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An integrated system of autotrophic Chlorella vulgaris cultivation using CO2 from the aerobic cultivation process of Rhodotorula glutinis
Journal of the Taiwan Institute of Chemical Engineers 62 (2016) 158–161
Contents lists available at ScienceDirect
Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
An integrated system of autotrophic Chlorella vulgaris cultivation using CO2 from the aerobic cultivation process of Rhodotorula glutinis Hong-Wei Yen∗, Chih-Yuan Hsu, Pin-Wen Chen Department of Chemical and Materials Engineering, Tunghai University, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 30 November 2015 Revised 26 January 2016 Accepted 27 January 2016 Available online 17 February 2016 Keywords: Integrated Microalgae Oleaginous Photosynthesis Biodiesel Carbon reduction
a b s t r a c t The production of microbial oils from oleaginous Rhodotorula glutinis is especially attractive with regard to the production of biodiesel. Nevertheless, a considerable amount of CO2 is generated during the aerobic cultivation process, which can have negative effects on the environment. An integrated system of autotrophic microalgae cultivation (Chlorella vulgaris) using CO2 from the aerobic R. glutinis bioreactor can efficiently reduce CO2 emissions in this regard. The results indicated about 2.5 ± 0.1% and 0.5 ± 0.15% of CO2 in the flue gas streams from the aerobic tank and from the photosynthesis bioreactor, respectively. It is estimated that about 80% of the CO2 generated in the cultivation of R. glutinis was fixed by C. vulgaris, with the integrated system producing biomass of about 20 g/L of R. glutinis and 1.2 g/L of C. vulgaris. The results of this work indicate that this integrated system can produce microbial oils without the high CO2 emissions seen with the standard cultivation system. Also, the high value compound produced in the microalgal biomass (e.g. lutein) can compensate the microbial oils production cost, which makes the commercialization of this integrated process more feasible. © 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction The limited supply of fossil-based energy resources and the pollutions associated with the consumption of such fuels has motivated researchers to explore the development of alternative, renewable energy resources, in addition to the processes of carbon capture and utilization. Among other options, biomass-derived fuels can play an important role in diversifying the energy supply and enhancing its security. One such fuel is biodiesel, which can be produced through the transesterificaiton of several oil-containing feedstocks, such as conventional oil crops, waste kitchen oils and microbial oils. Among all potential feedstocks, microbial oils from oleaginous microorganisms are especially attractive, as they can avoid the problem of competition with arable land use while using non-food carbon sources for the accumulation of microbial oils. Oleaginous microorganisms are defined as those species for which the dry biomass contains more than 20% of lipid content [1]. Numerous oleaginous yeasts and microalgae have been reported to be capable of accumulating large amounts of lipids, with some studies reporting a content of more than 70% [2–4]. The majority of these lipids are triacylglycerol (TAG) containing long-chain fatty acids, and the fatty acid profiles of most oleaginous microorganisms are comparable to those of conventional oil crops, which ∗
Corresponding author. Tel.: +886 4 23590262; fax: +886 4 23590 0 09. E-mail address: [email protected], [email protected] (H.-W. Yen).
are quite suitable for use as the feedstock for biodiesel production. More specifically, Rhodotorula glutinis is especially suitable for microbial oil production, due to its characteristics of rapid growth and high lipid content when using various carbon sources, such as crude glycerol and cellulosic hydrolysate [5,6]. Nevertheless, the cultivation of aerobic R. glutinis produces considerable CO2 emissions, working against the core concept of reducing such emissions by using renewable biofuels. One way to overcome this issue it to use photosynthetic microalgae to reduce the amount of CO2 emitted from the aerobic fermentation process. CO2 fixation by using microalgae has been widely explored in literature [7–9], and in addition to this property that microalgae are also a potential source of biofuels and numerous high value compounds, such as lutein and long chain fatty acids. Liquid fuels, such