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Effect of superficial gas velocity on growth of the green microalga Haematococcus pluvialis in airlift photobioreactor
159 Studies in Surface Science and Catalysis, volume 159 Hyun-Ku Rhee, In-Sik Nam and Jong Moon Park (Editors) All rights reserved reserved © 2006 Elsevier B.V. All
481 481
Effect of superficial gas velocity on growth of the green microalga Haematococcus pluvialis in airlift photobioreactor Sorawit Powtongsook ''2 Kamonpan Kaewpintong3 Artiwan Shotipruk3'* and Prasert Pavasant3 'National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathum Thani 12120, THAILAND 2
Center of Excellence for Marine Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, THAILAND department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330, THAILAND *e-mail:[email protected] ABSTRACT The green microalga Haematococcus pluvialis NIES144 was cultured in a 3 L airlift photobioreactor at 27+l°C and light intensity of 1,000 lux. The effect of different superficial gas velocities on growth of//, pluvialis was evaluated within the range between 0.4 and 3 cm-s"'. It was found that growth of//, pluvialis was strongly affected by the superficial gas velocity. The maximum cell density and specific growth rate were obtained in the culture with the superficial gas velocity of 0.4 cm-s"1. These values are 77 xlO4 cells mL"1 (2.79 g L"1 dry weight) and 0.45 day"1, respectively. Higher levels of gas velocity did not show any benefits, and in fact they were found to drastically slow down the growth, due to the increased shear stress. Further more an increase in superficial gas velocity could significantly change the cell morphology from motile vegetative cells to non-motile green cells or cysts. 1. INTRODUCTION Astaxanthin (or 3'3-dihydroxy-P,P'-carotene-4,4'-dione) is an important pigmentation source in aquaculture that has been shown to possess higher antioxidant activity than any other carotenoids [1]. The freshwater alga Haematococcus pluvialis is considered the most capable microorganism that produces the largest amount of astaxanthin compared to other astaxanthin producing microorganisms such as yeast and bacteria [2]. For this reason, the production of astaxanthin from H. pluvialis has gained a great deal of attention. Typical microalgal cultivation can usually be divided into open and close systems. A large-scale algal culture is usually an open tank or pond which is easy to operate and consumes less resource than a close system. Nevertheless, the difficulty in cultivation of H. pluvialis is its slow growth. The requirement for low growth temperature and the susceptibility to contamination also make it difficult to obtain optimum growth when cultivated in an open system [3]. As a result, cultivation in a closed system such as in a photobioreactor is required to achieve high cell densities. A variety of bioreactors including both mechanically and nonmechanically agitated systems have been used. However, mechanically stirred bioreactors are not suitable for the cultivation of//, pluvialis because the cells are sensitive to shear stress. On the other hand, mixing in nonmechanically agitated bioreactors such as bubble and airlift bioreactors is accomplished by aeration with
482
compressed air or using an air pump, thus the shear force is greatly reduced. Airlift bioreactor has particularly gained attention for cultivation of shear sensitive microorganisms as it offer several advantages such as well defined fluid flow pattern, low energy requirement, and simple construction [4]. The aims of the present work were to culture H. pluvialis in the airlift bioreactor in order to examine the effect of superficial gas velocities on growth of//, pluvialis. 2. MATERIALS AND METHODS 2.1 Source of the microalga and culture medium Haematococcus pluvialis NIES-144 was obtained from the National Institute of Environmental Studies, Tsukuba, Japan. The Fl medium [5] consisted of (per litre) 0.41 g KNO3, 0.03 g Na2HPO4, 9.78 mg CaCl2-2H2O, 2.46 mg MgSO4-7H2O, 0.008 mg CuSO4-5H2O, 0.08 mg Na2MoO4-2H2O, 0.66 mg MnCl2-4H2O, 0.05 mg Cr2O3, 0.0078 mg CoCl2-6H2O, 2.21 mg FeSO4-5H2O2, 0.03 mg SeO2, vitamin Bl 16 mg, vitamin B6 1.2 mg and vitamin B12 12 jig. The medium was autoclaved at 121 °C for 20 min prior to use. 2.2 Bioreactor design and culture condition The airlift photobioreactor used was simply a cylindrical acrylic bubble column with a draft tube that was centrally installed within the outer column having a working volume of 3 L. Both the draft tube and the outer column were made. The dimension of the outer tube was 60 cm in height and 10 cm in diameter, and that of the draft tube was 40 cm in height. The ratio between the downcomer and riser cross section areas (A
483
cell dry weight) and 0.45 day", respectively. Due to the equipment constraints, the air flow could not be accurately adjusted below 0.4 cm s"1. It was also difficult to adjust the air flow rate in the range between 0.4 and 2 cm s"1, therefore it could not be concluded at this point that this 0.4 cm s of superficial gas velocity was the optimal level. It should however be noticed that the maximum cell density obtained in this study was significantly higher than the cell densities reported in literatures as shown in Table 1. Further increase of superficial gas velocity to 0.4 cm s"1 gave no beneficial effect for growth. However, increase in superficial gas velocity in this study seemed not support growth.
