- Email: [email protected]
Repeated production of hydrogen by sulfate re-addition in sulfur deprived culture of Chlamydomonas reinhardtii
international journal of hydrogen energy 35 (2010) 13387–13391
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Repeated production of hydrogen by sulfate re-addition in sulfur deprived culture of Chlamydomonas reinhardtii Jun Pyo Kim a, Kyoung-Rok Kim a, Seung Phill Choi a, Se Jong Han b, Mi Sun Kim c, Sang Jun Sim a,* a
Department of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea Polar BioCenter, Korea Polar Research Institute, KORDI, Incheon 406-840, Republic of Korea c Biomass Research Team, Korea Institute of Energy Research, Daejeon 305-343, Republic of Korea b
article info
abstract
Article history:
Biological hydrogen production by the green alga, Chlamydomonas reinhardtii can be induced
Received 9 November 2009
in conditions of sulfur deprivation. In this study, we investigated the repeated and
Accepted 18 November 2009
enhanced hydrogen production afforded by the re-addition of sulfate with monitoring of
Available online 24 December 2009
pH and concentration of chlorophyll and sulfate. Without adjustment of the pH, the
Keywords:
re-addition of 30 mM of sulfate and the adjustment of the pH during 4 cycles of repeated
Repeated hydrogen production
production, we obtained the maximum amount of 789 ml H2 l1 culture, which is 3.4 times
Green algae
higher than that of one batch production without adjustment of pH, 236 ml H2 l1 culture.
Chlamydomonas reinhardtii
This means that the enhancement of the hydrogen production can be achieved by the
Sulfur deprivation
careful control of the sulfate re-addition and pH adjustment in the sulfur deprived culture.
Sulfate re-addition
ª 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
optimal concentration of re-added sulfate was 30 mM for the hydrogen production. By the
pH adjustment
1.
Introduction
Hydrogen is an attractive energy carrier, which is expected to replace fossil fuels in the near future, due to its clean and regenerative features. The combustion of hydrogen with oxygen produces only water vapor and is 50% more efficient than that of gasoline in automobiles. Hydrogen also has the highest gravimetric energy density of any known fuel. Although as a highly efficient and non-polluting fuel, hydrogen seems to be the perfect alternative, there is no real environmental benefit currently gained from its use, because most of it is still extracted from fossil fuels. In order to solve this problem, several researchers recently developed a biological method of producing hydrogen using green algae in order to reduce this dependence [1–3].
Green algae can generate hydrogen from light and water, both of which are plentiful resources in nature [4]. In green algae, hydrogen photoproduction is catalyzed by hydrogenase, an enzyme that exhibits activity only under anaerobic conditions [5]. Hydrogenase activation is prerequisite to hydrogen production by green algae, however, this enzyme is severely oxygen sensitive and easily inactivated by photosynthetic oxygen evolution. To overcome this problem, Melis et al. [1] proposed a method of partially inactivating the PSII activity to a point where all of the O2 evolved by photosynthesis is immediately uptaken by the respiratory activity of the culture. This method is based on the deprivation of sulfur from the culture medium and results in the temporal separation of the photosynthetic O2 and anaerobic H2 evolution activities in the green alga, Chlamydomonas reinhardtii (two
* Corresponding author. Tel.: þ82 31 290 7341; fax: þ82 31 290 7272. E-mail address: [email protected] (S.J. Sim). 0360-3199/$ – see front matter ª 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2009.11.113
13388
international journal of hydrogen energy 35 (2010) 13387–13391
stages process). In an effort to optimize this method, the influence of many different physiological factors has been studied, such as the effects of low residual sulfur at the start of the process [6], the re-addition of a little sulfate to the cultures [7], the use of light-synchronized cultures [8], the changes in the initial pH of the medium [9], the cell age at the start of sulfur deprivation [10], and various light intensities after sulfur deprivation [11–13]. Moreover, the most interesting discovery is that hydrogen and oxygen can be alternatively evolved by cycling a single C. reinhardtii culture between the two stages (oxygenic photosynthesis in the presence of sulfur and hydrogen production in its absence) [2,6]. Based on this phenomenon, we investigated the effects of sulfate modulation for the development of a continuous hydrogen production process using a single C. reinhardtii culture under sulfur deprived conditions. Various concentrations of sulfate ranging from 0 to 120 mM were re-added to the cell suspensions after sulfur deprivation, and these re-additions were performed 4 times in order to bring about the repeated production of hydrogen [7]. In order to optimize this system, various parameters were also studied after sulfur deprivation, such as the change of the chlorophyll concentration, residual sulfate consumption, and control of the pH by the addition of HEPES buffer.
