermentation alone (47.56 ± 7.31 g/L). The developed photosynthesis-fermentation hybrid system in this study was not only a feasible option to enhance microalgal lipid production, but also an environment-friendly approach to produce biofuel feedstock through concurrent utilization of ammonia nitrogen, CO2 , and organic carbons. Keywords: photosynthesis and fermentation; lipid and biofuel; Chlorella protothecoides; hybrid system; nitrogen removal
Introduction Microalgae are considered an ideal source of lipid feedstock for biofuel production as they have high lipid synthesis capacity and can be cultivated in bioreactors, without competing for arable land.[1] One challenge is that among photoautotrophic algae, rapidly growing cells generally contain fewer neutral lipids than slower-growing counterparts. For example, the traditional nitrogen starvation strategy may increase neutral lipid accumulation greatly in phototrophic algae but limits the cell growth, which eventually results in relatively lower lipid yield.[2,3] Even in the best photoautotrophic cultivation conditions, most species of microalgae hardly satisfy the biomass yields required for biofuel production. In order to obtain both fast cell growth rate and high lipid content, heterotrophic cultivation of Chlorella protothecoides 0710 for algal biofuels was evaluated. More than a 50-fold increase in lipid yield was reported in the previous studies in comparison with the photoautotrophic approach.[4–6] High cell density and high lipid content are considered two advantages for heterotrophic fermentation approach.[7] However, the caveats of such a cultivation method include the release of CO2 into the atmosphere and the consumption of organic carbon. To combine the advantages of CO2 fixation and solar energy conversion from photosynthesis with advantages of high cell density and high lipid content from fermentation, a photosynthesis–fermentation model (PFM) approach ∗ Corresponding
has been proposed. In this approach, the algal cells were cultivated photoautotrophically for CO2 fixation in the initial stage and then switched to heterotrophic fermentation in the second stage for lipid accumulation, leading to further enhancement of algal lipid synthesis. Experiments based on metabolic flux analysis (MFA) suggested that an increase in the conversion ratio of glucose to lipid in PFM resulted from Rubisco catalysing CO2 re-fixation in the second fermentation stage.[8] Therefore, PFM merges the beneficial features from photoautotrophic and heterotrophic approaches, thus producing more lipids, fixing more CO2 , and consuming less sugar. However in PFM, the algal cells from nitrogen-rich medium in the first photosynthetic stage have to be harvested by centrifugation or filtration before being transferred to nitrogen-deficient and glucose-rich medium to initiate the heterotrophic stage. Such kind of operations for medium replacement will not only increase the cost but also bring the risk of bacterial contamination in scale-up trials. On the other hand, to allow the cells to grow efficiently under heterotrophic conditions, the amount of nitrogen in the medium has to be reduced to a certain level.[9] Although various nitrogen sources such as nitrate, ammonium, urea, and glycine have been reported to be utilized by C. protothecoides without considerable differences,[10,11] few of them can be deprived technologically in medium by a simple and effective way. Studies in wastewater treatment
authors. Emails: [email protected], [email protected]
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suggested that ion exchange resin was effective in absorbing and removing ammonia nitrogen (NH+ 4 -N).[12] Therefore, if NH4 Cl was used as the sole nitrogen source in photoautotrophic cultivation, nitrogen could be removed by the resin for switching algae to heterotrophic growth. In order to mitigate the issues associated with the cell transfer in PFM, in this study, an integrated photosynthesisfermentation hybrid system was designed by using a nitrogen removal device to connect a large photobioreactor to a small fermentor. The nitrogen removal device with ion exchange resin column was used to effectively remove residual ammonia nitrogen from cell suspension and resulted in the switch of photoautotrophic cells to heterotrophic growth. CO2 released from heterotrophic cells in the fermentor was absorbed and recycled by photoautotrophic cells in the photobioreactor. Eventually, the photosynthesis-fermentation hybrid system produced more lipid in comparison with independent photobioreactor and fermentor. It provided a feasible approach of lipid production for algal biofuel in scale-up application.
Materials and methods Strain and medium The microalga C. protothecoides 0710 strain originally came from Culture Collection of Algae at the University of Texas (Austin, Texas, USA) and screened for high lipid yield in Algae Bioenergy Laboratory at Tsinghua University (Beijing, China). The composition of basal culture medium was: KH2 PO4 0.7 g/L, K2 HPO4 0.3 g/L, MgSO4 •7H2 O 0.3 g/L, FeSO4 •7H2 O 3 mg/L, vitamin B1 0.01 mg/L, and A5 trace mineral solution (H3 BO3 2.86 mg/L, ZnSO4 •7H2 O 0.222 mg/L, MnCl2 •4H2 O 1.81 mg/L, CuSO4 •5H2 O 0.074 mg/L, Na2 MoO4 •2H2 O 0.039 mg/L).[4] 8 g/L NH4 Cl was added in basal medium for photoautotrophic growth to obtain green cells and 30 g/L glucose was added in basal medium for heterotrophic growth to obtain yellow cells.
Photoautotrophic and heterotrophic batch cultures in shaking flasks The photoautotrophic and heterotrophic batch cultures in shaking flasks were two independent experiments with independent seed cells. Basal culture media with different concentrations of NH4 Cl (0, 4, 8, 12, 16, and 20 g/L, corresponding concentrations of NH+ 4 -N measured as 0, 1100, 2100, 2900, 3800, and 4900 mg/L, respectively) were used for testing photoautotrophic growth of C. protothecoides cells. Fifteen millilitres heterotrophic seed cells in exponential growth phase were inoculated into 600 mL medium in each 1 L glass flask, which then were incubated in an incubator shaker (ZWY-A2112B Incubator, ZHICHENG Analytical Instrument Manufacturing Co. Ltd, Shanghai,
China) under temperature at 28 ± 0.5◦ C, rotation speed at 220 rpm, and continuous artificial illumination at 100 μmol photon/m2 /s. The batch experiments of heterotrophic culture were conducted to investigate the effects of residual NH+ 4 -N on heterotrophic cell growth and lipid accumulation in darkness. After 8 days’ cultivation under the optimum conditions, photoautotrophic cell suspensions were centrifuged at 8000 g for 4 min (6–15 Centrifuge, Sigma, Germany). Cell pellets were washed twice by basal culture medium to remove the residual NH4 Cl and resuspended. Then the concentrated cells were transferred into 400 mL culture medium containing 30 g/L glucose and different concentrations of NH+ 4 -N (0, 20, 40, and 80 mg/L, respectively) in each 1 L shaking flask. The incubation temperature and rotation speed were the same as photoautotrophic cultivation above. Design of photosynthesis-fermentation hybrid system To switch C. protothecoides to heterotrophic fermentation after photoautotrophic cultivation and avoid potential bacterial contamination due to medium replacement, a 15 L cylindrical glass photobioreactor, a cell collection bottle, a nitrogen removal device, and a 1 L fermentor (Multifors, Infors Bio-Technology, Switzerland) were connected to form a single integrated system. The CO2 -rich off-gas derived from the fermentor was recycled for both agitating medium in the photobioreactor and providing CO2 for photoautotrophic cells (Figure 1). A device for ammonia nitrogen removal was designed and incorporated into the hybrid system (Figure 1). It is a stainless steel column (10.5 cm in diameter and 30.0 cm in length) filled with 0.5 L ion exchange resin (D61, Zhengguang Resin, China). At the end of log phase, phototrophic algal cells (green cells) were allowed to settle overnight. After discarding part of the supernatant, the concentrated photoautotrophic cells in 200 mL medium with residual nitrogen were pumped through the NH+ 4 -N removal device at 3 mL/min and subsequently pressed into a 1 L fermentor containing glucose-rich medium. By determining the concentration of NH+ 4 -N at the outlet of the resin column, the efficiency of NH+ 4 -N removal was calculated under different resin-to-medium ratios (v/v) or after different numbers of repeated usage. To reactivate the resin, the NH+ 4 -N removal device was rinsed with 1 M HCl for 8 h and washed by deionized water till the pH returned to 6.0. Operation conditions for the photosynthesis-fermentation hybrid system Cells in both photobioreactor and fermentor were cultivated at the same time at 28 ± 0.5◦ C. In photoautotrophic cultivation, 200 mL heterotrophic C. protothecoides suspensions were inoculated into 13 L medium in a cylindrical glass photobioreactor agitating by off-gas from a
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Figure 1. Schematic diagram of the photosynthesis-fermentation hybrid system.