4
-1
Haematococcus (x10 cells ml )
90 0.4 cm/s 2 cm/s 2.5 cm/s 3 cm/s
80 70 60 50 40 30 20 10 0 0
2
4
6
8
10
12
14
Day
Figure 1 Growth curve of//, pluvialis at different superficial gas velocities (Ug=0.4, 2, 2.5, 3 cm s" ) in 3L airlift bioreactor (Ad/Ar=3.2). Table 1 Comparison of the result from this study with published literatures. Reference
Reactor type
Reactor volume (ml)
Medium
Condition
Illumination (xlOOO lux)
Aeration (L/h)
Maximum density (xlO4 cell/ml)
[2] [6] [3] [1] This work
Tube Flask Stirred tank Airlift Airlift
70 200 3710 30000 3000
OHM Basal
Mixotrophic Mixotrophic Heterotrophic Autotrophic Autotrophic
2 8.6
15
37
100 80-90 27
51 14.5 25 77
Bold's Control
2.5 1
In general, increasing aeration rate induces mixing, liquid circulation and mass transfer between gas and liquid phases [7]. A higher mass transfer also facilitates the removal of gases such as oxygen, preventing the accumulation of these gases which might adversely affect the growth rate [8]. High superficial gas velocity was therefore expected to strongly influence mixing of the liquid medium, increasing the absorption of CO2 and improving light availability of the algal culture which probably stimulating photosynthesis and growth. However, the cell of//, pluvialis in the airlift system was negatively affected by an increase in superficial gas velocity. This was possibly due to the shear stress caused by the high aeration
484
rate. Hence, the cell of H. pluvialis was suppose to be highly shear sensitive and even with the shear caused by aeration could badly deteriorate the growth. As mentioned by Gudin and Chaumont [9], the key problem of microalgal cultivation in photobioreactors is cell damage due to shear stress. However, microscopic observation suggested that the inhibition of growth in this study was due to the transformation of vegetative cell into cyst, rather than cell death. To explain in more detail, H. pluvialis cells responded to high superficial gas velocity by releasing the flagella, altering metabolism, discontinuing cell division and finally transforming into resting stage aplanospore. The culture of green vegetative cell in exponential phase of growth usually requires a low liquid velocity because of the fragility [10]. The observation results from this study illustrated that an increase in superficial gas velocity (2-3 cm-s"1), induced the changed of cell morphology from oval-shape vegetative cells to round-shape non-motile green cells that affected to cell concentration. At superficial gas velocity of 0.4 cm-s"1, most of the algal cells were remain in the green vegetative cells (see Table 2) which were more productive in term of cell multiplication. Therefore, it would be difficult to obtain high cell density if the cell could not be maintained in vegetative form particularly in high shear stress condition. Although the non-motile green aplanospores can grow by increase in the size, their rate of cell division was very slow, this made the cell density almost constant. Table 2 Density of green vegetative cells and non-motile cysts (aplanospore) in batch cultures of H. pluvialis on day 7. % of cyst Superficial gas velocity Vegetative cell Cyst (cm sl) (xl0 4 cellmL"') (xl0 4 cellmL"') 0.4 56.17 2.04 1.17 2 6.67 19.67 74.68 