2.
Materials and methods
2.1.
Strain and culture conditions
The strain used in this study, C. reinhardtii UTEX 90, was obtained from UTEX, The Culture Collection of Algae at the University of Texas at Austin, in the USA. The cells were grown in 250 ml Erlenmeyer flasks which contained 120 ml of Tris–acetate–phosphate (TAP) medium [14] at pH 7.2 and 25 C with shaking at 150 rpm. The cells were subjected to alternate light (12 h) and dark (12 h) cycles using cool-white fluorescent lamps with an intensity of 60 mE m2 s1 [8]. The cell number was counted by an improved Neubauer ultraplane hemocytometer. The dry cell weight (DCW) was measured using a dried filter paper method at 80 C.
2.2. Repeated production of hydrogen by sulfate re-addition The algal cells were harvested in the late logarithmic phase after 4 h from the start of the light period for a high yield of hydrogen production (about 9.0 106 cells ml1) by centrifugation at 2000g for 5 min and washed seven times with sulfur omitted TAP medium (TAP-S medium) in order to obtain the sulfur deprived condition [8]. In the TAP-S medium, sulfate was substituted with an equivalent amount of chloride salts to satisfy the cation requirement [15]. The cells were re-suspended in the TAP-S medium with a concentration of about 1.3 107 cells ml1 (0.8 g DCW l1), and then sulfur was readded to the culture medium in the form of magnesium sulfate at different concentrations ranging from 0 to 120 mM 40 ml samples of the sulfur re-added cell suspensions were placed in 100 ml serum bottles and then argon (Ar) gas was purged to remove the oxygen in the headspace of each bottle before their
re-incubation. The re-addition of sulfate and purging with Ar were repeated up to 4 times at the end of each 140 h period of culture. The cells to which sulfate was re-added were cultivated under continuous fluorescent light intensity (200 mE m2 s1) at 25 C and 150 rpm for a total of 560 h. In order to fix the pH after sulfur deprivation, 11.92 g l1 of HEPES (2-[4(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) was only just added once with TAP-S at the beginning of the 1st cycle, and then the pH was adjusted to 7.2 using 1 N NaOH.
2.3.
Analytical methods
The light intensity at the culture surface was measured with an LI-250 quantum photometer (Li-Cor, Lincoln, NE, USA). For the chlorophyll measurements, cells were harvested by centrifugation at 3000g for 5 min, and the chlorophyll in the pellets was extracted with 95% ethanol (v/v). The cellular debris was removed by centrifugation at 15,000g for 30 s. The total chlorophyll concentration was calculated by measuring the light absorption at 649 and 665 nm, based on Spreitzer’s method [14]. A Hewlett–Packard 5890 gas chromatography system (Palo Alto, CA, USA) was used to determine the hydrogen concentration. A carboxen-1000 column (Supelco, Bellefonte, PA, USA) with Ar as the carrier gas was used to separate the H2 from the other gases in the headspace of the serum bottles. The signals were detected by a thermal conductivity detector and were calibrated with a known concentration of H2. The sulfate (SO2 4 ) concentration in the culture was measured by a DX-500 ion chromatograph (Dionex, Sunnyvale, CA, USA) with an IONPAC AS4 analytical column (Dionex). The detection limit of this instrument was 2 mg/l.
3.
