A Solar Photobioreactor for the Production of Biohydrogen from Microalgae

Luis Panta, Pedro Chveza, Daniel Robledob, Rodrigo Patio*a a Dpto. de Fsica Aplicada, Cinvestav-Mrida, AP 73, 97310 Cordemex, Mrida, Yuc., MXICO b Dpto. de Recursos del Mar, Cinvestav-Mrida, AP 73, 97310 Cordemex, Mrida, Yuc., MXICO *rtarkus@mda.cinvestav.mx; phone: +52(999)1242138; fax: +52(999)9309765
ABSTRACT
The green microalga Chlamydomonas reinhardtii is proposed to produce hydrogen in a low-cost system using the solar radiation in Yucatan, Mexico. A two-step process is necessary with a closed photobioreactor, in which the algae are firstly growth and then induced for hydrogen generation. Preliminary results are presented in this work with some planning for the future. Different culture broths, temperatures and light intensities were tested for biomass and hydrogen production in laboratory conditions. The first experiments in external conditions with solar radiation and without temperature control have been performed, showing the potential of this technique at larger scales. However, some additional work must be done in order to optimize the culture maintenance, particularly in relation with the temperature control, the light radiation and the carbon dioxide supply, with the idea of keeping an economic production. Keywords: Chlamydomonas reinhardtii, microalgae, photobioreactor, biohydrogen, solar energy

1. INTRODUCTION
Facing the problems of oil crisis1 and atmospheric pollution2, alternative fuels must be considered in the palette of renewable energies.3 Molecular hydrogen is a clean fuel but it is not found easily in the Earth. However, since water is an abundant resource, it may be proposed as the principal source of gaseous hydrogen, with the advantage of being regenerated after combustion for energy captation.4 Nevertheless, water splitting requires energy to free molecular hydrogen and solar energy is an alternative to explore, emulating the photosynthetic processes.5 Moreover, in stress conditions, some phototrophic bacteria and microalgae have shown their capacities to liberate hydrogen through the enzyme hydrolase.6-7 In the other hand, the use of bioreactors for the efficient growth of microalgae in controlled conditions has been proposed to perform better biomass yields and the biosynthesis of fine chemicals.8-10 These processes have been proposed both in laboratory and in open-air conditions. For external cultures, the solar radiation is used in a wide variety of forms, including open ponds and closed reactors. In addition, the techniques monitoring some culture properties as pH, dissolved oxygen, conductivity or even infrared spectroscopy have been tested for a better knowledge of the metabolic metabolism as well as to have a better control of the culture system.11-12 Chlamydomonas reinhardtii is a unicellular green alga isolated from natural ponds. In anaerobic conditions, with a medium lacking in sulfur, they are capable of producing hydrogen through metabolic alternatives in which photosynthesis is involved.13-16 The production is maintained until the hydrolases are inhibited with the generated hydrogen. These results have been reported for small culture volumes from experiments in laboratory controlled conditions. The aim of this work is the production of biomass and biohydrogen in external conditions using bioreactors illuminated with solar radiation in Yucatan peninsula, at the southeast of Mexico, a region characterized by high annual solar radiations. A low-cost process is expected for this system in order to be proposed as an alternative economic activity in rural communities. The first results are presented in this work, in which two culture media and different values of temperature and light radiation are tested for both the cellular growth and the hydrogen production.

Solar Hydrogen and Nanotechnology II, edited by Jinghua Guo, Proc. of SPIE Vol. 6650, 66500Z, (2007) 0277-786X/07/$18 doi: 10.1117/12.732468