as biodiesel, diesel, gasoline, and jet fuel, can also be produced from microalgal oils using existing technology. The source of CO2 needed to carry out photosynthesis is another factor affecting the growth rate of microalgae. The provision of sufficient light intensity and dissolved CO2 in the medium are both essential to attaining a high yield of microalgal biomass under autotrophic conditions. Many reports have explored the effects of the CO2 flow rate and percentage of CO2 in the inlet gas on the cultivation of microalgae [10,11]. In general, 2%–20% of CO2 is regarded as a suitable amount in the inlet gas for the cultivation of microalgae [12–14]. It was reported that an integrated system composed of Dunaliella tertiolecta microalgae using CO2 from the brewing fermentation
http://dx.doi.org/10.1016/j.jtice.2016.01.025 1876-1070/© 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
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process, and the effects of this CO2 on the production of lipid and carotenoid-rich D. tertiolecta were examined [15]. The results showed that the values from this system were almost twice as high as those observed for control cultivations in which atmospheric CO2 was used. This earlier work thus demonstrated that the integration of yeast fermenters and microalgae photo-bioreactors is an interesting approach to improve both biomass formation and product contents [15]. The purpose of the current study is to examine the effects of using CO2 from the aerobic cultivation of R. glutinis for the growth of C. vulgaris under autotrophic conditions. The growth of C. vulgaris using CO2 from the aerobic fermenter is compared to that seen with the batches using various CO2 percentages in the inlet air gas. The CO2 production profile in the aerobic fermentation process of R. glutinis is also examined. 2. Materials and methods 2.1. Microorganisms and medium The microalgae Chlorella vulgaris was generously provided by Prof. Jo-Shu Chang (National Cheng Kung University, Taiwan). A basal medium was used for autotrophic cultivation in flasks. Details of the basal medium preparation have been described in a previous work [16]. Freeze-dried Rhodotorula glutinis BCRC 21418 was obtained from the Bioresource Collection and Research Center, Taiwan (BCRC). The seed medium composition and the cultivation methods followed the suggestions provided by the BCRC. 2.2. Cultivation methods The batch autotrophic cultivation of C. vulgaris was performed at 25 ± 1°C, bubbled with 1 vvm air and supplemented with set CO2 percentages in a glass photosynthesis bioreactor (PBR) containing 3 l of medium. The glass column was continuously illuminated with regular fluorescent lights or white LED lights (model MR 16 with 7 Watts, Shianyih Electronic Industry Co., Taiwan) to provide sufficient light. The light intensity on the surface of the flask was measured by a light meter (LI 250, LI-COR, Inc., Lincoln, NE, USA), giving a value of 340 μmol/m2 s. Batch fermentation of R. glutinis was carried out in a 5-L agitation tank with a working volume of 3 L. All experiments were controlled at 24 ◦ C and the pH was controlled at 5.5 by using 1 N NaOH solution. The agitation rate was in the range of 100– 300 rpm to keep the DO over 20%, and the aeration rate was fixed at 1.0 vvm. The fermentation medium (per liter) comprised defined amounts of crude glycerol, 2 g of yeast extract, 2 g of (NH4 )2 SO4 , 1 g of KH2 PO4 , 0.5 g of MgSO4 ·7H2 O, 0.1 g of CaCl2 and 0.1 g of NaCl [17]. The integrated system comprised an agitation tank for the cultivation of R. glutinis and an autotrophic tank for the growth of C. vulgaris. The flue gas from the tank of R. glutinis was completely directed to the PBR of C. vulgaris, which achieved 1 vvm for the aeration rate in both tanks. A 0.45 μm sterilized air filter was installed in the air connection line between the agitation tank and the autotrophic PBR to avoid contamination of C. vulgaris cultivation. 2.3. Analytical methods An infrared balance (Denver Instrument, IR 35) was adopted to rapidly measure the biomass concentration. Five ml of broth was centrifuged at 70 0 0 rpm for 10 min. After removing the supernatant, about an equal volume of distilled water was added to eliminate impurities. This washing procedure was performed several times, and the final liquor was dried using the infrared balance at 150 °C to evaporate the water content.
Fig. 1. Time course of the percentage of CO2 and biomass growth of R. glutinis in a 5 L agitation fermenter.