sequential fermentor (Supplementary Figure 1, available online). Photoautotrophic cultivation lasted for 8 days with continuous illumination at 100 μmol photon/m2 /s. After cell sediment and nitrogen removal, the subsequent fermentation was controlled with pH at 6.2 ± 0.1, air flow rate at 2 L/min, and agitation speed at 200 rpm. A range of 20–50% dissolved oxygen associated with agitation speed was adjusted by computer. To obtain a high cell concentration, glucose and yeast extract (growth factor) was batch-fed to keep its concentration at about 30 g/L and 3 g/L, respectively.[4,13] After 8 days’ fermentation, heterotrophic cells were harvested by centrifugation at 6000 g for 4 min (Supplementary Figure 2, available online)
Analytical methods The concentration of ammonia nitrogen (NH+ 4 -N) was detected by water quality analyser (DR890, Hach, USA). To monitor the cell growth, algal cells in 1 mL suspension were harvested by centrifugation and the cell pellets were washed twice with distilled water, freezedried until constant weight, and weighed to determine dry cell weight (DCW).[14] The cellular lipid content was determined according to a developed method.[8] The glucose in culture medium was measured using an automatic glucose analyser (SBA-40C, manufactured by Biological Center of Shandong Sciences Academy, China). To measure
Figure 2. Effects of different amounts of ammonia nitrogen on microalga C. protothecoides growth in the shaking flasks. (a) Photoautotrophic cell growth and residual concentration of NH+ 4 -N under different concentrations of NH+ -N. (b) Heterotrophic cell 4 growth and lipid content under different concentrations of NH+ 4 -N.
chlorophyll content, the dry algal cells were boiled in N, N-Dimethylformamide (DMF) for 5 min. The chlorophyll content was calculated from the absorbance of the DMF extracts with the following formula: chlorophyll (mg/L) = 8.02 × OD663 + 20.2 × OD645 .[15] The concentration of CO2 at the inlet and outlet of the photobioreactor was determined with an online gas analyser (Tandem, Milligan Instrument, UK). Changes of subcellular structure were observed by transmission electron microscope (JEM-1230, Hitachi, Japan), which are detailed in a previous report.[16] All data presented in this article were averages of three replicates (average ± standard deviation). Results and discussion Effects of ammonia nitrogen on photoautotrophic and heterotrophic growth of C. protothecoides A previous study indicated that for the cultivation of photoautotrophic algae, adequate nitrogen enabled high cell growth rate and cell concentration.[17] However, too much
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NH+ 4 -N will result in decreasing of pH, which would inhibit the growth of algal cells and lead to excessive residual NH+ 4N in the medium, and, therefore, difficulties for nitrogen removal in the fermentation stage of the hybrid system. To optimize the application of NH4 Cl in this system, effects of the initial amount of NH+ 4 -N in culture medium on photoautotrophic growth of C. protothecoides were investigated and the residual NH+ 4 -N in the medium was measured after 8 days’ cultivation. As shown in Figure 2(a), the highest algal biomass concentration reached 0.92 ± 0.03 g/L and residual concentration of NH+ 4 -N was about 1000 ± 50 mg/L when initial concentration of NH+ 4 -N was set at 2100 mg/L, which equals 8 g/L NH4 Cl. Increase of the amount of NH4 Cl led to less biomass production, presumably due to the inhibitory effect of excessive NH+ 4 -N in the medium, and meanwhile higher residual NH+ 4 -N (Figure 2(a), white columns), which put even more burdens on NH+ 4 -N removal in the next stage. In biofuel production, the lipid yield (DCW g/L × lipid content %) was the key to microalgae cultivation. During the metabolic switch from photoautotrophy to heterotrophy, the amount of lipid within cells was tightly regulated by the availability of nitrogen in the medium. Nitrogen deficiency is the initial signal to channel the carbon excess towards lipid biosynthesis.[18] Once the nitrogen deficiency occurred, along with sufficient carbon source, high lipid yield could be obtained successfully.[9] So it is necessary to investigate effects of residual NH+ 4 -N on cell growth and lipid content in the fermentation stage. As shown in Figure 2(b), when cells were cultivated in heterotrophic medium without NH+ 4 -N, the highest biomass concentration of 14.53 ± 0.32 g/L (DCW) and the highest lipid of 59.62 ± 2.53% (w/w) were obtained, which were consistent with the previously optimized heterotrophic culture.[4] Thus, the residual NH+ 4 -N concentration of 1000 ± 50 mg/L at the end of the photosynthesis stage was too high for heterotrophic fermentation and would affect the final lipid yield significantly. Both biomass and lipid content decreased significantly even when the concentra+ tion of NH+ 4 -N was 80 mg/L. Therefore NH4 -N had great influence on the cell growth and lipid accumulation under heterotrophic condition and should be kept at 40 mg/L or below. Effects of resin volume and operation time on capacity of nitrogen removal As shown in Figure 2(b), low concentration of NH+ 4 -N at 40 mg/L or less enhanced the biomass concentration and lipid content in heterotrophic C. protothecoides. Therefore, the efficiency of different resin-to-medium ratios (v/v) and operation times on nitrogen removal were tested. Results in Figure 3(a) indicate that more than 99.5% of residual NH+ 4 -N was removed when the ratio of resin-to-medium (v/v) was set at 1:1 and operation time was at 60 min. In addition, it was observed that most green C. protothecoides
Figure 3. Tests for NH+ 4 -N removal capacity of the nitrogen removal device. (a) Effects of different ratios of resin volume to medium volume on NH+ 4 -N absorption. (b) Effects of repeated use of the nitrogen removal device on residual concentration of NH+ 4 -N.