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2. EXPERIMENTAL PROCEDURE
Conservation and preparation of the cultures. The microalga Chlamydomonas reinhardtii CC124 was from the Chlamydomonas Resource Center (Duke University, USA). It is normally maintained in the Sueoka's High Salt Medium,17 as recommended by the provider. Fluorescent lamps (200 uE.m-2.s-1) were used to illuminate the algae for maintenance at room temperature (24oC). The Tris-Acetate-Phosphate (TAP) Medium18 was also used, as reported in most of the previous works on hydrogen generation from Chlamydomonas. Both media were autoclaved and kept in refrigeration before use. Compressed air was used for continuous and gently bubbling in the cultures to induce a logarithmic cellular growth. Cellular density. The number of algal cells per volume unity was obtained by direct cellular count in a Neubauer cell with an optical microscope of phase contrast (Olympus CH-2). The algae were previously immobilized in a 4% formol solution. These measurements were correlated with the optical density of the culture. A Spectrophotometer (StellarNet, EPP2000) was used with a 2-cm length tip cell to obtain the optical density at 660 nm, using the corresponding medium as the blank and for dilution when necessary. A third technique was used to correlate with biomass in the cultures: the dry weight. For these determinations, a 10-mL sample of the algae cultures was filtered with vacuum. The weight of the dry filter was compared before and after filtration. The filters were dried in a microwave oven (15 min at 10 W) and kept in a desiccator (at least 30 min) before weighted in an analytic balance. Cellular growth. The algae were grown in 1-L ball flasks with 500 mL of culture medium and continuous air bubbling. Sueoka and TAP media were tested and compared at different values of temperature and irradiation. Light was supplied, in laboratory conditions, with two fluorescent lamps (continuously, 24h/24h) and, in external conditions, with direct sunlight (natural light/dark periods). In both cases, light intensity was reduced sometimes with a number of layers of a black-mesh tissue. In the laboratory, the illumination was measured with a radiometer (LI-COR L1-1000) and tested at three intensities: 65 8, 100 7 y 160 13 Em-2s-1. The room temperature was controlled at 23 1C; other temperature values were controlled with a circulating water bath: 26 1, 29 1, 32 1 and 35 1C. Then, a factorial experiment with 2 x 3 x 5 = 30 tests was performed in the laboratory to follow the culture growth during five days. In addition, some tests were performed in external conditions during 15 days without control of temperature and illumination, but with on-line monitoring of these variables using a data logger (HOBO Pendant). In the external experiments, a system for air humidification was used before bubbling in the cultures to avoid extreme evaporation of the culture media. Fig. 1 is a picture with an experimental test in external conditions.

;'

Fig. 1. A picture showing the cellular growth at external conditions

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Hydrogen generation. Algae were grown during six days in the Sueoka medium at 25 1 C and 160 13 Em-2s-1. Cultures were centrifuged at 3600 rpm for 10 min. The medium was thrown up and the cells were re-suspended in 250 mL of fresh medium in a 250-mL Kitazato flask with a top. The flask was connected through a rubber hose with the up side of a glass column. The down side of the column is connected through another hose with an open container. Between the column and the container, silicon oil was used to detect the level displacement due to the difference in pressures when gas is exchanged in the system (see Fig. 2a). The changes of the volume in the column were continuously registered during seven days. Hydrogen generation was confirmed at the end of the experiments using a hydrogen analyzer with a thermal conductivity sensor. A factorial experiment with 2 x 2 x 2 = 8 tests was performed in the laboratory to follow the gas exchange with two media (Sueoka and TAP), two temperatures (25 1 and 35 1 C) and two light intensities (65 8 and 160 13 Em-2s-1). Fig. 2b is an image showing the study for the production of biohydrogen from microalgae comparing Sueoka and TAP media.

b
Fig. 2. (a) The simple experimental system for trapping and quantifying biohydrogen from microalgal cultures, (b) A comparison of Sueoka (left) and TAP (right) medium for producing hydrogen at 35 1 C and 160 13 Em-2s-1.

Photobioreactor. A (50 cm x 50 cm x 10 cm = 25 L) flat panel reactor was constructed with Plexiglas. A rectangular tap was used on the top with an orifice to allow the exit of gases. A continuous air bubbling in the medium was assured with compressed air through small holes along two 50-cm plastic hoses fixed at the bottom of the reactor. The bubbling allowed also a good mixing of the culture. Some tests were performed using the reactor filled with water in order to avoid over-heating of the system with high solar radiations. The north-south and east-west positions were tested, as well as the use of insulating polyurethane on the narrowest walls of the reactor and the use of a black-mesh tissues and humidifiers to compensate evaporation of water from the medium. A data logger (HOBO Pendant) was used to follow the values of light radiation and temperature in the reactor, as compared with the values from another data logger at external conditions. An experiment was performed with 18 L of Sueoka medium and 2 L of culture. Biomass and chlorophyll were followed at different times during 15 days. A system for air humidification was also used to avoid high media evaporation. TAP medium will be tested for growing algae in the reactor at external conditions. A variety of diameters for tubular reactors is also planned to be compared with the flat panel in order to find the best system for biomass production. The same reactors are going to be tested for biohydrogen production at external conditions using solar radiation.

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3. RESULTS AND DISCUSSION

Table 1 shows a comparison of the molar composition between both media; the principal differences are given with the purely autotrophic Sueoka medium and the acetate in TAP medium as heterotrophic carbon source, in addition with the concentrations of phosphate, magnesium and calcium.
Table. 1. Molar composition of Sueoka and TAP media, the principal differences are in the source of carbon, phosphorous, magnesium and calcium. Although the original formulations include some sulphates, these were generally avoided using the corresponding chlorides (see metallic ions marked with *), specially when hydrogen generation is induced.