Results
3.1. Effect of re-added sulfate concentration on the repeated production of hydrogen The C. reinhardtii cells were cultured photoheterotrophically on the TAP medium under synchronized illumination (light 12 h: dark 12 h) at a light intensity of 60 mE m2 s1 for 5 days. The cells were harvested at the late exponential phase after 4 h from the start of the light period for a high yield of hydrogen production and re-suspended in TAP-S medium with a concentration of about 1.3 107 cells ml1. Sulfur was readded to the culture medium in the form of magnesium sulfate a total of four times at the end of each 140 h period during sulfur deprivation at different concentrations, viz. 0, 15, 30, 60 and 120 mM. Fig. 1 shows the repeated production of hydrogen afforded by sulfate re-addition after sulfur deprivation. The addition of small quantities of sulfate (up to 30 mM MgSO4 final concentration) to the sulfur deprived cell suspensions during 4 cycles resulted in an increase in the total volume of hydrogen (112 ml H2 l1 culture upon no re-addition of MgSO4; 479 ml H2 l1 culture at re-addition of 15 mM MgSO4; 605 ml H2 l1 culture at re-addition of 30 mM MgSO4; re-addition of 0 mM MgSO4 was the control experiment). On the other hand, the total volume of hydrogen decreased in the case of the re-addition of 60 mM sulfate (204 ml H2 l1 culture) and hydrogen production
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Hydrogen production (ml/l)
250
Residual sulfate concentration (mg/l)
international journal of hydrogen energy 35 (2010) 13387–13391
sulfate 0 µM sulfate 15 µM sulfate 30 µM sulfate 60 µM sulfate 120 µM
200
150
100
50
Sulfate 0 µM Sulfate 15 µM Sulfate 30 µM Sulfate 60 µM Sulfate 120 µM
12 10 8 6 4 2 0 0
0
100
200
300
400
500
Time after sulfur deprivation (h) 0
100
200
300
400
500
600
Time after sulfur deprivation (h)
Fig. 1 – Repeated production of hydrogen by re-addition of sulfate at various concentrations after sulfur deprived condition. The arrows represent the time of sulfate readdition depending on the sulfate concentration. (The error bars illustrate the relative standard deviation (RSD) of three replicates).
stopped when 120 mM of sulfate was re-added. The overall hydrogen production decreased gradually and the start of hydrogen production was delayed depending on the cycle number. In the case of the re-addition of 60 mM sulfate, the start of hydrogen production was delayed for 30 h at the first and second cycles and for 80 h at the third cycle in comparison with the cases of the re-addition of 15 and 30 mM sulfate, and hydrogen production stopped completely at the fourth cycle. The changes of the chlorophyll concentration and the consumption rate of residual sulfate were monitored to demonstrate the effect of sulfate re-addition after sulfate deprivation on the hydrogen production (Figs. 2 and 3). As shown in Fig. 2, the initial chlorophyll concentration was 27.7 mg l1 at the start of the sulfur deprivation phase. The chlorophyll concentration increased rapidly in proportion to the quantity of sulfate that was re-added (0–120 mM MgSO4) during the initial 24 h after sulfur deprivation, and then decreased gradually as time passed in all cases. These patterns were observed similarly during the 4 cycles in each
70
Chlorophyll concentration (mg/l)
14
sulfate 0 µM sulfate 15 µM sulfate 30 µM sulfate 60 µM sulfate 120 µM
60 50 40 30 20 10 0 0
100
200
300
400
500
600
Time after sulfur deprivation (h)
Fig. 2 – Changes in cellular total chlorophyll concentration by re-addition of sulfate at various concentrations after sulfur deprived condition. The arrows represent the time of sulfate re-addition depending on the sulfate concentration.
