- Email: [email protected]
Enhanced astaxanthin accumulation in Haematococcus pluvialis using high carbon dioxide concentration and light illumination
Accepted Manuscript Short Communication Enhanced Astaxanthin Accumulation in Haematococcus pluvialis Using High Carbon Dioxide Concentration and Light Illumination David Christian, Jun Zhang, Alicia J. Sawdon, Ching-An Peng PII: DOI: Reference:
S0960-8524(18)30271-2 https://doi.org/10.1016/j.biortech.2018.02.074 BITE 19584
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
20 December 2017 14 February 2018 16 February 2018
Please cite this article as: Christian, D., Zhang, J., Sawdon, A.J., Peng, C-A., Enhanced Astaxanthin Accumulation in Haematococcus pluvialis Using High Carbon Dioxide Concentration and Light Illumination, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.02.074
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Enhanced Astaxanthin Accumulation in Haematococcus pluvialis Using High Carbon Dioxide Concentration and Light Illumination
David Christian1,†, Jun Zhang1,†, Alicia J. Sawdon2 and Ching-An Peng1,* 1
Department of Biological Engineering, University of Idaho, Moscow, ID 83844 2
Department of Chemical Engineering, Michigan Technological University, Houghton, MI 49931
†
*
Equal contribution
Corresponding Author Ching-An Peng
Department of Biological Engineering Engineering Physics Building 421 875 Perimeter Drive MS 0904 Moscow, ID 83844-0904 Phone: 208-885-7461 E-mail: [email protected]
Abstract In this study, an economical two-stage method was proposed for the production of natural astaxanthin from Haematococcus pluvialis without a medium replacement step. In stage 1, H. pluvialis were grown under low light illumination until they reached optimal biomass. In stage 2, cells were switched to astaxanthin induction conditions utilizing the combination of high light illumination and elevated carbon dioxide levels (5 or 15%). The introduction of CO2 altered the C/N balance creating a nutrient deficiency without a change of media. The resulting astaxanthin yield was 2-3 times that of using either stressor alone. This astaxanthin induction method has many advantages over current methods including no medium replacement and a short induction time of less than four days. Keywords: Haematococcus pluvialis, Astaxanthin, Algal Biomass, Carbon Dioxide, Stress Induction
1. Introduction Astaxanthin is a high-value carotenoid pigment with applications in the nutraceutical, pharmaceutical and food industries (Guerin et al., 2003; Lorenz and Cysewski, 2000). While the production of synthetic astaxanthin is relatively inexpensive, its petroleum nature raises issues of food safety and pollution (Panis and Rosales Carreon, 2016), and is only approved for non-human use (Lorenz and Cysewski, 2000; Shah et al., 2016). Recently, some research projects aimed at developing the means to mass produce natural astaxanthin at a substantially lower cost than is currently possible (Panis and Rosales Carreon, 2016). Algae currently 1
account for less than 1% of commercial astaxanthin production. The major obstacles are the high cost of production and CO2 delivery (Zheng et al., 2016). The microalga, Haematococcus pluvialis, is one of the richest sources of natural astaxanthin (Lorenz and Cysewski, 2000), which naturally accumulates in H. pluvialis in response to adverse environmental conditions as part of the algae's survival strategy. The most common stressors used to trigger astaxanthin accumulation are nutrient starvation, high salinity, high temperature and high light intensity (Fabregas et al., 2003; He et al., 2007). In most H. pluvialis cultivation systems, this typically takes place in a two-stage process: first amassing suitable biomass, and then inducing astaxanthin accumulation. One method of achieving relative nitrogen starvation is altering the carbon/nitrogen (C/N) ratio by increasing carbon in the system as opposed to replacement with nitrogen deficient media (Orosa et al., 2001). Carbon dioxide (CO2) is a commonly used carbon source for aeration of H. pluvialis cultures with concentrations ranging from 1-5% (Kaewpintong et al., 2007). Cheng et al., (2016) observed the highest biomass production and astaxanthin induction with 6% CO 2 and high light intensity among the CO2 concentrations tested. Further increasing CO2 to 10% or 20% would lead to a decrease in cell growth (Chekanov et al., 2017). Recently, 15% CO2-tolerant H. pluvialis mutants have been developed to increase biomass dry weight as well as astaxanthin yield (Li et al., 2017). To this end, an alternative strategy is proposed to test the effect of high light illumination and elevated CO2 concentrations on astaxanthin induction in H. 2