Sueoka HCOOCH3 (mM) NH4Cl (mM) K2HPO4 (mM) KH2PO4 (mM) H3BO3 (M) EDTA (M) Mg(II) (M)* Zn(II) (M)* CaCl2 (M) MnCl2 (M) Fe(II) (M)* CoCl2 (M) Cu(II) (M)* (NH4)6Mo7O24 (M) 9.35 8.27 5.29 184 149 81.1 76.5 45.6 25.6 17.9 6.77 6.29 0.89

TAP 17.4 7.48 0.60 0.40 184 149 406 76.5 228 25.8 18.0 0.67 0.64 0.59

When laboratory conditions were imposed, the specific growth rate and the maximum of cellular concentrations were obtained for the 30 factorial different tests. Figures 3 and 4 show, respectively, a compilation of these results. It is possible to see that the Sueoka medium gives always better growth rates and cell concentrations than TAP medium. In fact, cultures with TAP medium at the higher temperatures showed contamination with a change in the color of the culture and a characteristic smell. This contamination is principally caused because acetate is a carbon source for a variety of heterotrophic microorganisms. In relation with the three light intensities, it is also seen that, in general, the best growth was with the highest light intensity. In fact, this growth diminishes with light intensity. In addition, although the best specific growth was reached at 29C, there are not big differences when the maximum cellular concentration is compared at the end of the experiments with the other temperatures. Indeed, from this analysis, microalgae cultures may be proposed to be grown in external conditions within the range of tested temperatures and with solar radiation.

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0.8

Specific growth rate (d )

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 23 26 29 32 35 SH SM SL TH TM TL

-1

Temperature (C)
Fig. 3. The specific growth of the microalgae cultures at different experimental conditions: medium (Sueoka and TAP), light intensity (65 8, 100 7 y 160 13 Em-2s-1) and temperature (23 1, 26 1, 29 1, 32 1 and 35 1C). The columns for every temperature are in the next order, from left to right: Sueoka medium (S) at the three light intensities high (H: 160 13 Em-2s-1), medium (M: 100 7 Em-2s-1) and low (L: 65 8 Em-2s-1), and TAP medium at the same three light intensities (TH, TM and TL, respectively). Medium TAP was contaminated after the first days when the cultures were grown at temperatures over 29C.

1600

Cellular density (cell/mL)

1400 1200 1000 800 600 400 200 0 23 26 29 32 35 SH SM SL TH TM TL

Temperature (C)
Fig. 4. The maximum cellular density after five days at different experimental conditions: medium (Sueoka and TAP), light intensity (65 8, 100 7 y 160 13 Em-2s-1) and temperature (23 1, 26 1, 29 1, 32 1 and 35 1C). The columns for every temperature are in the next order, from left to right: Sueoka medium (S) at the three light intensities high (H: 160 13 Em-2s-1), medium (M: 100 7 Em-2s-1) and low (L: 65 8 Em-2s-1), and TAP medium at the same three light intensities (TH, TM and TL, respectively). Medium TAP was contaminated after the first days when the cultures were grown at temperatures over 29C.

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The algal cultures in ball-flasks at external conditions were grown with Sueoka and TAP medium in spring days. The mean ambient temperature of the 15-day experiment was 33C in a range between 23 and 45C. The mean irradiance was 35 klux with a maximum of about 250 klux. This irradiance was reduced for the cultures using the dark-mesh tissues to a mean value of 17 klux with a maximum of about 200 klux. A graphic showing the biomass concentration with time is shown in Figure 5. It is possible to see that both the specific growth rate and the total biomass production are bigger in the TAP medium than in the Sueoka broth. This could be expected since mixotrophic growth with acetate may be faster and with better yields than just autotrophic cultures, but Sueoka medium appeared to be the best in laboratory conditions. It is possible that the solar radiation or the light/dark cycles favor TAP medium over Sueoka. However, a contamination in the TAP medium was observed and algae density fell down after 11 days of culture.

5000

Cellular density (cells/mL)

4000 3000 2000 1000 0 0 1 2 3

TAP Sueoka

10

11

12

13

14

15

16

17

Time (days)
Fig. 5. Cellular growth in ball flasks at external conditions with Sueoka and TAP media. The TAP culture was contaminated after 11 days of growing.