2.5 3.4 16.77 83.14 0.63 6.21 90.79 3 CONCLUSIONS This work demonstrated that an airlift system was suitable for the cultivation of Haematococcus pluvialis, one of the most effective microorganisms that could produce high potential antioxidant carotenoid, astaxanthin. Aeration was shown to be crucial for a proper growth of the alga in the airlift bioreactor, but it must be maintained at low level, and the most appropriate superficial velocity was found to be at the lower limit of the pump, i.e. 0.4 cms"1. REFERENCES [ 1 ] M. Harker, A. J. Tsavalos and A. J. Yong, J. Fermentation and Biotechnology., 82 (1996) 113-118 [2] J. Fabregas. A. Otero and A. Dominguez, J. Biotech., 89 (2001) 65-67. [3] F. Chen, H. Chen and X. Gong, J. Biores. Tech., 62 (1997) 19-24. [4] J. A. Asenjo and J. C. Merchuk (eds), Bioreactor system design, New York: Dekker, 1995. [5] J. Fabregas, A. Dominquez, D.G. Alvarez, T. Lamela and A. Otero, Biotech. Lett. 20 (1998) 623626. [6] A. Tjahjono, T. Kakizono, Y. Hayama, N. Nishio and S. Nagai, J. Fermentation and Bioengineering, 77 (1994) 352-357. [7] J. C. Merchuk and Y. Stein, J. AIChE., 27 (1981) 377-388. [8] H. L. Tung, C.C. Tu, Y. Y. Chang and W. T. Wu, Bioproc. Eng., 18(1998) 323-328. [9] C. Gudin and D. Chaumont, J. Biores. Tech., 38 (1991) 145-151. [10] N. Hata, J. C. Ogbonna, Y. Hasegawa, H. Taroda and H. Tanaka, J. Appl. PhycoL, 13 (2001) 395402.
481 481
Effect of superficial gas velocity on growth of the green microalga Haematococcus pluvialis in airlift photobioreactor Sorawit Powtongsook ''2 Kamonpan Kaewpintong3 Artiwan Shotipruk3'* and Prasert Pavasant3 'National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathum Thani 12120, THAILAND 2
Center of Excellence for Marine Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, THAILAND department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330, THAILAND *e-mail:[email protected] ABSTRACT The green microalga Haematococcus pluvialis NIES144 was cultured in a 3 L airlift photobioreactor at 27+l°C and light intensity of 1,000 lux. The effect of different superficial gas velocities on growth of//, pluvialis was evaluated within the range between 0.4 and 3 cm-s"'. It was found that growth of//, pluvialis was strongly affected by the superficial gas velocity. The maximum cell density and specific growth rate were obtained in the culture with the superficial gas velocity of 0.4 cm-s"1. These values are 77 xlO4 cells mL"1 (2.79 g L"1 dry weight) and 0.45 day"1, respectively. Higher levels of gas velocity did not show any benefits, and in fact they were found to drastically slow down the growth, due to the increased shear stress. Further more an increase in superficial gas velocity could significantly change the cell morphology from motile vegetative cells to non-motile green cells or cysts. 