The dry biomass was first ground into a fine powder; 0.05 g of the powder was then blended with 5 ml chloroform/methanol (2:1), and subsequently agitated for 20 min at room temperature in an orbital shaker. The solvent phase was recovered by centrifugation at 70 0 0 rpm for 10 min. The same process was repeated twice, and the whole solvent was evaporated and dried under vacuum conditions. The glycerol concentration was measured by HPLC (Agilent series 1100, Agilent Technologies, Santa Clara, CA) with a refractive index detector, while the analysis was performed in a C-18 column (Vercopak N5ODS, 250 mm ×4.6 mm, Taiwan). The mobile phase was composed of 0.01 N H2 SO4 with a flow rate of 0.4 ml/min [18]. All shaker conditions were performed in triplicate to obtain the mean ± standard deviation. 3. Results and discussion 3.1. Time course of CO2 generation in the batch of R. glutinis The cultivation of R. glutinis is a typical aerobic fermentation process, and requires a considerable amount of dissolved oxygen to obtain a large amount of high biomass [19,20]. The use of crude glycerol for the cultivation of R. glutinis was performed in an agitation tank at the aeration rate of 1 vvm. The time courses of the biomass productions, glycerol consumption and CO2 generation are shown in Fig. 1. It can be seen that the maximum biomass was 12.4 g/L after 5 days’ cultivation. The initial glycerol was about 21 g/L, and this decreased to less than 1 g/L after 5 days’ cultivation. The maximum biomass growth rate was about 4.7 g/L day, which led to the maximum CO2 generation percentage of 1.8% observed in the flue gas. During the exponential growth phase, the rapid growth of biomass lead to the maximum CO2 percentage observed in the flue gas. The CO2 generation curve reached its maximum after 1 day cultivation, and fell to a stable level of about 0.5% while the cell growth entered the stationary stage. The results indicated that the CO2 generation was strongly related to the growth of biomass. A higher biomass would produce a higher CO2 percentage in the flue gas. To confirm this, the effects of biomass on CO2 generation were examined by controlling the dissolved oxygen (DO) at high (>30%) and low (<30%) levels. The DO control strategy was achieved by automatically adjusting the agitation rate to provide the required DO level, and the results are shown in Fig. 2. It can be seen that the batch with high DO (denoted as high) produced more biomass than the low DO batch (denoted as low). Besides the higher biomass obtained in the high batch, a higher CO2
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Fig. 2. The comparison of CO2 generation in a 5 L agitation fermenter with high and low biomass R. glutinis cultivation. Fig. 3. The effects of CO2 concentration on the growth of Chlorella vulgaris.
percentage was also observed. Similar to the results found with the low biomass batch, in the one with high biomass the greatest CO2 percentage was found after 24 h’ cultivation. However, no significant decrease in CO2 generation was observed in the batch with a high biomass. The CO2 generation rates were 3.07 and 1.84 mmol/g DCW h for the growth of R. glutinis in the batches with high and low biomass, respectively. As compared to the 11.8–18.6 mmol/g DCW h for the cultivation of E. coli reported in the literature [21], the CO2 secretion rate in the cultivation of yeast was significantly lower. This implies that the growth rate of E. coli should be higher than that of R. glutinis. The CO2 secretion rate in the cultivation of R. glutinis represents a valuable carbon sink, as its stable CO2 generation rate can be used as the carbon source for the growth of microalgae. 3.2. The growth of Chlorella vulgaris under different concentrations of CO2 As shown in the previous section, the CO2 percentage would be about 2.5% of the flue gas in the batch with the biomass concentration of about 20 g/L, with the CO2 being utilized as the carbon source for the cultivation of autotrophic microalgae. Therefore, the effects of the CO2 concentration in the inlet gas on the growth of microalgae (Chlorella vulgaris) were examined. A CO2 concentration in the range of 0.03 (using air) to 60% was used in the cultivation of C. vulgaris, with the results shown in Fig. 3. It can be seen that the CO2 concentration of 2.5%–40% can be regarded as a suitable range for the cultivation of C. vulgaris. However, the low CO2 percentage in the pure air cannot provide enough source for the growth of C. vulgaris and the generation of large amounts of biomass. When the CO2 percentage was over 40% (such as 50 and 60%), this then led to a decrease in the pH and a reduction in the growth of biomass. The results of the current work indicate that 2.5% of CO2 in the flue gas emitted from the tank of R. glutinis can be a good carbon source for C. vulgaris cultivation. To further confirm this, a comparison of using pure air, 2.5% CO2 supplemented in the air and the flue gas emitted from the R. glutinis cultivation tank for the growth of C. vulgaris was performed, with the results shown in Fig. 4. As seen in the figure, the batch using flue gas emitted from the aerobic fermenter had a higher growth rate than that purging with 2.5% of CO2 and the pure air. One possible reason for this is a high CO2 percentage (2.5%–3.0%) provided at the beginning of exponential growth phase of R. glutinis, which might provide more carbon source for the growth of C. vulgaris. Nevertheless, the results indicated that the flue gas emitted from the
Fig. 4. Comparison of Chlorella vulgaris cultivation using 2.5% CO2 and the flue gas from the fermenter of R. glutinis.