in the medium could pass through the resin column with only few cells left in the device. Thus, the nitrogen removal device could provide a quick and efficient method for deprivation of NH+ 4 -N, which was effective to switch green cells to the next heterotrophic stage and obtain high lipid yield (Figure 2(b)). To test the capacity of the nitrogen removal device for cost saving, a nutrient recycling strategy was adopted without reactivating the resin. As shown in Figure 3(b), the concentration of NH+ 4 -N at the outlet of the nitrogen removal device exceeded 40 mg/L only after the device was used continuously four times, suggesting that the resin could be used multiple times before reactivation. Furthermore NH+ 4 -N eluted from the resin column during reactivation could be recycled for preparing the photoautotrophic medium. Therefore, a connective device based on the ion exchange column for nitrogen removal between photobioreactor and fermentor was a feasible
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and flexible option to expand the application of the PFM approach.
Test of the photosynthesis-fermentation hybrid system for lipid production The photosynthesis-fermentation hybrid system was set up in lab-scale and operated for at least three cycles. C. protothecoides were cultivated in both photobioreactor and fermentor at the same time. Since the cell density after heterotrophic fermentation was much higher than that of green cells after photoautotrophic cultivation, the use of heterotrophic cells as seeds in the photosynthesis stage could save cultivation time (at least 4 days) and enhance the biomass concentration in the photosynthesis stage.[19] Therefore, at the beginning of the operation, 200 mL heterotrophic cells were inoculated into 13 L photoautotrophic medium in the photobioreactor. After being cultured photoautotrophically for 184 h, cells reached 1.03 ± 0.07 g/L (DCW) in the photobioreactor (Figure 4(a)) and the
Figure 4. Profiles of the photosynthesis stage of microalga C. protothecoides in the photosynthesis-fermentation hybrid system. (a) Photoautotrophic cell growth and NH+ 4 -N utilization during incubation. (b) CO2 absorption in a 15 L photobioreactor during incubation.
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concentration of NH+ 4 -N decreased from 2400 ± 200 to 1000 ± 50 mg/L. Since there was no other gas or air supply except a rubber tube connecting the fermentor to the close photobioreactor, CO2 in the off-gas from fermentation could be utilized by green algae cells in the photobioreactor. The fixation of CO2 was approximately 10.53 ± 0.73 g based on Figure 4(b) and the following calculation: C = F × T × D × (Cin − Cout ),
(1)
where C is the total CO2 fixed (g), F is the flow rate of offgas (L/min), T is the incubation time (min), D is the density of CO2 (g/L), Cin is the concentration of CO2 (%) at the inlet of the photobioreactor, and Cout is the concentration of CO2 (%) at the outlet of the photobioreactor. After cultivation in the photobioreactor, the cells were sedimented and most of the supernatant was removed. And then the concentrated green cells were transferred into a collection bottle, where they were pumped through the nitrogen removal device. The sequential fermentation was conducted for about 180 h and phenotypically the colour of algal cells completely turned from green to yellow (Supplementary Figures 1 and 2, available online). C. protothecoides cells in the fermentation stage grew vigorously with fast glucose consumption (Figure 5(a)). The final cell concentration reached up to 100.51 ± 2.03 g/L, which was 100.5 times higher than that in the photobioreactor (Figures 4(a) and 5(a)). Meanwhile, the final intracellular lipid reached 60.05 ± 1.38% (w/w), which was 7.23 times higher than that in the photobioreactor (Figure 5(b)). Together, as much as 60.36 ± 2.63 g/L in lipid yield (DCW g/L × lipid content %) was obtained by using the photosynthesisfermentation hybrid system in one cycle. Furthermore, in the hybrid system, harvest of yellow cells and inoculation for photoautotrophic growth might be operated simultaneously after the concentrated green cells were transferred to the collection bottle. By means of green and yellow cells being switched to each other, the biomass production could be conducted in the photosynthesis-fermentation hybrid system for multiple cycles. To verify the efficiency of switch from photoautotrophic to heterotrophic growth in the photosynthesis-fermentation hybrid system, transmission electron microscopy was used to monitor the change of subcellular structures (Figure 6). The membrane structures of photosynthetic lamellae in chloroplasts were clearly visible in cells collected from the photobioreactor (Figure 6(a) and 6(b)). In addition, a few starch granules were observed in these lamellaes. On the other hand, thylakoid membranes completely disappeared in cells collected from the fermentor (Figure 6(c) and 6(d)). Instead, the cytoplasm was filled with large lipid droplets and each was over 1 μm in diameter. Together, biochemical and cell structural evidences (Figures 5 and 6) confirmed that biodegradation of photosynthetic organelles provided enough space for lipid droplets in the heterotrophic C. protothecoides cell.[16] It was obvious that
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Figure 5. Profiles of the fermentation stage of microalga C. protothecoides in the photosynthesis-fermentation hybrid system. (a) Heterotrophic cell growth and glucose consumption during incubation. (b) Lipid accumulation and chlorophyll degradation during incubation.