1. INTRODUCTION Astaxanthin (or 3'3-dihydroxy-P,P'-carotene-4,4'-dione) is an important pigmentation source in aquaculture that has been shown to possess higher antioxidant activity than any other carotenoids [1]. The freshwater alga Haematococcus pluvialis is considered the most capable microorganism that produces the largest amount of astaxanthin compared to other astaxanthin producing microorganisms such as yeast and bacteria [2]. For this reason, the production of astaxanthin from H. pluvialis has gained a great deal of attention. Typical microalgal cultivation can usually be divided into open and close systems. A large-scale algal culture is usually an open tank or pond which is easy to operate and consumes less resource than a close system. Nevertheless, the difficulty in cultivation of H. pluvialis is its slow growth. The requirement for low growth temperature and the susceptibility to contamination also make it difficult to obtain optimum growth when cultivated in an open system [3]. As a result, cultivation in a closed system such as in a photobioreactor is required to achieve high cell densities. A variety of bioreactors including both mechanically and nonmechanically agitated systems have been used. However, mechanically stirred bioreactors are not suitable for the cultivation of//, pluvialis because the cells are sensitive to shear stress. On the other hand, mixing in nonmechanically agitated bioreactors such as bubble and airlift bioreactors is accomplished by aeration with
482
compressed air or using an air pump, thus the shear force is greatly reduced. Airlift bioreactor has particularly gained attention for cultivation of shear sensitive microorganisms as it offer several advantages such as well defined fluid flow pattern, low energy requirement, and simple construction [4]. The aims of the present work were to culture H. pluvialis in the airlift bioreactor in order to examine the effect of superficial gas velocities on growth of//, pluvialis. 2. MATERIALS AND METHODS 2.1 Source of the microalga and culture medium Haematococcus pluvialis NIES-144 was obtained from the National Institute of Environmental Studies, Tsukuba, Japan. The Fl medium [5] consisted of (per litre) 0.41 g KNO3, 0.03 g Na2HPO4, 9.78 mg CaCl2-2H2O, 2.46 mg MgSO4-7H2O, 0.008 mg CuSO4-5H2O, 0.08 mg Na2MoO4-2H2O, 0.66 mg MnCl2-4H2O, 0.05 mg Cr2O3, 0.0078 mg CoCl2-6H2O, 2.21 mg FeSO4-5H2O2, 0.03 mg SeO2, vitamin Bl 16 mg, vitamin B6 1.2 mg and vitamin B12 12 jig. The medium was autoclaved at 121 °C for 20 min prior to use. 2.2 Bioreactor design and culture condition The airlift photobioreactor used was simply a cylindrical acrylic bubble column with a draft tube that was centrally installed within the outer column having a working volume of 3 L. Both the draft tube and the outer column were made. The dimension of the outer tube was 60 cm in height and 10 cm in diameter, and that of the draft tube was 40 cm in height. The ratio between the downcomer and riser cross section areas (A
483
cell dry weight) and 0.45 day", respectively. Due to the equipment constraints, the air flow could not be accurately adjusted below 0.4 cm s"1. It was also difficult to adjust the air flow rate in the range between 0.4 and 2 cm s"1, therefore it could not be concluded at this point that this 0.4 cm s of superficial gas velocity was the optimal level. It should however be noticed that the maximum cell density obtained in this study was significantly higher than the cell densities reported in literatures as shown in Table 1. Further increase of superficial gas velocity to 0.4 cm s"1 gave no beneficial effect for growth. However, increase in superficial gas velocity in this study seemed not support growth.