cultivation of R. glutinis was a suitable CO2 source that can be used for the growth of C. vulgaris. 3.3. An integrated system of R. glutinis cultivation coupled with C. vulgaris cultivation An integrated system of aerobic R. glutinis fermentation coupled with autotrophic C. vulgaris cultivation was built to examine the feasibility of using the CO2 generated from R. glutinis for the growth of C. vulgaris, with the results shown in Fig. 5. It can be seen that the CO2 generation rapidly goes up to the maximum level after about 18 h’ cultivation in the flue gas of the R. glutinis tank, and the CO2 concentration also reaches its maximum level at the same time. It is thus clear that the growth rate of R. glutinis is faster than that of C. vulgaris. Therefore, the amount of CO2 purging into the tank of microalgae cannot be fixed efficiently at the beginning of C. vulgaris cultivation due to the low biomass density. Nevertheless, as the cultivation time increases more microalgal biomass is produced in the tank, and this can efficiently reduce the CO2 concentration, thus leading to a decrease in the CO2 concentration observed in the flue gas emitted from the microalgae tank. After 20 h’ cultivation, the CO2 concentration was stable at about 2.5 ± 0.1 and 0.5 ± 0.15 in the flue gas emitted from the
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(MOST) under grant numbers 104-2621-M-029 -004 and 104-3113E-0 06-0 03. References
Fig. 5. An integrated system of R. glutinis growth coupled with CO2 -fixation by using photosynthetic cultivation of Chlorella vulgaris.
tanks of R. glutinis and C. vulgaris, respectively. The results indicate that about 80% of the CO2 generated in the cultivation process of R. glutinis can be utilized by C. vulgaris through photosynthesis, which can produce about 20 g/L of R. glutinis and 1.2 g/L of C. vulgaris. In this integrated cultivation system, CO2 generation from the aerobic tank can be assimilated by autotrophic microalgae. Thus, a higher microalgal cells density might provide a higher carbon reduction efficiency. If some high value compounds can be extracted from microalgae (e.g. lutein, ω-3 fatty acid etc.), which the economic benefits can contribute to the overall process and reduce the microbial oils production cost. Besides, the assimilation of CO2 derived from the aerobic yeast by using microalgae can make this microbial oils production a so-called sustainable process, which yield the potential of commercialized production of microbial oils. 4. Conclusion The production of microbial oils using oleaginous R. glutinis as the feedstock for biodiesel is an attractive idea. Nevertheless, a considerable amount of CO2 is generated during the fermentation process, making this approach less environmentally feasible. The results of this study indicate that coupling an autotrophic microalgae PBR with the aerobic fermenter can efficiently reduce 80% of CO2 generated in the flue gas of R. glutinis cultivation. This integrated system proves the production of microbial oils can be a green process, with lower CO2 emissions. Acknowledgments The authors gratefully acknowledge the financial support this study received from Taiwan’s Ministry of Science and Technology
[1] Ageitos JM, Vallejo JA, Veiga-Crespo P, Villa TG. Oily yeasts as oleaginous cell factories. Appl Microbiol Biotechnol 2011;90:1219–27. [2] Meng X, Yang J, Xu X, Zhang L, Nie Q, Xian M. Biodiesel production from oleaginous microorganisms. Renewable Energy 2009;34:1–5. [3] Chisti Y. Biodiesel from microalgae. Biotechnol Adv 2007;25:294–306. [4] Demirbas A. Biodiesel from oilgae, biofixation of carbon dioxide by microalgae: a solution to pollution problems. Appl Energy 2011;88:3541–7. [5] Li M, Liu G-L, Chi Z, Chi Z-M. Single cell oil production from hydrolysate of cassava starch by marine-derived yeast Rhodotorula mucilaginosa TJY15a. Biomass Bioenergy 2010;34:101–7. [6] Zhao C-H, Zhang T, Li M, Chi Z-M. Single cell oil production from hydrolysates of inulin and extract of tubers of Jerusalem artichoke by Rhodotorula mucilaginosa TJY15a. Process Biochem 2010;45(7):1121–6. [7] Devi MP, Mohan SV. CO2 supplementation to domestic wastewater enhances microalgae lipid accumulation under mixotrophic microenvironment: Effect of sparging period and interval. Bioresource Technol 2012;112:116–23. [8] Ho S-H, Chen C-Y, Lee D-J, Chang J-S. Perspectives on microalgal CO2 emission mitigation systems — a review. Biotechnol Adv 2011;29:189–98. [9] Martínez L, Redondas V, García A-I, Morán A. Optimization of growth operational conditions for CO2 biofixation by native Synechocystis sp. J Chem Technol Biotechnol 2011;86:681–90. [10] Gao K, Yu A. Influence of CO2 , light and watering on growth of Nostoc flagelliforme mats. J Appl Phycology 20 0 0;12:185–9. [11] Gordillo FJL, Jim´enez C, Figueroa FeL, Niell FX. Effects of increased atmospheric CO2 and N supply on photosynthesis, growth and cell composition of the cyanobacterium Spirulina platensis (Arthrospira). J Appl Phycology 1999;10:461–9. [12] Sforza E, Cipriani R, Morosinotto T, Bertucco A, Giacometti GM. Excess CO2 supply inhibits mixotrophic growth