pluvialis cells. It is hypothesized that the infusion of additional carbon into the culture system will shift the C/N balance and create a relative nutrient deficiency which will enhance astaxanthin accumulation without a separation step and change of media between biomass accumulation and astaxanthin induction. This two-step process allows the algal cells to grow to late exponential growth phase at low light intensity, then astaxanthin production is induced by introducing high levels of CO2 with a high light intensity. This novel two-step process does not require a change of culture medium, thus simplifying and shortening previously used induction methods. 2. Materials and Methods 2.1 Strain and cultivation conditions The microalgae H. pluvialis (strain 2505, UTEX) was maintained at room temperature in MES-Volvox culture medium by sub-culturing under continuous illumination of 80 µmol m-2s-1. The light was provided by cool white full spectrum fluorescent bulbs as measured by a light meter (Fisher Scientific, Hampton, NH). 2.2 Experimental conditions and astaxanthin content determination A H. pluvialis culture was grown to near the end of the log phase, approximately 1x106 cells/mL, and then moved to stress conditions. Induction experiments were done with 30 mL H. pluvialis cultures in 50 mL flasks. There was no change of media or effort to control pH during the experiment. The flasks were placed at two distances from a light source (20 W compact fluorescent bulb with 1300 lumens), providing the cells with low light (80 µmol m-2s-1) and high light intensities (300 µmol m-2s-1). Sterilized tubing was inserted into the flasks to 3
provide the cells with either air (0.04% CO2), 5% or 15% CO2 balanced with air, with a mean flow rate of ~7 mL/min. Two controls were also run under low and high light intensity conditions without the addition of air or CO2. During the 4-day induction period, algal cell numbers were counted daily to determine the growth kinetics. pH of the culture media was measured and cells from each flask were imaged by a light microscope. After 4 days, cells were collected and freeze-dried to determine the final biomass dry weight. According to the report by Boussiba et al. (1992), the dried cell pellet was resuspended in 5 mL of 5% (w/v) KOH in 30% methanol and held at 70oC for 5 min to remove chlorophyll. Cells were centrifuged down and resuspended in 10 mL DMSO and bath sonicated for 2 min three times with intervals of 30 sec. The cells were then repeatedly heated in a 70oC water bath for 5 min to recover astaxanthin until cell debris was almost colorless. The absorbance of the extract was determined with
= 490 nm
on a SpectraMax M2e Microplate Reader (Molecular Devices, Sunnyvale, CA). A standard curve for astaxanthin concentration (mg/mL) was generated using 1 mg of astaxanthin (Sigma-Aldrich, St. Louis, MO) serially diluted with 1 mL of DMSO. Astaxanthin concentration was multiplied by the sample volume then divided by the dry cell mass to determine the astaxanthin yield (mg/g). 3. Results and discussion Microalgae, H. pluvialis, was grown to the late log phase with a density of 106 cells/mL in ~8 days, and then split into individual flasks for exposure to different induction conditions. Within one day of being shifted to stress conditions, divergent 4
responses were apparent. The cultures exposed to 5 or 15% CO2 and high light intensity had begun to turn to orange color, with the cells becoming less active and settling to the bottom of the flask (Fig. S1). By day two, these same cultures were already red in color, indicating the accumulation of astaxanthin. This is consistent with what Li et al., (2017) observed with a CO2 tolerant mutant. They reported cellular changes in stress induced cells at 6 h, and astaxanthin accumulation beginning by 24 h. The decrease in activity and the settling of cells were expected when the population shifted from predominantly flagellated motile cells to palmella and aplanospores. This shift in the population was also reflected in decreased cell counts during the 4-day induction period (Fig. 1). The above changes were also observed in the cultures exposed to high levels of CO2 and low light intensity as early as day one, although these cultures progressed towards red senescence more slowly, not becoming orange or red until three or four days after induction. The macroscopic observations of the cultures under different induction conditions were further confirmed by light microscopy. The size of green motile cells was ~10 m. After reddening of the cells, the size increased to up to ~50 m after 4 days of infusing 5% or 15% CO2 (Fig. S2). Introducing CO2 led to a drop in pH, from ~7 to ~6.2 when given 15% CO2, and to ~6.7 when infused with 5% CO2 (Table S1). Whether or not the change in pH alone could account for astaxanthin accumulation or whether the effect was due to CO2 itself was tested. The pH of H. pluvialis cultures were adjusted to several pHs ranging from 5 to 8 using HCl and KOH, and these cultures were 5