the occurrence of chlorophyll breakdown and degradation of the photosynthetic system was associated with lipogenesis during fermentation in the hybrid system too. Comparison of lipid yield among photobioreactor, fermentor, and the hybrid system In order to evaluate the photosynthesis-fermentation hybrid system, the biomass concentration and lipid content were measured either after heterotrophic fermentation of C. protothecoides in an independent fermentor or under photoautotrophic cultivation in a photobioreactor. Comparison of the lipid yield in an independent photobioreactor, an independent fermentor, and the integrated hybrid system is shown in Table 1. The lipid yield at the end of the fermentation stage of the integrated hybrid system was over 700 times higher than that in the photobioreactor, and it was even higher than that of fermentation alone. Why was the lipid yield in the photosynthesisfermentation hybrid system higher than that in both the photobioreactor and the independent fermentor? One possible
explanation was given by MFA (Supplementary Figure 3, available online) based on our previous studies on elementary flux modes.[9] For photosynthetic carbon metabolism in the photobioreactor, glucose, starch, and cellulose are the major end products from CO2 by light reaction and the Calvin cycle. Fatty acids are not the preferred end products because to generate one molecule of stearic acid (C18:0 ), it requires 71 ATP, 52 NADH, and one NADPH.[8] Sunlight could not afford so much energy for lipid synthesis during photosynthesis. Therefore, most plants, including algae, synthesize sugars rather than lipids as their main product (Supplementary Figure 3(a), available online). For heterotrophic carbon metabolism in the fermentor, glucose is first degraded to pyruvate via glycolysis, and then transformed to acetyl-CoA. Two carbons in acetyl-CoA are added to the fatty acid chain step by step for lipid synthesis. Generation of one molecule of twocarbon acetyl-CoA from three-carbon pyruvate releases one molecule of CO2 , leading to the theoretical maximum carbon ratio from glucose to lipid as much as 66.7% (Supplementary Figure 3(b), available online). While in the photosynthesis-fermentation system, it was suggested that CO2 released from pyruvate might be re-fixed through non-oxidative pentose pathway during transferring green cells to heterotrophic fermentation. It provides more CO2 for extra lipid synthesis, which resulted in the theoretical maximum carbon ratios (83.33–100%) from glucose to lipid, much higher than that in independent fermentation (66.7%) (Supplementary Figure 3(c), available online). It was also indicated that in the fermentation stage after photosynthesis, CO2 fixation catalysed by the remaining Rubisco continued after feeding glucose in fermentation.[8] It might possibly explain why the hybrid system produced more lipid in comparison with the independent fermentor. In the photosynthesis-fermentation hybrid system, CO2 released from fermentation was not only fixed through the non-oxidative pentose pathway in yellow cells but also was absorbed by green cells in the photobioreactor by transporting off-gas through a tube from the fermentor to the photobioreactor (Supplementary Figures 1 and 2, available online). In the hybrid system, the volume of the photobioreactor (15 L) was much larger than that of the fermentor (1 L) (Figure 1). The large volume of green cells means that more CO2 could be fixed and more energy from sunlight could be absorbed, which together enhanced the final biomass and lipid yield. Meanwhile, the smaller volume in the fermentor for cultivation of concentrated green cells reduced cost in fermentation processing including less consumption of water, power, agitation-gas, and human labours. In addition, smaller volume of yellow cells with high cell density and lipid content would reduce the harvest cost at the end. All these benefits (Table 1) suggested that the photosynthesis-fermentation hybrid system was a feasible system of lipid production for algal biofuel with potential scale-up application.
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Figure 6. Images of transmission electron microscopy of microalga C. protothecoides cells. (a) Photoautotrophic cell, 25,000×. (b) Photoautotrophic cells, 10,000×. (c) Heterotrophic cell, 25,000×. (d) Heterotrophic cells, 10,000×. Close triangle: 1 = Chloroplast, 2 = Starch granule, 3 = Lipid droplet. Table 1. Comparison of independent photobioreactor and independent fermentor with the integrated hybrid system.
Cell concentration (DCW g/L) Lipid content (%) Lipid yield (g/L)
Hybrid system
Photobioreactor
Fermentor
100.51 ± 2.03 60.05 ± 1.38 60.36 ± 2.63
1.03 ± 0.07 8.30 ± 2.61 0.085 ± 0.034
80.03 ± 6.99 59.43 ± 3.63 47.56 ± 7.31
Note: All data presented in the above table are averages of three replicates (average ± standard deviation).
Conclusion A photosynthesis-fermentation hybrid system was designed, constructed and tested in this study. After photoautotrophic cultivation of C. protothecoides in a large
photobioreactor, concentrated green cells in collection bottle were pumped through a nitrogen removal device and then to a small fermentor for heterotrophic cultivation. 10.53 ± 0.73 g CO2 released from fermentation was
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absorbed and recycled by green cells in the photobioreactor. The nitrogen removal device with ion exchange resin column was effective to remove residual ammonia nitrogen from cell suspension and resulted in the switch of green cells to heterotrophic growth in the fermentor. The final dry cell weight and lipid content could reach as high as 100.51 ± 2.03 g/L and 60.05 ± 1.38%, respectively, in the photosynthesis-fermentation hybrid system. The lipid yield (60.36 ± 2.63 g/L) in the hybrid system was over 700 times higher than that in a photobioreactor and exceeded that of fermentation alone (47.56 ± 7.31 g/L). Therefore, the photosynthesis-fermentation hybrid system was considered a feasible integrated system with higher lipid yield, more CO2 fixation, and less cost for algal biofuel production. Acknowledgements This study was supported by NSFC project 41030210, MOST project 2011BAD14B05 and 2011CB808804.
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[7] Chi Z, Liu Y, Frear C, Chen S. Study of a two-stage growth of DHA-producing marine algae Schizochytrium limacinum SR21 with shifting dissolved oxygen level. Appl Microbiol Biotechnol. 2009;81:1141–1148. [8] Xiong W, Gao C, Yan D, Wu C, Wu Q. Double CO(2) fixation in photosynthesis-fermentation model enhances algal lipid synthesis for biodiesel production. Bioresource Technol. 2010;101:2287–2293. [9] Xiong W, Liu L, Wu C, Yang C, Wu Q. 13Ctracer and gas chromatography-mass spectrometry analyses reveal metabolic flux distribution in the oleaginous microalga Chlorella protothecoides. Plant Physiol. 2010;154: 1001–1011. [10] Shi X, Zhang X, Chen F. Heterotrophic production of biomass and lutein by Chlorella protothecoides on various nitrogen sources. Enzyme Microb Technol. 2000;27: 312–318. [11] Li X, Xu H, Wu Q. Large-scale biodiesel production from microalga Chlorella protothecoides through heterotrophic cultivation in bioreactors. Biotechnol Bioeng. 2007;98: 764–771. [12] Mori M, Tanaka K, Helaleh MI, Xu Q, Ikedo M, Ogura Y, Sato S, Hu W, Hasebe K. Selective determination of ammonium ions by high-speed ion-exclusion chromatography on a weakly basic anion-exchange resin column. J Chromatogr A. 2003;997:191–197. [13] Shen Y, Yuan W, Pei Z, Mao E. Heterotrophic culture of Chlorella protothecoides in various nitrogen sources for lipid production. Appl Biochem Biotechnol. 2010;160: 1674–1684. [14] Lee JY, Yoo C, Jun SY, Ahn CY, Oh HM. Comparison of several methods for effective lipid extraction from microalgae. Bioresour Technol. 2010;101:S75–S77. [15] MacKinney G. Absorption of light by chlorophyll solutions. J Biol Chem. 1941;140:315–322. [16] Jiang Q, Zhao L, Dai J, Wu Q. Analysis of autophagy genes in microalgae: chlorella as a potential model to study mechanism of autophagy. PLoS One. 2012;7:e41826. [17] Jin HF, Lim BR, Lee K. Influence of nitrate feeding on carbon dioxide fixation by microalgae. J Environ Sci Health A Tox Hazard Subst Environ Eng. 2006;41:2813–2824. [18] Yan D, Dai J, Wu Q. Characterization of an ammonium transporter in the oleaginous alga Chlorella protothecoides. Appl Microbiol Biotechnol. 2013;97:919–928. [19] Han F, Huang J, Li Y, Wang W, Wang J, Fan J, Shen G. Enhancement of microalgal biomass and lipid productivities by a model of photoautotrophic culture with heterotrophic cells as seed. Bioresour Technol. 2012;118:431–437.