of Chlorella protothecoides and Nannochloropsis salina. Bioresource Technol 2011;104:523–9. [13] Yang Y, Gao K. Effects of CO2 concentrations on the freshwater microalgae, Chlamydomonas reinhardtii, Chlorella pyrenoidosa and Scenedesmus obliquus (Chlorophyta). J Appl Phycology 20 03;0 0:1–11. [14] Yen HW, Hu IC, Chen CY, Ho SH, Lee DJ, Chang JS. Microalgae-based biorefinery-from biofuels to natural products. Bioresour Technol 2013;135:166– 74. [15] Chagas AL, Rios AO, Jarenkow A, Marcílio NR, Ayub MAZ, Rech R. Production of carotenoids and lipids by Dunaliella tertiolecta using CO2 from beer fermentation. Process Biochem 2015;50(6):981–8. [16] Yen H-W, Chiang W-C. Effects of mutual shading, pressurization and oxygen partial pressure on the autotrophical cultivation of Scenedesmus obliquus. J Taiwan Institute Chem Eng 2012;43:820–4. [17] Kim BK, Park PK, Chae HJ, Kim EY. Effect of Phenol on β -carotene content in total carotenoids production in cultivation of Rhodotorula glutinis. Korean J Chem Eng. 2004;21:689–92. [18] Athalye SK, Garcia RA, Wen Z. Use of biodiesel-derived crude glycerol for producing eicosapentaenoic acid (epa) by the fungus Pythium irregulare. J Agric Food Chem. 2009;57:2739–44. [19] Yen HW, Zhang Z. Effects of dissolved oxygen level on cell growth and total lipid accumulation in the cultivation of Rhodotorula glutinis. J Biosci Bioeng 2011;112(1):71–4. [20] Yen H-W, Liu YX. Application of airlift bioreactor for the cultivation of aerobic oleaginous yeast Rhodotorula glutinis with different aeration rates. J Biosci Bioeng 2014;118:195–8. [21] Gong F, Liu G, Zhai X, Zhou J, Cai Z, Li Y. Quantitative analysis of an engineered CO2 -fixing Escherichia coli reveals great potential of heterotrophic CO2 fixation. Biotechnol Biofuels 2015;8:86.
Contents lists available at ScienceDirect
Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
An integrated system of autotrophic Chlorella vulgaris cultivation using CO2 from the aerobic cultivation process of Rhodotorula glutinis Hong-Wei Yen∗, Chih-Yuan Hsu, Pin-Wen Chen Department of Chemical and Materials Engineering, Tunghai University, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 30 November 2015 Revised 26 January 2016 Accepted 27 January 2016 Available online 17 February 2016 Keywords: Integrated Microalgae Oleaginous Photosynthesis Biodiesel Carbon reduction
a b s t r a c t The production of microbial oils from oleaginous Rhodotorula glutinis is especially attractive with regard to the production of biodiesel. Nevertheless, a considerable amount of CO2 is generated during the aerobic cultivation process, which can have negative effects on the environment. An integrated system of autotrophic microalgae cultivation (Chlorella vulgaris) using CO2 from the aerobic R. glutinis bioreactor can efficiently reduce CO2 emissions in this regard. The results indicated about 2.5 ± 0.1% and 0.5 ± 0.15% of CO2 in the flue gas streams from the aerobic tank and from the photosynthesis bioreactor, respectively. It is estimated that about 80% of the CO2 generated in the cultivation of R. glutinis was fixed by C. vulgaris, with the integrated system producing biomass of about 20 g/L of R. glutinis and 1.2 g/L of C. vulgaris. The results of this work indicate that this integrated system can produce microbial oils without the high CO2 emissions seen with the standard cultivation system. Also, the high value compound produced in the microalgal biomass (e.g. lutein) can compensate the microbial oils production cost, which makes the commercialization of this integrated process more feasible. © 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction The limited supply of fossil-based energy resources and the pollutions associated with the consumption of such fuels has motivated researchers to explore the development of alternative, renewable energy resources, in addition to the processes of carbon capture and utilization. Among other options, biomass-derived fuels can play an important role in diversifying the energy supply and enhancing its security. One such fuel is biodiesel, which can be produced through the transesterificaiton of several oil-containing feedstocks, such as conventional oil crops, waste kitchen oils and microbial oils. Among all potential feedstocks, microbial oils from oleaginous microorganisms are especially attractive, as they can avoid the problem of competition with arable land use while using non-food carbon sources for the accumulation of microbial oils. Oleaginous microorganisms are defined as those species for which the dry biomass contains more than 20% of lipid content [1]. Numerous oleaginous yeasts and microalgae have been reported to be capable of accumulating large amounts of lipids, with some studies reporting a content of more than 70% [2–4]. The majority of these lipids are triacylglycerol (TAG) containing long-chain fatty acids, and the fatty acid profiles of most oleaginous microorganisms are comparable to those of conventional oil crops, which ∗
Corresponding author. Tel.: +886 4 23590262; fax: +886 4 23590 0 09. E-mail address: [email protected], [email protected] (H.-W. Yen).