exposed to both high and low light intensities. No apparent color change or astaxanthin accumulation was observed in any of the cultures exposed to either low or high light intensities in 5 days. The cells remained green and motile. The non-stressed controls (with no air or CO2 introduced) exhibited a similar response to that of the cultures exposed to bubbled air at low and high light intensity (Table 1). This would indicate that the bubbling of gases was not an additional stress factor. The cultures exposed to high light intensity, whether given air or static, began to change color around day three or four, at which point light stress may have begun to become a factor. Biomass accumulation was greater when the cultures were infused with CO2 (Table 1, Fig. 2). The carbon added to the system stimulated biomass accumulation by at least 2X when exposed to low light conditions and 3.5X with high light exposure. There was no significant difference in biomass accumulation between the cultures receiving 5% or 15% CO2, although there was a noticeable increase with the increased light intensity (Fig. 2). Chekanov et al., (2017) likewise observed biomass stimulation when introducing 5% and 10% CO2, although this entailed an increase in zoospores with 5% CO2, but a decrease at 10%. The most critical difference observed was in the amount of astaxanthin accumulation. The controls and the cultures receiving infused air all accumulated a small amount of astaxanthin, ~9 mg/g dry weight (Table 1). The cultures infused with CO2 and exposed to low light intensity accumulated more, ~22 mg/g, approximately twice that of the control and air infused cultures. The cultures 6
infused with CO2 and exposed to high light intensity accumulated somewhat more, up to 36 mg/g, which is about four times that of the control. Kang et al. (2005) reported 18 mg/g when infused with air, and ~80 mg/g with 5% CO2. The 20 mg/g astaxanthin yield they reported with 10% CO2 is comparable to that observed here. Cheng et al., (2016) reported 9 mg/L with air (compared to our 9 mg/g (3 mg/L)) and 20 mg/L with 6% CO2 (33 mg/g (90 mg/L) herein). Shah et al., (2016) reported astaxanthin accumulation ranging from 3 to 5% of total cell weight. The 3% astaxanthin accumulation reported here is at the low end of this range. However, most of these reported induction experiments were conducted over two weeks or longer (Table S2). The amount of astaxanthin accumulation inferred from these studies after 4 days of induction was comparable with that reported here. Comparisons are difficult due to differences in the H. pluvialis strain used as well as the choice of media for experiments. The accumulation of inhibitory compounds in batch culturing must also be taken into consideration (Park et al., 2014). The combination of stressors in this system had greater effect than either stressor alone (Fig. 2). Increasing light intensity without the addition of carbon to the system had little ability to induce astaxanthin production. The introduction of CO2 doubled the amount of astaxanthin generated at a normal light level. Increasing the light intensity along with CO2 boosted the astaxanthin level even higher. There was little difference in the amount of astaxanthin produced whether the cultures received 5% or 15% CO2. Similar results were observed in the effects on biomass production. This is understandable, as it is well-known that astaxanthin 7
plays a role in protecting the algae's photosynthetic processes from light induced stresses, specifically, photo-oxidative stress (Chekanov et al., 2016; Imamoglu et al., 2009; Kobayashi, 2000). The potential of eliminating the separation step and change out of media could reduce the time and energy involved in natural astaxanthin production. By growing cells until the late log phase and allowing them to deplete nutrients naturally, then shifting the C/N balance by the introduction of CO2, could be an economical and efficient approach to induce astaxanthin accumulation in H. pluvialis culture systems. 4. Conclusion A process which bypasses a separation and media change step between the biomass accumulation and induction stages would greatly improve the efficiency and economics of astaxanthin production. The induction of astaxanthin in H. pluvialis was accomplished by high light illumination and elevated CO 2 conditions, without change-out of media. The culture of H. pluvialis infused with 15% CO2 balanced with air and high light illumination induced up to 36 mg/g astaxanthin accumulation. The potential use of this simplified induction process in industrial applications warrants further investigation.
Appendix A. Supplementary data Supplementary data can be found in online version of the paper at xxxxx.
8
References 1. Boussiba, S., Fan, L., Vonshak, A. 1992. Enhancement and determination of astaxanthin accumulation in green alga Haematococcus pluvialis. in: Methods in Enzymology, (Ed.) P. Lester, Vol. Volume 213, Academic Press, pp. 386-391. 2. Chekanov, K., Lukyanov, A., Boussiba, S., Aflalo, C., Solovchenko, A., 2016. Modulation of photosynthetic activity and photoprotection in Haematococcus pluvialis cells during their conversion into haematocysts and back. Photosynth. Res., 128, 313-23. 3. Chekanov, K., Schastnaya, E., Solovchenko, A., Lobakova, E., 2017. Effects of CO2 enrichment on primary photochemistry, growth and astaxanthin accumulation in the chlorophyte Haematococcus pluvialis. J Photochem Photobiol B, 171, 58-66. 4. Cheng, J., Li, K., Yang, Z., Zhou, J., Cen, K., 2016. Enhancing the growth rate and astaxanthin yield of Haematococcus pluvialis by nuclear irradiation and high concentration of carbon dioxide stress. Bioresour. Technol., 204, 49-54. 5. Fabregas, J., Dominguez, A., Maseda, A., Otero, A., 2003. Interactions between irradiance and nutrient availability during astaxanthin accumulation and degradation in Haematococcus pluvialis. Appl. Microbiol. Biotechnol., 61, 545-51.