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Photosynthesis-fermentation hybrid system to produce lipid feedstock for algal biofuel a
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School of Life Sciences , Tsinghua University , Beijing , 100084 , People's Republic of China Published online: 08 Oct 2013.
To cite this article: Yue Lu , Junbiao Dai & Qingyu Wu (2013) Photosynthesis-fermentation hybrid system to produce lipid feedstock for algal biofuel, Environmental Technology, 34:13-14, 1869-1876, DOI: 10.1080/09593330.2013.824011 To link to this article: http://dx.doi.org/10.1080/09593330.2013.824011
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Environmental Technology, 2013 Vol. 34, Nos. 13–14, 1869–1876, http://dx.doi.org/10.1080/09593330.2013.824011
Photosynthesis-fermentation hybrid system to produce lipid feedstock for algal biofuel Yue Lu, Junbiao Dai∗ and Qingyu Wu∗ School of Life Sciences, Tsinghua University, Beijing 100084, People’s Republic of China
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(Received 29 March 2013; final version received 1 July 2013 ) To avoid bacterial contamination due to medium replacement in the expanded application of a photosynthesis–fermentation model, an integrated photosynthesis-fermentation hybrid system was set up and evaluated for algal lipid production using Chlorella protothecoides. In this system, the CO2 -rich off-gas from the fermentation process was recycled to agitate medium in the photobioreactor, which could provide initial cells for the heterotrophic fermentation. The cell concentration reached 1.03 ± 0.07 g/L during photoautotrophic growth and then the concentrated green cells were switched to heterotrophic fermentation after removing over 99.5% of nitrogen in the medium by a nitrogen removal device. At the end of fermentation in the system, the cell concentration could reach as high as 100.51 ± 2.03 g/L, and 60.05 ± 1.38% lipid content was achieved simultaneously. The lipid yield (60.36 ± 2.63 g/L) in the hybrid system was over 700 times higher than that in a photobioreactor and exceeded that by fermentation alone (47.56 ± 7.31 g/L). The developed photosynthesis-fermentation hybrid system in this study was not only a feasible option to enhance microalgal lipid production, but also an environment-friendly approach to produce biofuel feedstock through concurrent utilization of ammonia nitrogen, CO2 , and organic carbons. Keywords: photosynthesis and fermentation; lipid and biofuel; Chlorella protothecoides; hybrid system; nitrogen removal
Introduction Microalgae are considered an ideal source of lipid feedstock for biofuel production as they have high lipid synthesis capacity and can be cultivated in bioreactors, without competing for arable land.[1] One challenge is that among photoautotrophic algae, rapidly growing cells generally contain fewer neutral lipids than slower-growing counterparts. For example, the traditional nitrogen starvation strategy may increase neutral lipid accumulation greatly in phototrophic algae but limits the cell growth, which eventually results in relatively lower lipid yield.[2,3] Even in the best photoautotrophic cultivation conditions, most species of microalgae hardly satisfy the biomass yields required for biofuel production. In order to obtain both fast cell growth rate and high lipid content, heterotrophic cultivation of Chlorella protothecoides 0710 for algal biofuels was evaluated. More than a 50-fold increase in lipid yield was reported in the previous studies in comparison with the photoautotrophic approach.[4–6] High cell density and high lipid content are considered two advantages for heterotrophic fermentation approach.[7] However, the caveats of such a cultivation method include the release of CO2 into the atmosphere and the consumption of organic carbon. To combine the advantages of CO2 fixation and solar energy conversion from photosynthesis with advantages of high cell density and high lipid content from fermentation, a photosynthesis–fermentation model (PFM) approach ∗ Corresponding
has been proposed. In this approach, the algal cells were cultivated photoautotrophically for CO2 fixation in the initial stage and then switched to heterotrophic fermentation in the second stage for lipid accumulation, leading to further enhancement of algal lipid synthesis. Experiments based on metabolic flux analysis (MFA) suggested that an increase in the conversion ratio of glucose to lipid in PFM resulted from Rubisco catalysing CO2 re-fixation in the second fermentation stage.[8] Therefore, PFM merges the beneficial features from photoautotrophic and heterotrophic approaches, thus producing more lipids, fixing more CO2 , and consuming less sugar. However in PFM, the algal cells from nitrogen-rich medium in the first photosynthetic stage have to be harvested by centrifugation or filtration before being transferred to nitrogen-deficient and glucose-rich medium to initiate the heterotrophic stage. Such kind of operations for medium replacement will not only increase the cost but also bring the risk of bacterial contamination in scale-up trials. On the other hand, to allow the cells to grow efficiently under heterotrophic conditions, the amount of nitrogen in the medium has to be reduced to a certain level.[9] Although various nitrogen sources such as nitrate, ammonium, urea, and glycine have been reported to be utilized by C. protothecoides without considerable differences,[10,11] few of them can be deprived technologically in medium by a simple and effective way. Studies in wastewater treatment
authors. Emails: [email protected], [email protected]
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suggested that ion exchange resin was effective in absorbing and removing ammonia nitrogen (NH+ 4 -N).[12] Therefore, if NH4 Cl was used as the sole nitrogen source in photoautotrophic cultivation, nitrogen could be removed by the resin for switching algae to heterotrophic growth. In order to mitigate the issues associated with the cell transfer in PFM, in this study, an integrated photosynthesisfermentation hybrid system was designed by using a nitrogen removal device to connect a large photobioreactor to a small fermentor. The nitrogen removal device with ion exchange resin column was used to effectively remove residual ammonia nitrogen from cell suspension and resulted in the switch of photoautotrophic cells to heterotrophic growth. CO2 released from heterotrophic cells in the fermentor was absorbed and recycled by photoautotrophic cells in the photobioreactor. Eventually, the photosynthesis-fermentation hybrid system produced more lipid in comparison with independent photobioreactor and fermentor. It provided a feasible approach of lipid production for algal biofuel in scale-up application.