Fig. 3 – Changes in the concentration of residual sulfate in the culture after sulfate re-addition. There is a specific maximum sulfate concentration (2 mg lL1, i.e. 20.8 mM) below which sulfur deprivation is induced. The arrows represent the time of sulfate re-addition depending on the sulfate concentration.
case. The final chlorophyll concentrations after 4 cycles were different in all cases (5.5 mg l1 after the re-addition of 15 mM MgSO4; 10.7 mg l1 after the re-addition of 30 mM MgSO4; 32.8 mg l1 after re-addition of 60 mM MgSO4). Fig. 3 shows the consumption rate of residual sulfate after sulfur deprivation. Sulfate was gradually exhausted during every cycle after its re-addition and hydrogen production was started when the sulfate concentration decreased to a specific level, namely about 2 mg l1 (Fig. 3). The consumption rate of residual sulfate was different in each case and the residual sulfate was consumed slowly as the re-added sulfate concentration increased. In the case of the re-addition of 120 mM sulfate, the concentration of residual sulfate remained above 2 mg l1 and no hydrogen was produced.
3.2. Hydrogen production by control of pH during four cycles Generally, the C. reinhardtii cells were grown and maintained in the TAP medium at an initial pH of 7.2. However, the pH of the culture increased to 8.0–8.5 during the four cycles after sulfur deprivation in all cases of sulfate re-addition (Fig. 4 (A)). Thus, we used HEPES buffer in order to stabilize the pH value for hydrogen production in the case of 30 mM sulfate re-addition. The pH of the culture increased from 7.2 to 7.5 during the initial 24 h, and then remained at about 7.5 until the end of hydrogen production (Fig. 4(B)). The total volume of hydrogen produced was 789 ml H2 l1 culture and the amount of hydrogen produced during each cycle was similarly sustained up to the third cycle with HEPES. However, it decreased abruptly at the fourth cycle (Fig. 5). The changes of the chlorophyll concentration in the sulfur deprived culture with or without HEPES are shown in Fig. 6. In the case of the sulfur deprived culture without HEPES, the final chlorophyll concentrations were 33.3 mg l1 at the first cycle, 30.1 mg l1 at the second cycle, 20.9 mg l1 at the third cycle, and 9.7 mg l1 at the fourth cycle. However, in the case of the sulfur deprived culture with HEPES, the final chlorophyll concentrations were 36.8 mg l1 at the first cycle, 35.3 mg l1 at the second cycle,
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international journal of hydrogen energy 35 (2010) 13387–13391
8.8 8.6 8.4
pH
8.2 8.0 7.8 7.6
sulfate 0 µM sulfate 15 µM sulfate 30 µM sulfate 60 µM sulfate 120 µM
7.4 7.2
Chlorophyll concentration (mg/l)
70
A
Without HEPES buffer With HEPES buffer
60 50 40 30 20 10 0
0
100
200
300
400
500
600
Time after sulfur deprivation (h)
7.0 0
100
200
300
400
500
9.0
B 8.5
Fig. 6 – Changes in cellular total chlorophyll concentration in the case of the re-addition of 30 mM sulfate under sulfur deprived culture with and without HEPES buffer. The arrows represent the time of sulfate re-addition.
pH
8.0
4.
7.5
7.0
6.5
6.0 0
100
200
300
400
500
Time after sulfur deprivation (h)
Fig. 4 – Changes of pH in sulfur deprived TAP-S medium (A) without HEPES buffer according to the re-addition of various sulfate concentrations and (B) with HEPES buffer according to the re-addition of 30 mM sulfate. The arrows represent the time of sulfate re-addition.
26.5 mg l1 at the third cycle, and 15.2 mg l1 at the fourth cycle. As shown in the above results, the chlorophyll concentrations remained higher in the sulfur deprived culture with HEPES than that without HEPES.
Hydrogen production (ml/l)
300 With HEPES buffer Without HEPES buffer
250 200 150 100 50 0
0
100
200
300
400
500
Time after sulfur deprivation (h)
Fig. 5 – Comparison of hydrogen production by re-addition of 30 mM sulfate in sulfur deprived culture with and without HEPES buffer. The arrows represent the time of sulfate re-addition.