9
6. He, P., Duncan, J., Barber, J., 2007. Astaxanthin Accumulation in the Green Alga Haematococcus pluvialis: Effects of Cultivation Parameters. J. Integr. Plant. Biol., 49, 447-451. 7. Imamoglu, E., Dalay, M.C., Sukan, F.V., 2009. Influences of different stress media and high light intensities on accumulation of astaxanthin in the green alga Haematococcus pluvialis. N. Biotechnol., 26, 199-204. 8. Kaewpintong, K., Shotipruk, A., Powtongsook, S., Pavasant, P., 2007. Photoautotrophic high-density cultivation of vegetative cells of Haematococcus pluvialis in airlift bioreactor. Bioresour. Technol., 98, 28895. 9. Kang, C.D., Lee, J.S., Park, T.H., Sim, S.J. 2005. Comparison of heterotrophic and photoautotrophic induction on astaxanthin production by Haematococcus pluvialis. Appl. Microbiol. Biotechnol., 68, 237-241. 10. Kobayashi, M., 2000. In vivo antioxidant role of astaxanthin under oxidative stress in the green alga Haematococcus pluvialis. Appl. Microbiol. Biotechnol., 54, 550-555. 11. Li, K., Cheng, J., Ye, Q., He, Y., Zhou, J., Cen, K., 2017. In vivo kinetics of lipids and astaxanthin evolution in Haematococcus pluvialis mutant under 15% CO2 using Raman microspectroscopy. Bioresour Technol, 244, 14391444.
10
12. Lorenz, R.T., Cysewski, G.R., 2000. Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends Biotechnol., 18, 160-167. 13. Orosa, M., Franqueira, D., Cid, A., Abalde, J., 2001. Carotenoid accumulation in Haematococcus pluvialis in mixotrophic growth. Biotechnol Lett., 23, 373-378. 14. Panis, G., Rosales Carreon, J., 2016. Commercial astaxanthin production derived by green alga Haematococcus pluvialis: A microalgae process model and a techno-economic assessment all through production line. Algal Res., 18, 175-190. 15. Park, J.C., Choi, S.P., Hong, M.E., Sim, S.J., 2014. Enhanced astaxanthin production from microalga, Haematococcus pluvialis by two-stage perfusion culture with stepwise light irradiation. Bioprocess Biosyst. Eng., 37, 20392047. 16. Shah, M.M., Liang, Y., Cheng, J.J., Daroch, M., 2016. AstaxanthinProducing Green Microalga Haematococcus pluvialis: From Single Cell to High Value Commercial Products. Front Plant Sci., 7, 531. 17. Zheng, Q., Martin, G.J.O., Kentish, S.E., 2016. Energy efficient transfer of carbon dioxide from flue gases to microalgal systems. Energy Environ. Sci., 9, 1074-1082.
11
Figure Legends Fig. 1. Cell growth kinetics on a logarithmic scale of H. pluvialis grown in MES Volvox media at room temperature and with 80 µmol m-2s-1 (low light) continuous illumination and following shift to stress conditions at day 8. The cultures were then exposed to elevated CO2 and exposure to 300 µmol m-2s-1 (high light) for 4 days. The control was maintained under low light intensity without the addition of air or CO2. Fig. 2. Normalized H. pluvialis biomass (mg dry wt; ■) and astaxanthin yield (mg/g dry wt;
) after 4-day induction with exposure to 300 µmol m-2s-1 (high light) or 80
µmol m-2s-1 (low light) illumination, and air, 15% or 5% CO2. Biomass and astaxanthin data was normalized to the low light control in each experimental run and averaged over 2 – 3 experiment repetitions. All experiments were performed with triplicate samples.