Materials and methods Strain and medium The microalga C. protothecoides 0710 strain originally came from Culture Collection of Algae at the University of Texas (Austin, Texas, USA) and screened for high lipid yield in Algae Bioenergy Laboratory at Tsinghua University (Beijing, China). The composition of basal culture medium was: KH2 PO4 0.7 g/L, K2 HPO4 0.3 g/L, MgSO4 •7H2 O 0.3 g/L, FeSO4 •7H2 O 3 mg/L, vitamin B1 0.01 mg/L, and A5 trace mineral solution (H3 BO3 2.86 mg/L, ZnSO4 •7H2 O 0.222 mg/L, MnCl2 •4H2 O 1.81 mg/L, CuSO4 •5H2 O 0.074 mg/L, Na2 MoO4 •2H2 O 0.039 mg/L).[4] 8 g/L NH4 Cl was added in basal medium for photoautotrophic growth to obtain green cells and 30 g/L glucose was added in basal medium for heterotrophic growth to obtain yellow cells.
Photoautotrophic and heterotrophic batch cultures in shaking flasks The photoautotrophic and heterotrophic batch cultures in shaking flasks were two independent experiments with independent seed cells. Basal culture media with different concentrations of NH4 Cl (0, 4, 8, 12, 16, and 20 g/L, corresponding concentrations of NH+ 4 -N measured as 0, 1100, 2100, 2900, 3800, and 4900 mg/L, respectively) were used for testing photoautotrophic growth of C. protothecoides cells. Fifteen millilitres heterotrophic seed cells in exponential growth phase were inoculated into 600 mL medium in each 1 L glass flask, which then were incubated in an incubator shaker (ZWY-A2112B Incubator, ZHICHENG Analytical Instrument Manufacturing Co. Ltd, Shanghai,
China) under temperature at 28 ± 0.5◦ C, rotation speed at 220 rpm, and continuous artificial illumination at 100 μmol photon/m2 /s. The batch experiments of heterotrophic culture were conducted to investigate the effects of residual NH+ 4 -N on heterotrophic cell growth and lipid accumulation in darkness. After 8 days’ cultivation under the optimum conditions, photoautotrophic cell suspensions were centrifuged at 8000 g for 4 min (6–15 Centrifuge, Sigma, Germany). Cell pellets were washed twice by basal culture medium to remove the residual NH4 Cl and resuspended. Then the concentrated cells were transferred into 400 mL culture medium containing 30 g/L glucose and different concentrations of NH+ 4 -N (0, 20, 40, and 80 mg/L, respectively) in each 1 L shaking flask. The incubation temperature and rotation speed were the same as photoautotrophic cultivation above. Design of photosynthesis-fermentation hybrid system To switch C. protothecoides to heterotrophic fermentation after photoautotrophic cultivation and avoid potential bacterial contamination due to medium replacement, a 15 L cylindrical glass photobioreactor, a cell collection bottle, a nitrogen removal device, and a 1 L fermentor (Multifors, Infors Bio-Technology, Switzerland) were connected to form a single integrated system. The CO2 -rich off-gas derived from the fermentor was recycled for both agitating medium in the photobioreactor and providing CO2 for photoautotrophic cells (Figure 1). A device for ammonia nitrogen removal was designed and incorporated into the hybrid system (Figure 1). It is a stainless steel column (10.5 cm in diameter and 30.0 cm in length) filled with 0.5 L ion exchange resin (D61, Zhengguang Resin, China). At the end of log phase, phototrophic algal cells (green cells) were allowed to settle overnight. After discarding part of the supernatant, the concentrated photoautotrophic cells in 200 mL medium with residual nitrogen were pumped through the NH+ 4 -N removal device at 3 mL/min and subsequently pressed into a 1 L fermentor containing glucose-rich medium. By determining the concentration of NH+ 4 -N at the outlet of the resin column, the efficiency of NH+ 4 -N removal was calculated under different resin-to-medium ratios (v/v) or after different numbers of repeated usage. To reactivate the resin, the NH+ 4 -N removal device was rinsed with 1 M HCl for 8 h and washed by deionized water till the pH returned to 6.0. Operation conditions for the photosynthesis-fermentation hybrid system Cells in both photobioreactor and fermentor were cultivated at the same time at 28 ± 0.5◦ C. In photoautotrophic cultivation, 200 mL heterotrophic C. protothecoides suspensions were inoculated into 13 L medium in a cylindrical glass photobioreactor agitating by off-gas from a
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Figure 1. Schematic diagram of the photosynthesis-fermentation hybrid system.
sequential fermentor (Supplementary Figure 1, available online). Photoautotrophic cultivation lasted for 8 days with continuous illumination at 100 μmol photon/m2 /s. After cell sediment and nitrogen removal, the subsequent fermentation was controlled with pH at 6.2 ± 0.1, air flow rate at 2 L/min, and agitation speed at 200 rpm. A range of 20–50% dissolved oxygen associated with agitation speed was adjusted by computer. To obtain a high cell concentration, glucose and yeast extract (growth factor) was batch-fed to keep its concentration at about 30 g/L and 3 g/L, respectively.[4,13] After 8 days’ fermentation, heterotrophic cells were harvested by centrifugation at 6000 g for 4 min (Supplementary Figure 2, available online)
Analytical methods The concentration of ammonia nitrogen (NH+ 4 -N) was detected by water quality analyser (DR890, Hach, USA). To monitor the cell growth, algal cells in 1 mL suspension were harvested by centrifugation and the cell pellets were washed twice with distilled water, freezedried until constant weight, and weighed to determine dry cell weight (DCW).[14] The cellular lipid content was determined according to a developed method.[8] The glucose in culture medium was measured using an automatic glucose analyser (SBA-40C, manufactured by Biological Center of Shandong Sciences Academy, China). To measure
Figure 2. Effects of different amounts of ammonia nitrogen on microalga C. protothecoides growth in the shaking flasks. (a) Photoautotrophic cell growth and residual concentration of NH+ 4 -N under different concentrations of NH+ -N. (b) Heterotrophic cell 4 growth and lipid content under different concentrations of NH+ 4 -N.