Discussion
The repeated production of hydrogen by sulfur deprived C. reinhardtii culture depended on the re-addition of micromolar concentrations of sulfur. It was observed that an increase in the re-added sulfur concentration of up to 30 mM resulted in a gradual increase in the total hydrogen production after sulfur deprivation (Fig. 1). The chlorophyll concentration also increased as the re-added sulfur concentration was increased up to 30 mM after sulfur deprivation (Fig. 2). According to the report of Kosourov et al. [7], the study of H2 photoproduction in sulfur deprived algal cells in the presence of PSII electron transport inhibitors revealed that at least 80% of the electrons required for H2 production originate from the residual water-oxidation activity in PSII (residual PSII activity). In green algae, chlorophyll is the pigment primarily responsible for harvesting the light energy used in photosynthesis. When light is absorbed by the antenna chlorophyll in the photosynthetic membranes of C. reinhardtii, electrons are released from an oxygen-evolving complex through the watersplitting reaction [16], and then hydrogen is synthesized from the two electrons plus two protons by hydrogenase under anaerobic conditions. Therefore, more hydrogen was produced in the case of the re-addition of a higher sulfate concentration, owing to the conversion of the greater number electrons that were released at a high chlorophyll concentration [7]. On the other hand, the re-addition of sulfate at concentrations higher than 60 mM resulted in a significant decrease in the total amount of hydrogen produced, even though the increase in the chlorophyll concentration was greater than that observed after the re-addition of 30 mM sulfate. Moreover, in the case of the re-addition of 120 mM sulfate, hydrogen production was stopped altogether. These results were related to the consumption rate of residual sulfate after sulfur deprivation (Fig. 3). In fact, we observed that sulfur deprived conditions occurred below a specific level of sulfate concentration, viz. 2 mg sulfate/l culture, not at a sulfate concentration of zero. In the case of the re-addition of 60 mM sulfate, the consumption rate of residual sulfate declined gradually with increasing cycle number, which resulted in a decrease of the
international journal of hydrogen energy 35 (2010) 13387–13391
hydrogen productivity (Fig. 3). When the sulfur deprived condition was more quickly induced, hydrogenase was activated more quickly, because the culture conditions became anaerobic more rapidly, and the synthesis of hydrogen by the activated hydrogenase began sooner [7]. In the case of the readdition of 60 mM sulfate at the fourth cycle and 120 mM sulfate during first cycle, H2 production stopped because the PSII repair cycle was reactivated and the resultant O2 production inactivated hydrogenase function [7]. Therefore, a major requisite for enhancing hydrogen productivity by sulfur deprived single C. reinhardtii culture is the application of an appropriate sulfate concentration. In the current study, we expected that the level of hydrogen productivity would be maintained by the re-addition of sulfate after sulfur deprivation in every cycle. However, the amount of hydrogen produced decreased gradually whenever sulfate was re-added in the cases of the re-addition of 15–60 mM of sulfate and the pH then increased continuously in all cases of sulfate re-addition. Kosourov et al. [9] reported that an optimal pH existed for hydrogen production in sulfur deprived culture. Accordingly, we performed hydrogen production by the readdition of 30 mM sulfate in sulfur deprived culture with HEPES buffer, in order to maintain the hydrogen productivity during all 4 cycles. The total volume of hydrogen produced in the culture with HEPES buffer was 1.3 times higher than that without HEPES buffer (Fig. 5). The maintenance of the pH after sulfur deprivation played an important role in maintaining the residual PSII activity required for hydrogen production [9]. The chlorophyll concentration with HEPES buffer remained higher than that without HEPES buffer, even though the overall chlorophyll concentration decreased gradually during the culture after sulfur deprivation (Fig. 6). Therefore, the hydrogen productivity increased in the sulfur deprived culture with HEPES buffer, due to the higher residual PSII activity induced by the higher concentration of chlorophyll. However, the hydrogen production decreased sharply at the fourth cycle, because the reduction of chlorophyll occurred in the culture due to the application of a strong light intensity (200 mE/m2s) for a long period of time (560 h) [13].