12
Table 1. H. pluvialis biomass and astaxanthin accumulation after four days of induction
Induction Condition
Biomass (mg/L)
control low light
246±50
9.89±0.90
air low light
222±19
8.87±2.69
air high light
200±33
9.27±1.00
5% CO2 low light
607±78
22.86±2.20
5% CO2 high light
945±146
32.74±1.89
15% CO2 low light
517±47
21.68±5.77
15% CO2 high light
878±62
36.23±5.48
13
Astaxanthin Yield (mg/g)
Fig. 1
14
Fig. 2
15
Highlights
Astaxanthin production in H. pluvialis induced by high light intensity and 15% CO2
Effective astaxanthin induction achieved without using nutrient deficient media
The reported astaxanthin production approach has economical and efficient merits
Graphical Abstract
16
S0960-8524(18)30271-2 https://doi.org/10.1016/j.biortech.2018.02.074 BITE 19584
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
20 December 2017 14 February 2018 16 February 2018
Please cite this article as: Christian, D., Zhang, J., Sawdon, A.J., Peng, C-A., Enhanced Astaxanthin Accumulation in Haematococcus pluvialis Using High Carbon Dioxide Concentration and Light Illumination, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.02.074
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Enhanced Astaxanthin Accumulation in Haematococcus pluvialis Using High Carbon Dioxide Concentration and Light Illumination
David Christian1,†, Jun Zhang1,†, Alicia J. Sawdon2 and Ching-An Peng1,* 1
Department of Biological Engineering, University of Idaho, Moscow, ID 83844 2
Department of Chemical Engineering, Michigan Technological University, Houghton, MI 49931
†
*
Equal contribution
Corresponding Author Ching-An Peng
Department of Biological Engineering Engineering Physics Building 421 875 Perimeter Drive MS 0904 Moscow, ID 83844-0904 Phone: 208-885-7461 E-mail: [email protected]
Abstract In this study, an economical two-stage method was proposed for the production of natural astaxanthin from Haematococcus pluvialis without a medium replacement step. In stage 1, H. pluvialis were grown under low light illumination until they reached optimal biomass. In stage 2, cells were switched to astaxanthin induction conditions utilizing the combination of high light illumination and elevated carbon dioxide levels (5 or 15%). The introduction of CO2 altered the C/N balance creating a nutrient deficiency without a change of media. The resulting astaxanthin yield was 2-3 times that of using either stressor alone. This astaxanthin induction method has many advantages over current methods including no medium replacement and a short induction time of less than four days. Keywords: Haematococcus pluvialis, Astaxanthin, Algal Biomass, Carbon Dioxide, Stress Induction
1. Introduction Astaxanthin is a high-value carotenoid pigment with applications in the nutraceutical, pharmaceutical and food industries (Guerin et al., 2003; Lorenz and Cysewski, 2000). While the production of synthetic astaxanthin is relatively inexpensive, its petroleum nature raises issues of food safety and pollution (Panis and Rosales Carreon, 2016), and is only approved for non-human use (Lorenz and Cysewski, 2000; Shah et al., 2016). Recently, some research projects aimed at developing the means to mass produce natural astaxanthin at a substantially lower cost than is currently possible (Panis and Rosales Carreon, 2016). Algae currently 1
account for less than 1% of commercial astaxanthin production. The major obstacles are the high cost of production and CO2 delivery (Zheng et al., 2016). The microalga, Haematococcus pluvialis, is one of the richest sources of natural astaxanthin (Lorenz and Cysewski, 2000), which naturally accumulates in H. pluvialis in response to adverse environmental conditions as part of the algae's survival strategy. The most common stressors used to trigger astaxanthin accumulation are nutrient starvation, high salinity, high temperature and high light intensity (Fabregas et al., 2003; He et al., 2007). In most H. pluvialis cultivation systems, this typically takes place in a two-stage process: first amassing suitable biomass, and then inducing astaxanthin accumulation. One method of achieving relative nitrogen starvation is altering the carbon/nitrogen (C/N) ratio by increasing carbon in the system as opposed to replacement with nitrogen deficient media (Orosa et al., 2001). Carbon dioxide (CO2) is a commonly used carbon source for aeration of H. pluvialis cultures with concentrations ranging from 1-5% (Kaewpintong et al., 2007). Cheng et al., (2016) observed the highest biomass production and astaxanthin induction with 6% CO 2 and high light intensity among the CO2 concentrations tested. Further increasing CO2 to 10% or 20% would lead to a decrease in cell growth (Chekanov et al., 2017). Recently, 15% CO2-tolerant H. pluvialis mutants have been developed to increase biomass dry weight as well as astaxanthin yield (Li et al., 2017). To this end, an alternative strategy is proposed to test the effect of high light illumination and elevated CO2 concentrations on astaxanthin induction in H. 2