chlorophyll content, the dry algal cells were boiled in N, N-Dimethylformamide (DMF) for 5 min. The chlorophyll content was calculated from the absorbance of the DMF extracts with the following formula: chlorophyll (mg/L) = 8.02 × OD663 + 20.2 × OD645 .[15] The concentration of CO2 at the inlet and outlet of the photobioreactor was determined with an online gas analyser (Tandem, Milligan Instrument, UK). Changes of subcellular structure were observed by transmission electron microscope (JEM-1230, Hitachi, Japan), which are detailed in a previous report.[16] All data presented in this article were averages of three replicates (average ± standard deviation). Results and discussion Effects of ammonia nitrogen on photoautotrophic and heterotrophic growth of C. protothecoides A previous study indicated that for the cultivation of photoautotrophic algae, adequate nitrogen enabled high cell growth rate and cell concentration.[17] However, too much
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NH+ 4 -N will result in decreasing of pH, which would inhibit the growth of algal cells and lead to excessive residual NH+ 4N in the medium, and, therefore, difficulties for nitrogen removal in the fermentation stage of the hybrid system. To optimize the application of NH4 Cl in this system, effects of the initial amount of NH+ 4 -N in culture medium on photoautotrophic growth of C. protothecoides were investigated and the residual NH+ 4 -N in the medium was measured after 8 days’ cultivation. As shown in Figure 2(a), the highest algal biomass concentration reached 0.92 ± 0.03 g/L and residual concentration of NH+ 4 -N was about 1000 ± 50 mg/L when initial concentration of NH+ 4 -N was set at 2100 mg/L, which equals 8 g/L NH4 Cl. Increase of the amount of NH4 Cl led to less biomass production, presumably due to the inhibitory effect of excessive NH+ 4 -N in the medium, and meanwhile higher residual NH+ 4 -N (Figure 2(a), white columns), which put even more burdens on NH+ 4 -N removal in the next stage. In biofuel production, the lipid yield (DCW g/L × lipid content %) was the key to microalgae cultivation. During the metabolic switch from photoautotrophy to heterotrophy, the amount of lipid within cells was tightly regulated by the availability of nitrogen in the medium. Nitrogen deficiency is the initial signal to channel the carbon excess towards lipid biosynthesis.[18] Once the nitrogen deficiency occurred, along with sufficient carbon source, high lipid yield could be obtained successfully.[9] So it is necessary to investigate effects of residual NH+ 4 -N on cell growth and lipid content in the fermentation stage. As shown in Figure 2(b), when cells were cultivated in heterotrophic medium without NH+ 4 -N, the highest biomass concentration of 14.53 ± 0.32 g/L (DCW) and the highest lipid of 59.62 ± 2.53% (w/w) were obtained, which were consistent with the previously optimized heterotrophic culture.[4] Thus, the residual NH+ 4 -N concentration of 1000 ± 50 mg/L at the end of the photosynthesis stage was too high for heterotrophic fermentation and would affect the final lipid yield significantly. Both biomass and lipid content decreased significantly even when the concentra+ tion of NH+ 4 -N was 80 mg/L. Therefore NH4 -N had great influence on the cell growth and lipid accumulation under heterotrophic condition and should be kept at 40 mg/L or below. Effects of resin volume and operation time on capacity of nitrogen removal As shown in Figure 2(b), low concentration of NH+ 4 -N at 40 mg/L or less enhanced the biomass concentration and lipid content in heterotrophic C. protothecoides. Therefore, the efficiency of different resin-to-medium ratios (v/v) and operation times on nitrogen removal were tested. Results in Figure 3(a) indicate that more than 99.5% of residual NH+ 4 -N was removed when the ratio of resin-to-medium (v/v) was set at 1:1 and operation time was at 60 min. In addition, it was observed that most green C. protothecoides
Figure 3. Tests for NH+ 4 -N removal capacity of the nitrogen removal device. (a) Effects of different ratios of resin volume to medium volume on NH+ 4 -N absorption. (b) Effects of repeated use of the nitrogen removal device on residual concentration of NH+ 4 -N.
in the medium could pass through the resin column with only few cells left in the device. Thus, the nitrogen removal device could provide a quick and efficient method for deprivation of NH+ 4 -N, which was effective to switch green cells to the next heterotrophic stage and obtain high lipid yield (Figure 2(b)). To test the capacity of the nitrogen removal device for cost saving, a nutrient recycling strategy was adopted without reactivating the resin. As shown in Figure 3(b), the concentration of NH+ 4 -N at the outlet of the nitrogen removal device exceeded 40 mg/L only after the device was used continuously four times, suggesting that the resin could be used multiple times before reactivation. Furthermore NH+ 4 -N eluted from the resin column during reactivation could be recycled for preparing the photoautotrophic medium. Therefore, a connective device based on the ion exchange column for nitrogen removal between photobioreactor and fermentor was a feasible
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and flexible option to expand the application of the PFM approach.
Test of the photosynthesis-fermentation hybrid system for lipid production The photosynthesis-fermentation hybrid system was set up in lab-scale and operated for at least three cycles. C. protothecoides were cultivated in both photobioreactor and fermentor at the same time. Since the cell density after heterotrophic fermentation was much higher than that of green cells after photoautotrophic cultivation, the use of heterotrophic cells as seeds in the photosynthesis stage could save cultivation time (at least 4 days) and enhance the biomass concentration in the photosynthesis stage.[19] Therefore, at the beginning of the operation, 200 mL heterotrophic cells were inoculated into 13 L photoautotrophic medium in the photobioreactor. After being cultured photoautotrophically for 184 h, cells reached 1.03 ± 0.07 g/L (DCW) in the photobioreactor (Figure 4(a)) and the
Figure 4. Profiles of the photosynthesis stage of microalga C. protothecoides in the photosynthesis-fermentation hybrid system. (a) Photoautotrophic cell growth and NH+ 4 -N utilization during incubation. (b) CO2 absorption in a 15 L photobioreactor during incubation.
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concentration of NH+ 4 -N decreased from 2400 ± 200 to 1000 ± 50 mg/L. Since there was no other gas or air supply except a rubber tube connecting the fermentor to the close photobioreactor, CO2 in the off-gas from fermentation could be utilized by green algae cells in the photobioreactor. The fixation of CO2 was approximately 10.53 ± 0.73 g based on Figure 4(b) and the following calculation: C = F × T × D × (Cin − Cout ),
(1)
where C is the total CO2 fixed (g), F is the flow rate of offgas (L/min), T is the incubation time (min), D is the density of CO2 (g/L), Cin is the concentration of CO2 (%) at the inlet of the photobioreactor, and Cout is the concentration of CO2 (%) at the outlet of the photobioreactor. After cultivation in the photobioreactor, the cells were sedimented and most of the supernatant was removed. And then the concentrated green cells were transferred into a collection bottle, where they were pumped through the nitrogen removal device. The sequential fermentation was conducted for about 180 h and phenotypically the colour of algal cells completely turned from green to yellow (Supplementary Figures 1 and 2, available online). C. protothecoides cells in the fermentation stage grew vigorously with fast glucose consumption (Figure 5(a)). The final cell concentration reached up to 100.51 ± 2.03 g/L, which was 100.5 times higher than that in the photobioreactor (Figures 4(a) and 5(a)). Meanwhile, the final intracellular lipid reached 60.05 ± 1.38% (w/w), which was 7.23 times higher than that in the photobioreactor (Figure 5(b)). Together, as much as 60.36 ± 2.63 g/L in lipid yield (DCW g/L × lipid content %) was obtained by using the photosynthesisfermentation hybrid system in one cycle. Furthermore, in the hybrid system, harvest of yellow cells and inoculation for photoautotrophic growth might be operated simultaneously after the concentrated green cells were transferred to the collection bottle. By means of green and yellow cells being switched to each other, the biomass production could be conducted in the photosynthesis-fermentation hybrid system for multiple cycles. To verify the efficiency of switch from photoautotrophic to heterotrophic growth in the photosynthesis-fermentation hybrid system, transmission electron microscopy was used to monitor the change of subcellular structures (Figure 6). The membrane structures of photosynthetic lamellae in chloroplasts were clearly visible in cells collected from the photobioreactor (Figure 6(a) and 6(b)). In addition, a few starch granules were observed in these lamellaes. On the other hand, thylakoid membranes completely disappeared in cells collected from the fermentor (Figure 6(c) and 6(d)). Instead, the cytoplasm was filled with large lipid droplets and each was over 1 μm in diameter. Together, biochemical and cell structural evidences (Figures 5 and 6) confirmed that biodegradation of photosynthetic organelles provided enough space for lipid droplets in the heterotrophic C. protothecoides cell.[16] It was obvious that
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Figure 5. Profiles of the fermentation stage of microalga C. protothecoides in the photosynthesis-fermentation hybrid system. (a) Heterotrophic cell growth and glucose consumption during incubation. (b) Lipid accumulation and chlorophyll degradation during incubation.