4
-1
Haematococcus (x10 cells ml )
90 0.4 cm/s 2 cm/s 2.5 cm/s 3 cm/s
80 70 60 50 40 30 20 10 0 0
2
4
6
8
10
12
14
Day
Figure 1 Growth curve of//, pluvialis at different superficial gas velocities (Ug=0.4, 2, 2.5, 3 cm s" ) in 3L airlift bioreactor (Ad/Ar=3.2). Table 1 Comparison of the result from this study with published literatures. Reference
Reactor type
Reactor volume (ml)
Medium
Condition
Illumination (xlOOO lux)
Aeration (L/h)
Maximum density (xlO4 cell/ml)
[2] [6] [3] [1] This work
Tube Flask Stirred tank Airlift Airlift
70 200 3710 30000 3000
OHM Basal
Mixotrophic Mixotrophic Heterotrophic Autotrophic Autotrophic
2 8.6
15
37
100 80-90 27
51 14.5 25 77
Bold's Control
2.5 1
In general, increasing aeration rate induces mixing, liquid circulation and mass transfer between gas and liquid phases [7]. A higher mass transfer also facilitates the removal of gases such as oxygen, preventing the accumulation of these gases which might adversely affect the growth rate [8]. High superficial gas velocity was therefore expected to strongly influence mixing of the liquid medium, increasing the absorption of CO2 and improving light availability of the algal culture which probably stimulating photosynthesis and growth. However, the cell of//, pluvialis in the airlift system was negatively affected by an increase in superficial gas velocity. This was possibly due to the shear stress caused by the high aeration
484
rate. Hence, the cell of H. pluvialis was suppose to be highly shear sensitive and even with the shear caused by aeration could badly deteriorate the growth. As mentioned by Gudin and Chaumont [9], the key problem of microalgal cultivation in photobioreactors is cell damage due to shear stress. However, microscopic observation suggested that the inhibition of growth in this study was due to the transformation of vegetative cell into cyst, rather than cell death. To explain in more detail, H. pluvialis cells responded to high superficial gas velocity by releasing the flagella, altering metabolism, discontinuing cell division and finally transforming into resting stage aplanospore. The culture of green vegetative cell in exponential phase of growth usually requires a low liquid velocity because of the fragility [10]. The observation results from this study illustrated that an increase in superficial gas velocity (2-3 cm-s"1), induced the changed of cell morphology from oval-shape vegetative cells to round-shape non-motile green cells that affected to cell concentration. At superficial gas velocity of 0.4 cm-s"1, most of the algal cells were remain in the green vegetative cells (see Table 2) which were more productive in term of cell multiplication. Therefore, it would be difficult to obtain high cell density if the cell could not be maintained in vegetative form particularly in high shear stress condition. Although the non-motile green aplanospores can grow by increase in the size, their rate of cell division was very slow, this made the cell density almost constant. Table 2 Density of green vegetative cells and non-motile cysts (aplanospore) in batch cultures of H. pluvialis on day 7. % of cyst Superficial gas velocity Vegetative cell Cyst (cm sl) (xl0 4 cellmL"') (xl0 4 cellmL"') 0.4 56.17 2.04 1.17 2 6.67 19.67 74.68 2.5 3.4 16.77 83.14 0.63 6.21 90.79 3 CONCLUSIONS This work demonstrated that an airlift system was suitable for the cultivation of Haematococcus pluvialis, one of the most effective microorganisms that could produce high potential antioxidant carotenoid, astaxanthin. Aeration was shown to be crucial for a proper growth of the alga in the airlift bioreactor, but it must be maintained at low level, and the most appropriate superficial velocity was found to be at the lower limit of the pump, i.e. 0.4 cms"1. REFERENCES [ 1 ] M. Harker, A. J. Tsavalos and A. J. Yong, J. Fermentation and Biotechnology., 82 (1996) 113-118 [2] J. Fabregas. A. Otero and A. Dominguez, J. Biotech., 89 (2001) 65-67. [3] F. Chen, H. Chen and X. Gong, J. Biores. Tech., 62 (1997) 19-24. [4] J. A. Asenjo and J. C. Merchuk (eds), Bioreactor system design, New York: Dekker, 1995. [5] J. Fabregas, A. Dominquez, D.G. Alvarez, T. Lamela and A. Otero, Biotech. Lett. 20 (1998) 623626. [6] A. Tjahjono, T. Kakizono, Y. Hayama, N. Nishio and S. Nagai, J. Fermentation and Bioengineering, 77 (1994) 352-357. [7] J. C. Merchuk and Y. Stein, J. AIChE., 27 (1981) 377-388. [8] H. L. Tung, C.C. Tu, Y. Y. Chang and W. T. Wu, Bioproc. Eng., 18(1998) 323-328. [9] C. Gudin and D. Chaumont, J. Biores. Tech., 38 (1991) 145-151. [10] N. Hata, J. C. Ogbonna, Y. Hasegawa, H. Taroda and H. Tanaka, J. Appl. PhycoL, 13 (2001) 395402.