pluvialis cells. It is hypothesized that the infusion of additional carbon into the culture system will shift the C/N balance and create a relative nutrient deficiency which will enhance astaxanthin accumulation without a separation step and change of media between biomass accumulation and astaxanthin induction. This two-step process allows the algal cells to grow to late exponential growth phase at low light intensity, then astaxanthin production is induced by introducing high levels of CO2 with a high light intensity. This novel two-step process does not require a change of culture medium, thus simplifying and shortening previously used induction methods. 2. Materials and Methods 2.1 Strain and cultivation conditions The microalgae H. pluvialis (strain 2505, UTEX) was maintained at room temperature in MES-Volvox culture medium by sub-culturing under continuous illumination of 80 µmol m-2s-1. The light was provided by cool white full spectrum fluorescent bulbs as measured by a light meter (Fisher Scientific, Hampton, NH). 2.2 Experimental conditions and astaxanthin content determination A H. pluvialis culture was grown to near the end of the log phase, approximately 1x106 cells/mL, and then moved to stress conditions. Induction experiments were done with 30 mL H. pluvialis cultures in 50 mL flasks. There was no change of media or effort to control pH during the experiment. The flasks were placed at two distances from a light source (20 W compact fluorescent bulb with 1300 lumens), providing the cells with low light (80 µmol m-2s-1) and high light intensities (300 µmol m-2s-1). Sterilized tubing was inserted into the flasks to 3
provide the cells with either air (0.04% CO2), 5% or 15% CO2 balanced with air, with a mean flow rate of ~7 mL/min. Two controls were also run under low and high light intensity conditions without the addition of air or CO2. During the 4-day induction period, algal cell numbers were counted daily to determine the growth kinetics. pH of the culture media was measured and cells from each flask were imaged by a light microscope. After 4 days, cells were collected and freeze-dried to determine the final biomass dry weight. According to the report by Boussiba et al. (1992), the dried cell pellet was resuspended in 5 mL of 5% (w/v) KOH in 30% methanol and held at 70oC for 5 min to remove chlorophyll. Cells were centrifuged down and resuspended in 10 mL DMSO and bath sonicated for 2 min three times with intervals of 30 sec. The cells were then repeatedly heated in a 70oC water bath for 5 min to recover astaxanthin until cell debris was almost colorless. The absorbance of the extract was determined with
= 490 nm
on a SpectraMax M2e Microplate Reader (Molecular Devices, Sunnyvale, CA). A standard curve for astaxanthin concentration (mg/mL) was generated using 1 mg of astaxanthin (Sigma-Aldrich, St. Louis, MO) serially diluted with 1 mL of DMSO. Astaxanthin concentration was multiplied by the sample volume then divided by the dry cell mass to determine the astaxanthin yield (mg/g). 3. Results and discussion Microalgae, H. pluvialis, was grown to the late log phase with a density of 106 cells/mL in ~8 days, and then split into individual flasks for exposure to different induction conditions. Within one day of being shifted to stress conditions, divergent 4
responses were apparent. The cultures exposed to 5 or 15% CO2 and high light intensity had begun to turn to orange color, with the cells becoming less active and settling to the bottom of the flask (Fig. S1). By day two, these same cultures were already red in color, indicating the accumulation of astaxanthin. This is consistent with what Li et al., (2017) observed with a CO2 tolerant mutant. They reported cellular changes in stress induced cells at 6 h, and astaxanthin accumulation beginning by 24 h. The decrease in activity and the settling of cells were expected when the population shifted from predominantly flagellated motile cells to palmella and aplanospores. This shift in the population was also reflected in decreased cell counts during the 4-day induction period (Fig. 1). The above changes were also observed in the cultures exposed to high levels of CO2 and low light intensity as early as day one, although these cultures progressed towards red senescence more slowly, not becoming orange or red until three or four days after induction. The macroscopic observations of the cultures under different induction conditions were further confirmed by light microscopy. The size of green motile cells was ~10 m. After reddening of the cells, the size increased to up to ~50 m after 4 days of infusing 5% or 15% CO2 (Fig. S2). Introducing CO2 led to a drop in pH, from ~7 to ~6.2 when given 15% CO2, and to ~6.7 when infused with 5% CO2 (Table S1). Whether or not the change in pH alone could account for astaxanthin accumulation or whether the effect was due to CO2 itself was tested. The pH of H. pluvialis cultures were adjusted to several pHs ranging from 5 to 8 using HCl and KOH, and these cultures were 5