the occurrence of chlorophyll breakdown and degradation of the photosynthetic system was associated with lipogenesis during fermentation in the hybrid system too. Comparison of lipid yield among photobioreactor, fermentor, and the hybrid system In order to evaluate the photosynthesis-fermentation hybrid system, the biomass concentration and lipid content were measured either after heterotrophic fermentation of C. protothecoides in an independent fermentor or under photoautotrophic cultivation in a photobioreactor. Comparison of the lipid yield in an independent photobioreactor, an independent fermentor, and the integrated hybrid system is shown in Table 1. The lipid yield at the end of the fermentation stage of the integrated hybrid system was over 700 times higher than that in the photobioreactor, and it was even higher than that of fermentation alone. Why was the lipid yield in the photosynthesisfermentation hybrid system higher than that in both the photobioreactor and the independent fermentor? One possible
explanation was given by MFA (Supplementary Figure 3, available online) based on our previous studies on elementary flux modes.[9] For photosynthetic carbon metabolism in the photobioreactor, glucose, starch, and cellulose are the major end products from CO2 by light reaction and the Calvin cycle. Fatty acids are not the preferred end products because to generate one molecule of stearic acid (C18:0 ), it requires 71 ATP, 52 NADH, and one NADPH.[8] Sunlight could not afford so much energy for lipid synthesis during photosynthesis. Therefore, most plants, including algae, synthesize sugars rather than lipids as their main product (Supplementary Figure 3(a), available online). For heterotrophic carbon metabolism in the fermentor, glucose is first degraded to pyruvate via glycolysis, and then transformed to acetyl-CoA. Two carbons in acetyl-CoA are added to the fatty acid chain step by step for lipid synthesis. Generation of one molecule of twocarbon acetyl-CoA from three-carbon pyruvate releases one molecule of CO2 , leading to the theoretical maximum carbon ratio from glucose to lipid as much as 66.7% (Supplementary Figure 3(b), available online). While in the photosynthesis-fermentation system, it was suggested that CO2 released from pyruvate might be re-fixed through non-oxidative pentose pathway during transferring green cells to heterotrophic fermentation. It provides more CO2 for extra lipid synthesis, which resulted in the theoretical maximum carbon ratios (83.33–100%) from glucose to lipid, much higher than that in independent fermentation (66.7%) (Supplementary Figure 3(c), available online). It was also indicated that in the fermentation stage after photosynthesis, CO2 fixation catalysed by the remaining Rubisco continued after feeding glucose in fermentation.[8] It might possibly explain why the hybrid system produced more lipid in comparison with the independent fermentor. In the photosynthesis-fermentation hybrid system, CO2 released from fermentation was not only fixed through the non-oxidative pentose pathway in yellow cells but also was absorbed by green cells in the photobioreactor by transporting off-gas through a tube from the fermentor to the photobioreactor (Supplementary Figures 1 and 2, available online). In the hybrid system, the volume of the photobioreactor (15 L) was much larger than that of the fermentor (1 L) (Figure 1). The large volume of green cells means that more CO2 could be fixed and more energy from sunlight could be absorbed, which together enhanced the final biomass and lipid yield. Meanwhile, the smaller volume in the fermentor for cultivation of concentrated green cells reduced cost in fermentation processing including less consumption of water, power, agitation-gas, and human labours. In addition, smaller volume of yellow cells with high cell density and lipid content would reduce the harvest cost at the end. All these benefits (Table 1) suggested that the photosynthesis-fermentation hybrid system was a feasible system of lipid production for algal biofuel with potential scale-up application.
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Figure 6. Images of transmission electron microscopy of microalga C. protothecoides cells. (a) Photoautotrophic cell, 25,000×. (b) Photoautotrophic cells, 10,000×. (c) Heterotrophic cell, 25,000×. (d) Heterotrophic cells, 10,000×. Close triangle: 1 = Chloroplast, 2 = Starch granule, 3 = Lipid droplet. Table 1. Comparison of independent photobioreactor and independent fermentor with the integrated hybrid system.
Cell concentration (DCW g/L) Lipid content (%) Lipid yield (g/L)
Hybrid system
Photobioreactor
Fermentor
100.51 ± 2.03 60.05 ± 1.38 60.36 ± 2.63
1.03 ± 0.07 8.30 ± 2.61 0.085 ± 0.034
80.03 ± 6.99 59.43 ± 3.63 47.56 ± 7.31
Note: All data presented in the above table are averages of three replicates (average ± standard deviation).
Conclusion A photosynthesis-fermentation hybrid system was designed, constructed and tested in this study. After photoautotrophic cultivation of C. protothecoides in a large
photobioreactor, concentrated green cells in collection bottle were pumped through a nitrogen removal device and then to a small fermentor for heterotrophic cultivation. 10.53 ± 0.73 g CO2 released from fermentation was
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absorbed and recycled by green cells in the photobioreactor. The nitrogen removal device with ion exchange resin column was effective to remove residual ammonia nitrogen from cell suspension and resulted in the switch of green cells to heterotrophic growth in the fermentor. The final dry cell weight and lipid content could reach as high as 100.51 ± 2.03 g/L and 60.05 ± 1.38%, respectively, in the photosynthesis-fermentation hybrid system. The lipid yield (60.36 ± 2.63 g/L) in the hybrid system was over 700 times higher than that in a photobioreactor and exceeded that of fermentation alone (47.56 ± 7.31 g/L). Therefore, the photosynthesis-fermentation hybrid system was considered a feasible integrated system with higher lipid yield, more CO2 fixation, and less cost for algal biofuel production. Acknowledgements This study was supported by NSFC project 41030210, MOST project 2011BAD14B05 and 2011CB808804.
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