Biohydrogen generation was performed in laboratory conditions with both Sueoka and TAP media, at two temperatures and two light radiations. Figure 6 resume the profiles of gas displacement during the experiments. As can be seen, Sueoka medium showed small quantities of hydrogen produced during the first day for all the cases. When low light radiation was used, a second period for hydrogen production appeared after two days. In the other way, TAP medium showed a significant production of hydrogen only at 25C and 160 Em-2s-1. With high temperatures or low light radiations, there was not apparent hydrogen production. Further tests should be performed in order to confirm these results. With reference to the flat panel bioreactor, some characterization tests were performed in order to avoid overheating of the culture medium. This impact was obtained comparing the maximum of temperatures among the ambiance and the water in the reactor during some days. The values of these temperature differences, T, are resumed in table 2. When the biggest walls of the reactor are east-west oriented, the temperature differences are small, indicating that the reactor is heated easily. If these walls are north-south oriented, the temperature differences are bigger, meaning that the reactor is less heated with the solar radiation. Using air bubbling in the water and polyurethane taps for the smallest walls of the reactor, including the top, a reduction in the heating of the reactor is achieved. Finally, the use of one black-mesh tissue is not really efficient to avoid the heating of the bioreactor; however, when two tissues are used, a significant reduction of the reactor heating is possible. At the moment of the writing of this manuscript, an experiment with Sueoka medium is being performed to grow the microalgae in the reactor.

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DEZPLAZAMI(NTOSVOLUMETRICOS MEDID SUEOKA

EXP 1-T:25C, 1:159 pEm-2s-1)

6
S S

EXP 2-(T;25C, I; 67 pErn-2s-1)

EXP 3-(T:3SC, 1:159 pEm-2s-1)

x EXP 4-(T:35C 1:67 iErn-2s-1)

4 3 2 1 0

to
1

60

SD

100

123

Volume cm3

-2

12 11

id
9 8 6
S

TIEMPO horas)

DEZPLAZAMIEFJTOS VOtUM TRlCOS

M EDIO T4P

EXP 1-(T:25C, I:159pEm-2s-1

EXP 2-(T:25C, 1:67 kIEm-2s-1
EXP 3- T:35C, 1:159 p[m-2s-1)
x EXP 4-(T:35C, 1:67 kIEm-2s-1)
N.

i3 u2
a1
luG

03 >
-4 -5 -6 -7 -8 -9 -10 -11 -12

-1 -2

10

50

60

10

100

110

TIEMPO (horas)

Time / h

Fig. 6. Volume displacement for gas exchange during experiments for hydrogen generation. Different experimental conditions were performed: Sueoka and TAP media, temperature values at 25C (EXP 1 and EXP 2) and 35C (EXP 3 and EXP 4) and light irradiance values at 160 Em-2s-1 (EXP 1 and EXP 3) and at 65 Em-2s-1 (EXP 2 and EXP 4).

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Table 2. The differences of maxima temperatures among the reactor and the ambiance, T, as a function of some design factors to improve the performance of the bioreactor. The mean values during some days are reported, the incertitude is the standard deviation of the mean. Experiment 1 2 3 4 5 6 Orientation East-West East-West East-West North-South North-South North-South Air bubbling No Yes Yes Yes Yes Yes Polyurethane taps No Yes Yes Yes Yes Yes Black-mesh tissues 0 0 1 0 1 2 T/K 2.3 0.6 5.4 2.3 3.8 0.2 7.3 0.8 6.9 0.8 10.6 2.1

4. CONCLUSIONS
A number of experiments have been performed in order to test the biomass and biohydrogen production from Chlamydomonas reinhardtii microalgae at different experimental laboratory conditions. Sueoka and TAP media showed important differences in the performance of the algae. Besides possible contaminations, temperature was not a significant factor but light radiation is a key variable to improve the biomass growth and the biohydrogen generation. These former results were confirmed when the algae were successfully grown in external conditions with solar radiation. In the future work, the optimization of the cellular growth in the reactor is considered, as well as the first tests for biohydrogen generation with solar radiation.

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13. A.A. Tsigankov, S.N. Kosourov, I.V. Tolstygina, M.L. Ghirardi, M. Seibert, Hydrogen production by sulfurdeprived Chlamydomonas reinhardtii under photoautotrophic conditions, International Journal of Hydrogen Energy 31, 1574-1584 (2006). 14. J.P. Kim, C.D. Kang, T.H. Park, M.S. Kim and S.J. Sim, Enhanced hydrogen production by controlling light intensity in sulfur-deprived Chlamydomonas reinhardtii cultures, International Journal of Hydrogen Energy 31: 1585-1590 (2006). 15. T.V. Laurinavichene, T.V.; A.S. Fedorov, M.L. Ghirardi, M. Seibert and A.A. Tsygankov, Demonstration of sustained hydrogen photoproduction by immobilized, sulfur-deprived Chlamydomonas reinhardtii cells, International Journal of Hydrogen Energy 31, 659-667 (2006). 16. J.H. Jo, D.S. Lee and J.M. Park, Modeling and optimization of photosynthetic hydrogen gas production by green alga Chlamydomonas reinhardtii in sulfur-deprived circumstance, Biotechnol. Prog. 22, 431-437 (2006). 17. N. Sueoka, Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardtii, Proc. Natl. Acad. Sci. 46, 83-91 (1960). 18. E.H. Harris, The Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use, Academic Press, San Diego, 1989.

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