exposed to both high and low light intensities. No apparent color change or astaxanthin accumulation was observed in any of the cultures exposed to either low or high light intensities in 5 days. The cells remained green and motile. The non-stressed controls (with no air or CO2 introduced) exhibited a similar response to that of the cultures exposed to bubbled air at low and high light intensity (Table 1). This would indicate that the bubbling of gases was not an additional stress factor. The cultures exposed to high light intensity, whether given air or static, began to change color around day three or four, at which point light stress may have begun to become a factor. Biomass accumulation was greater when the cultures were infused with CO2 (Table 1, Fig. 2). The carbon added to the system stimulated biomass accumulation by at least 2X when exposed to low light conditions and 3.5X with high light exposure. There was no significant difference in biomass accumulation between the cultures receiving 5% or 15% CO2, although there was a noticeable increase with the increased light intensity (Fig. 2). Chekanov et al., (2017) likewise observed biomass stimulation when introducing 5% and 10% CO2, although this entailed an increase in zoospores with 5% CO2, but a decrease at 10%. The most critical difference observed was in the amount of astaxanthin accumulation. The controls and the cultures receiving infused air all accumulated a small amount of astaxanthin, ~9 mg/g dry weight (Table 1). The cultures infused with CO2 and exposed to low light intensity accumulated more, ~22 mg/g, approximately twice that of the control and air infused cultures. The cultures 6
infused with CO2 and exposed to high light intensity accumulated somewhat more, up to 36 mg/g, which is about four times that of the control. Kang et al. (2005) reported 18 mg/g when infused with air, and ~80 mg/g with 5% CO2. The 20 mg/g astaxanthin yield they reported with 10% CO2 is comparable to that observed here. Cheng et al., (2016) reported 9 mg/L with air (compared to our 9 mg/g (3 mg/L)) and 20 mg/L with 6% CO2 (33 mg/g (90 mg/L) herein). Shah et al., (2016) reported astaxanthin accumulation ranging from 3 to 5% of total cell weight. The 3% astaxanthin accumulation reported here is at the low end of this range. However, most of these reported induction experiments were conducted over two weeks or longer (Table S2). The amount of astaxanthin accumulation inferred from these studies after 4 days of induction was comparable with that reported here. Comparisons are difficult due to differences in the H. pluvialis strain used as well as the choice of media for experiments. The accumulation of inhibitory compounds in batch culturing must also be taken into consideration (Park et al., 2014). The combination of stressors in this system had greater effect than either stressor alone (Fig. 2). Increasing light intensity without the addition of carbon to the system had little ability to induce astaxanthin production. The introduction of CO2 doubled the amount of astaxanthin generated at a normal light level. Increasing the light intensity along with CO2 boosted the astaxanthin level even higher. There was little difference in the amount of astaxanthin produced whether the cultures received 5% or 15% CO2. Similar results were observed in the effects on biomass production. This is understandable, as it is well-known that astaxanthin 7
plays a role in protecting the algae's photosynthetic processes from light induced stresses, specifically, photo-oxidative stress (Chekanov et al., 2016; Imamoglu et al., 2009; Kobayashi, 2000). The potential of eliminating the separation step and change out of media could reduce the time and energy involved in natural astaxanthin production. By growing cells until the late log phase and allowing them to deplete nutrients naturally, then shifting the C/N balance by the introduction of CO2, could be an economical and efficient approach to induce astaxanthin accumulation in H. pluvialis culture systems. 4. Conclusion A process which bypasses a separation and media change step between the biomass accumulation and induction stages would greatly improve the efficiency and economics of astaxanthin production. The induction of astaxanthin in H. pluvialis was accomplished by high light illumination and elevated CO 2 conditions, without change-out of media. The culture of H. pluvialis infused with 15% CO2 balanced with air and high light illumination induced up to 36 mg/g astaxanthin accumulation. The potential use of this simplified induction process in industrial applications warrants further investigation.
Appendix A. Supplementary data Supplementary data can be found in online version of the paper at xxxxx.
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References 1. Boussiba, S., Fan, L., Vonshak, A. 1992. Enhancement and determination of astaxanthin accumulation in green alga Haematococcus pluvialis. in: Methods in Enzymology, (Ed.) P. Lester, Vol. Volume 213, Academic Press, pp. 386-391. 2. Chekanov, K., Lukyanov, A., Boussiba, S., Aflalo, C., Solovchenko, A., 2016. Modulation of photosynthetic activity and photoprotection in Haematococcus pluvialis cells during their conversion into haematocysts and back. Photosynth. Res., 128, 313-23. 3. Chekanov, K., Schastnaya, E., Solovchenko, A., Lobakova, E., 2017. Effects of CO2 enrichment on primary photochemistry, growth and astaxanthin accumulation in the chlorophyte Haematococcus pluvialis. J Photochem Photobiol B, 171, 58-66. 4. Cheng, J., Li, K., Yang, Z., Zhou, J., Cen, K., 2016. Enhancing the growth rate and astaxanthin yield of Haematococcus pluvialis by nuclear irradiation and high concentration of carbon dioxide stress. Bioresour. Technol., 204, 49-54. 5. Fabregas, J., Dominguez, A., Maseda, A., Otero, A., 2003. Interactions between irradiance and nutrient availability during astaxanthin accumulation and degradation in Haematococcus pluvialis. Appl. Microbiol. Biotechnol., 61, 545-51.