5.
Conclusion
This study shows that there is a good possibility of developing a continuous process for the enhanced production of hydrogen by sulfate re-addition using a single cell culture under sulfur deprived condition. Hydrogenase activity and reduce equivalents from the residual PSII H2O-oxidation are the key factor for H2 production by C. reinhardtii. After sulfur deprivation, the readdition of a small amount of sulfate intensified the hydrogen productivity, due to the enhancement of the residual PSII activity, and it was found that there is an optimal sulfate concentration. The adjustment of the pH with HEPES buffer makes it possible to maintain the level of hydrogen productivity, due to the preservation of the residual PSII activity. Therefore, the repeated and increased production of hydrogen can be achieved by controlling the amount of re-added sulfur and adjusting the pH in the sulfur deprived culture.
13391
Acknowledgments This Research was performed for the Hydrogen Energy R&D Center, one of the 21st Century Frontier R&D Programs, funded by the Ministry of Science and Technology of Korea.
references
[1] Melis A, Zhang L, Forestier M, Ghirardi ML, Seibert M. Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. Plant Physiol 2000;122:127–35. [2] Ghirardi ML, Zhang L, Lee JW, Flynn T, Seibert M, Greenbaum E, et al. Microalgae: a green source of renewable H2. Trends Biotechnol 2000;18:506–11. [3] Sim SJ, Gong GT, Kim MS, Park TH. Dark hydrogen production by a green microalga, Chlamydomonas reinhardtii UTEX 90. J Microbiol Biotechnol 2005;15:1159–63. [4] Kim M, Chang M. Enhanced production of Phaeodactylum tricornutum (marine diatoms) cultured on a new medium with swine wastewater fermented by soil bacteria. J Microbiol Biotechnol 2006;16:1947–53. [5] Baek J, Choi E, Yun Y, Kim S, Kim M. Comparison of hydrogenases from Clostridium butyricum and Thiocapsa roseopersicina: hydrogenases of C. butyricum and T. roseopersicina. J Microbiol Biotechnol 2006;16:1210–5. [6] Zhang L, Happe T, Melis A. Biochemical and morphological characterization of sulfur-deprived and H2-producing Chlamydomonas reinhardtii (green alga). Planta 2002;214: 552–61. [7] Kosourov S, Tsygankov A, Seibert M, Ghirardi ML. Sustained hydrogen photoproduction by Chlamydomonas reinhardtii: effects of culture parameters. Biotechnol Bioeng 2002;78: 731–40. [8] Tsygankov A, Kosourov S, Seibert M, Ghirardi ML. Hydrogen photoproduction under continuous illumination by sulfurdeprived, synchronous Chlamydomonas reinhardtii cultures. Int J Hydrogen Energy 2002;27:1239–44. [9] Kosourov S, Seibert M, Ghirardi ML. Effect of extracellular pH on the metabolic pathways in sulfur-deprived, H2-producing Chlamydomonas reinhardtii cultures. Plant Cell Physiol 2003;44: 146–55. [10] Kim JP, Kang CD, Sim SJ, Kim MS, Park TH, Lee DH, et al. Cell age optimization for hydrogen production induced by sulfur deprivation using a green alga Chlamydomonas reinhardtii UTEX 90. J Microbiol Biotechnol 2005;15:131–5. [11] Hahn JJ, Ghirardi ML, Jacoby WA. Effect of process variable on photosynthetic algal hydrogen production. Biotechnol Prog 2004;20:989–91. [12] Kim JP, Kang CD, Park TH, Kim MS, Sim SJ. Enhanced hydrogen production by controlling light intensity in sulfurdeprived Chlamydomonas reinhardtii culture. Int J Hydrogen Energy 2006;31:1585–90. [13] Laurinavichene T, Tolstygina I, Tsygankov A. The effect of light intensity on hydrogen production by sulfur-deprived Chlamydomonas reinhardtii. J Biotechnol 2004;114:143–51. [14] Harris EH. The chlamydomonas sourcebook. San Diego: Academic Press Inc.; 1989. p. 607–8. [15] Davies JP, Yildiz FH, Grossman AR. Sac1, a putative regulator that is critical for survival of Chlamydomonas reinhardtii during sulfur deprivation. EMBO J 1996;15:2150–9. [16] Hopkins WG, Hu¨ner NPA. Introduction to plant physiology. New Jersey: Wiley & Johnsons, Inc.; 2004. p. 63–166.