are quite suitable for use as the feedstock for biodiesel production. More specifically, Rhodotorula glutinis is especially suitable for microbial oil production, due to its characteristics of rapid growth and high lipid content when using various carbon sources, such as crude glycerol and cellulosic hydrolysate [5,6]. Nevertheless, the cultivation of aerobic R. glutinis produces considerable CO2 emissions, working against the core concept of reducing such emissions by using renewable biofuels. One way to overcome this issue it to use photosynthetic microalgae to reduce the amount of CO2 emitted from the aerobic fermentation process. CO2 fixation by using microalgae has been widely explored in literature [7–9], and in addition to this property that microalgae are also a potential source of biofuels and numerous high value compounds, such as lutein and long chain fatty acids. Liquid fuels, such as biodiesel, diesel, gasoline, and jet fuel, can also be produced from microalgal oils using existing technology. The source of CO2 needed to carry out photosynthesis is another factor affecting the growth rate of microalgae. The provision of sufficient light intensity and dissolved CO2 in the medium are both essential to attaining a high yield of microalgal biomass under autotrophic conditions. Many reports have explored the effects of the CO2 flow rate and percentage of CO2 in the inlet gas on the cultivation of microalgae [10,11]. In general, 2%–20% of CO2 is regarded as a suitable amount in the inlet gas for the cultivation of microalgae [12–14]. It was reported that an integrated system composed of Dunaliella tertiolecta microalgae using CO2 from the brewing fermentation
http://dx.doi.org/10.1016/j.jtice.2016.01.025 1876-1070/© 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
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process, and the effects of this CO2 on the production of lipid and carotenoid-rich D. tertiolecta were examined [15]. The results showed that the values from this system were almost twice as high as those observed for control cultivations in which atmospheric CO2 was used. This earlier work thus demonstrated that the integration of yeast fermenters and microalgae photo-bioreactors is an interesting approach to improve both biomass formation and product contents [15]. The purpose of the current study is to examine the effects of using CO2 from the aerobic cultivation of R. glutinis for the growth of C. vulgaris under autotrophic conditions. The growth of C. vulgaris using CO2 from the aerobic fermenter is compared to that seen with the batches using various CO2 percentages in the inlet air gas. The CO2 production profile in the aerobic fermentation process of R. glutinis is also examined. 2. Materials and methods 2.1. Microorganisms and medium The microalgae Chlorella vulgaris was generously provided by Prof. Jo-Shu Chang (National Cheng Kung University, Taiwan). A basal medium was used for autotrophic cultivation in flasks. Details of the basal medium preparation have been described in a previous work [16]. Freeze-dried Rhodotorula glutinis BCRC 21418 was obtained from the Bioresource Collection and Research Center, Taiwan (BCRC). The seed medium composition and the cultivation methods followed the suggestions provided by the BCRC. 2.2. Cultivation methods The batch autotrophic cultivation of C. vulgaris was performed at 25 ± 1°C, bubbled with 1 vvm air and supplemented with set CO2 percentages in a glass photosynthesis bioreactor (PBR) containing 3 l of medium. The glass column was continuously illuminated with regular fluorescent lights or white LED lights (model MR 16 with 7 Watts, Shianyih Electronic Industry Co., Taiwan) to provide sufficient light. The light intensity on the surface of the flask was measured by a light meter (LI 250, LI-COR, Inc., Lincoln, NE, USA), giving a value of 340 μmol/m2 s. Batch fermentation of R. glutinis was carried out in a 5-L agitation tank with a working volume of 3 L. All experiments were controlled at 24 ◦ C and the pH was controlled at 5.5 by using 1 N NaOH solution. The agitation rate was in the range of 100– 300 rpm to keep the DO over 20%, and the aeration rate was fixed at 1.0 vvm. The fermentation medium (per liter) comprised defined amounts of crude glycerol, 2 g of yeast extract, 2 g of (NH4 )2 SO4 , 1 g of KH2 PO4 , 0.5 g of MgSO4 ·7H2 O, 0.1 g of CaCl2 and 0.1 g of NaCl [17]. The integrated system comprised an agitation tank for the cultivation of R. glutinis and an autotrophic tank for the growth of C. vulgaris. The flue gas from the tank of R. glutinis was completely directed to the PBR of C. vulgaris, which achieved 1 vvm for the aeration rate in both tanks. A 0.45 μm sterilized air filter was installed in the air connection line between the agitation tank and the autotrophic PBR to avoid contamination of C. vulgaris cultivation. 2.3. Analytical methods An infrared balance (Denver Instrument, IR 35) was adopted to rapidly measure the biomass concentration. Five ml of broth was centrifuged at 70 0 0 rpm for 10 min. After removing the supernatant, about an equal volume of distilled water was added to eliminate impurities. This washing procedure was performed several times, and the final liquor was dried using the infrared balance at 150 °C to evaporate the water content.