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6. He, P., Duncan, J., Barber, J., 2007. Astaxanthin Accumulation in the Green Alga Haematococcus pluvialis: Effects of Cultivation Parameters. J. Integr. Plant. Biol., 49, 447-451. 7. Imamoglu, E., Dalay, M.C., Sukan, F.V., 2009. Influences of different stress media and high light intensities on accumulation of astaxanthin in the green alga Haematococcus pluvialis. N. Biotechnol., 26, 199-204. 8. Kaewpintong, K., Shotipruk, A., Powtongsook, S., Pavasant, P., 2007. Photoautotrophic high-density cultivation of vegetative cells of Haematococcus pluvialis in airlift bioreactor. Bioresour. Technol., 98, 28895. 9. Kang, C.D., Lee, J.S., Park, T.H., Sim, S.J. 2005. Comparison of heterotrophic and photoautotrophic induction on astaxanthin production by Haematococcus pluvialis. Appl. Microbiol. Biotechnol., 68, 237-241. 10. Kobayashi, M., 2000. In vivo antioxidant role of astaxanthin under oxidative stress in the green alga Haematococcus pluvialis. Appl. Microbiol. Biotechnol., 54, 550-555. 11. Li, K., Cheng, J., Ye, Q., He, Y., Zhou, J., Cen, K., 2017. In vivo kinetics of lipids and astaxanthin evolution in Haematococcus pluvialis mutant under 15% CO2 using Raman microspectroscopy. Bioresour Technol, 244, 14391444.
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12. Lorenz, R.T., Cysewski, G.R., 2000. Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends Biotechnol., 18, 160-167. 13. Orosa, M., Franqueira, D., Cid, A., Abalde, J., 2001. Carotenoid accumulation in Haematococcus pluvialis in mixotrophic growth. Biotechnol Lett., 23, 373-378. 14. Panis, G., Rosales Carreon, J., 2016. Commercial astaxanthin production derived by green alga Haematococcus pluvialis: A microalgae process model and a techno-economic assessment all through production line. Algal Res., 18, 175-190. 15. Park, J.C., Choi, S.P., Hong, M.E., Sim, S.J., 2014. Enhanced astaxanthin production from microalga, Haematococcus pluvialis by two-stage perfusion culture with stepwise light irradiation. Bioprocess Biosyst. Eng., 37, 20392047. 16. Shah, M.M., Liang, Y., Cheng, J.J., Daroch, M., 2016. AstaxanthinProducing Green Microalga Haematococcus pluvialis: From Single Cell to High Value Commercial Products. Front Plant Sci., 7, 531. 17. Zheng, Q., Martin, G.J.O., Kentish, S.E., 2016. Energy efficient transfer of carbon dioxide from flue gases to microalgal systems. Energy Environ. Sci., 9, 1074-1082.
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Figure Legends Fig. 1. Cell growth kinetics on a logarithmic scale of H. pluvialis grown in MES Volvox media at room temperature and with 80 µmol m-2s-1 (low light) continuous illumination and following shift to stress conditions at day 8. The cultures were then exposed to elevated CO2 and exposure to 300 µmol m-2s-1 (high light) for 4 days. The control was maintained under low light intensity without the addition of air or CO2. Fig. 2. Normalized H. pluvialis biomass (mg dry wt; ■) and astaxanthin yield (mg/g dry wt;
) after 4-day induction with exposure to 300 µmol m-2s-1 (high light) or 80
µmol m-2s-1 (low light) illumination, and air, 15% or 5% CO2. Biomass and astaxanthin data was normalized to the low light control in each experimental run and averaged over 2 – 3 experiment repetitions. All experiments were performed with triplicate samples.
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Table 1. H. pluvialis biomass and astaxanthin accumulation after four days of induction
Induction Condition
Biomass (mg/L)
control low light
246±50
9.89±0.90
air low light
222±19
8.87±2.69
air high light
200±33
9.27±1.00
5% CO2 low light
607±78
22.86±2.20
5% CO2 high light
945±146
32.74±1.89
15% CO2 low light
517±47
21.68±5.77
15% CO2 high light
878±62
36.23±5.48
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Astaxanthin Yield (mg/g)
Fig. 1
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Fig. 2
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Highlights
Astaxanthin production in H. pluvialis induced by high light intensity and 15% CO2
Effective astaxanthin induction achieved without using nutrient deficient media
The reported astaxanthin production approach has economical and efficient merits
Graphical Abstract
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