Fig. 1. Time course of the percentage of CO2 and biomass growth of R. glutinis in a 5 L agitation fermenter.
The dry biomass was first ground into a fine powder; 0.05 g of the powder was then blended with 5 ml chloroform/methanol (2:1), and subsequently agitated for 20 min at room temperature in an orbital shaker. The solvent phase was recovered by centrifugation at 70 0 0 rpm for 10 min. The same process was repeated twice, and the whole solvent was evaporated and dried under vacuum conditions. The glycerol concentration was measured by HPLC (Agilent series 1100, Agilent Technologies, Santa Clara, CA) with a refractive index detector, while the analysis was performed in a C-18 column (Vercopak N5ODS, 250 mm ×4.6 mm, Taiwan). The mobile phase was composed of 0.01 N H2 SO4 with a flow rate of 0.4 ml/min [18]. All shaker conditions were performed in triplicate to obtain the mean ± standard deviation. 3. Results and discussion 3.1. Time course of CO2 generation in the batch of R. glutinis The cultivation of R. glutinis is a typical aerobic fermentation process, and requires a considerable amount of dissolved oxygen to obtain a large amount of high biomass [19,20]. The use of crude glycerol for the cultivation of R. glutinis was performed in an agitation tank at the aeration rate of 1 vvm. The time courses of the biomass productions, glycerol consumption and CO2 generation are shown in Fig. 1. It can be seen that the maximum biomass was 12.4 g/L after 5 days’ cultivation. The initial glycerol was about 21 g/L, and this decreased to less than 1 g/L after 5 days’ cultivation. The maximum biomass growth rate was about 4.7 g/L day, which led to the maximum CO2 generation percentage of 1.8% observed in the flue gas. During the exponential growth phase, the rapid growth of biomass lead to the maximum CO2 percentage observed in the flue gas. The CO2 generation curve reached its maximum after 1 day cultivation, and fell to a stable level of about 0.5% while the cell growth entered the stationary stage. The results indicated that the CO2 generation was strongly related to the growth of biomass. A higher biomass would produce a higher CO2 percentage in the flue gas. To confirm this, the effects of biomass on CO2 generation were examined by controlling the dissolved oxygen (DO) at high (>30%) and low (<30%) levels. The DO control strategy was achieved by automatically adjusting the agitation rate to provide the required DO level, and the results are shown in Fig. 2. It can be seen that the batch with high DO (denoted as high) produced more biomass than the low DO batch (denoted as low). Besides the higher biomass obtained in the high batch, a higher CO2
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Fig. 2. The comparison of CO2 generation in a 5 L agitation fermenter with high and low biomass R. glutinis cultivation. Fig. 3. The effects of CO2 concentration on the growth of Chlorella vulgaris.
percentage was also observed. Similar to the results found with the low biomass batch, in the one with high biomass the greatest CO2 percentage was found after 24 h’ cultivation. However, no significant decrease in CO2 generation was observed in the batch with a high biomass. The CO2 generation rates were 3.07 and 1.84 mmol/g DCW h for the growth of R. glutinis in the batches with high and low biomass, respectively. As compared to the 11.8–18.6 mmol/g DCW h for the cultivation of E. coli reported in the literature [21], the CO2 secretion rate in the cultivation of yeast was significantly lower. This implies that the growth rate of E. coli should be higher than that of R. glutinis. The CO2 secretion rate in the cultivation of R. glutinis represents a valuable carbon sink, as its stable CO2 generation rate can be used as the carbon source for the growth of microalgae. 3.2. The growth of Chlorella vulgaris under different concentrations of CO2 As shown in the previous section, the CO2 percentage would be about 2.5% of the flue gas in the batch with the biomass concentration of about 20 g/L, with the CO2 being utilized as the carbon source for the cultivation of autotrophic microalgae. Therefore, the effects of the CO2 concentration in the inlet gas on the growth of microalgae (Chlorella vulgaris) were examined. A CO2 concentration in the range of 0.03 (using air) to 60% was used in the cultivation of C. vulgaris, with the results shown in Fig. 3. It can be seen that the CO2 concentration of 2.5%–40% can be regarded as a suitable range for the cultivation of C. vulgaris. However, the low CO2 percentage in the pure air cannot provide enough source for the growth of C. vulgaris and the generation of large amounts of biomass. When the CO2 percentage was over 40% (such as 50 and 60%), this then led to a decrease in the pH and a reduction in the growth of biomass. The results of the current work indicate that 2.5% of CO2 in the flue gas emitted from the tank of R. glutinis can be a good carbon source for C. vulgaris cultivation. To further confirm this, a comparison of using pure air, 2.5% CO2 supplemented in the air and the flue gas emitted from the R. glutinis cultivation tank for the growth of C. vulgaris was performed, with the results shown in Fig. 4. As seen in the figure, the batch using flue gas emitted from the aerobic fermenter had a higher growth rate than that purging with 2.5% of CO2 and the pure air. One possible reason for this is a high CO2 percentage (2.5%–3.0%) provided at the beginning of exponential growth phase of R. glutinis, which might provide more carbon source for the growth of C. vulgaris. Nevertheless, the results indicated that the flue gas emitted from the
Fig. 4. Comparison of Chlorella vulgaris cultivation using 2.5% CO2 and the flue gas from the fermenter of R. glutinis.
cultivation of R. glutinis was a suitable CO2 source that can be used for the growth of C. vulgaris. 3.3. An integrated system of R. glutinis cultivation coupled with C. vulgaris cultivation An integrated system of aerobic R. glutinis fermentation coupled with autotrophic C. vulgaris cultivation was built to examine the feasibility of using the CO2 generated from R. glutinis for the growth of C. vulgaris, with the results shown in Fig. 5. It can be seen that the CO2 generation rapidly goes up to the maximum level after about 18 h’ cultivation in the flue gas of the R. glutinis tank, and the CO2 concentration also reaches its maximum level at the same time. It is thus clear that the growth rate of R. glutinis is faster than that of C. vulgaris. Therefore, the amount of CO2 purging into the tank of microalgae cannot be fixed efficiently at the beginning of C. vulgaris cultivation due to the low biomass density. Nevertheless, as the cultivation time increases more microalgal biomass is produced in the tank, and this can efficiently reduce the CO2 concentration, thus leading to a decrease in the CO2 concentration observed in the flue gas emitted from the microalgae tank. After 20 h’ cultivation, the CO2 concentration was stable at about 2.5 ± 0.1 and 0.5 ± 0.15 in the flue gas emitted from the
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(MOST) under grant numbers 104-2621-M-029 -004 and 104-3113E-0 06-0 03. References
Fig. 5. An integrated system of R. glutinis growth coupled with CO2 -fixation by using photosynthetic cultivation of Chlorella vulgaris.
tanks of R. glutinis and C. vulgaris, respectively. The results indicate that about 80% of the CO2 generated in the cultivation process of R. glutinis can be utilized by C. vulgaris through photosynthesis, which can produce about 20 g/L of R. glutinis and 1.2 g/L of C. vulgaris. In this integrated cultivation system, CO2 generation from the aerobic tank can be assimilated by autotrophic microalgae. Thus, a higher microalgal cells density might provide a higher carbon reduction efficiency. If some high value compounds can be extracted from microalgae (e.g. lutein, ω-3 fatty acid etc.), which the economic benefits can contribute to the overall process and reduce the microbial oils production cost. Besides, the assimilation of CO2 derived from the aerobic yeast by using microalgae can make this microbial oils production a so-called sustainable process, which yield the potential of commercialized production of microbial oils. 4. Conclusion The production of microbial oils using oleaginous R. glutinis as the feedstock for biodiesel is an attractive idea. Nevertheless, a considerable amount of CO2 is generated during the fermentation process, making this approach less environmentally feasible. The results of this study indicate that coupling an autotrophic microalgae PBR with the aerobic fermenter can efficiently reduce 80% of CO2 generated in the flue gas of R. glutinis cultivation. This integrated system proves the production of microbial oils can be a green process, with lower CO2 emissions. Acknowledgments The authors gratefully acknowledge the financial support this study received from Taiwan’s Ministry of Science and Technology
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