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Repeated production of hydrogen by sulfate re-addition in sulfur deprived culture of Chlamydomonas reinhardtii Jun Pyo Kim a, Kyoung-Rok Kim a, Seung Phill Choi a, Se Jong Han b, Mi Sun Kim c, Sang Jun Sim a,* a
Department of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea Polar BioCenter, Korea Polar Research Institute, KORDI, Incheon 406-840, Republic of Korea c Biomass Research Team, Korea Institute of Energy Research, Daejeon 305-343, Republic of Korea b
article info
abstract
Article history:
Biological hydrogen production by the green alga, Chlamydomonas reinhardtii can be induced
Received 9 November 2009
in conditions of sulfur deprivation. In this study, we investigated the repeated and
Accepted 18 November 2009
enhanced hydrogen production afforded by the re-addition of sulfate with monitoring of
Available online 24 December 2009
pH and concentration of chlorophyll and sulfate. Without adjustment of the pH, the
Keywords:
re-addition of 30 mM of sulfate and the adjustment of the pH during 4 cycles of repeated
Repeated hydrogen production
production, we obtained the maximum amount of 789 ml H2 l1 culture, which is 3.4 times
Green algae
higher than that of one batch production without adjustment of pH, 236 ml H2 l1 culture.
Chlamydomonas reinhardtii
This means that the enhancement of the hydrogen production can be achieved by the
Sulfur deprivation
careful control of the sulfate re-addition and pH adjustment in the sulfur deprived culture.
Sulfate re-addition
ª 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
optimal concentration of re-added sulfate was 30 mM for the hydrogen production. By the
pH adjustment
1.
Introduction
Hydrogen is an attractive energy carrier, which is expected to replace fossil fuels in the near future, due to its clean and regenerative features. The combustion of hydrogen with oxygen produces only water vapor and is 50% more efficient than that of gasoline in automobiles. Hydrogen also has the highest gravimetric energy density of any known fuel. Although as a highly efficient and non-polluting fuel, hydrogen seems to be the perfect alternative, there is no real environmental benefit currently gained from its use, because most of it is still extracted from fossil fuels. In order to solve this problem, several researchers recently developed a biological method of producing hydrogen using green algae in order to reduce this dependence [1–3].
Green algae can generate hydrogen from light and water, both of which are plentiful resources in nature [4]. In green algae, hydrogen photoproduction is catalyzed by hydrogenase, an enzyme that exhibits activity only under anaerobic conditions [5]. Hydrogenase activation is prerequisite to hydrogen production by green algae, however, this enzyme is severely oxygen sensitive and easily inactivated by photosynthetic oxygen evolution. To overcome this problem, Melis et al. [1] proposed a method of partially inactivating the PSII activity to a point where all of the O2 evolved by photosynthesis is immediately uptaken by the respiratory activity of the culture. This method is based on the deprivation of sulfur from the culture medium and results in the temporal separation of the photosynthetic O2 and anaerobic H2 evolution activities in the green alga, Chlamydomonas reinhardtii (two
* Corresponding author. Tel.: þ82 31 290 7341; fax: þ82 31 290 7272. E-mail address: [email protected] (S.J. Sim). 0360-3199/$ – see front matter ª 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2009.11.113
13388
international journal of hydrogen energy 35 (2010) 13387–13391
stages process). In an effort to optimize this method, the influence of many different physiological factors has been studied, such as the effects of low residual sulfur at the start of the process [6], the re-addition of a little sulfate to the cultures [7], the use of light-synchronized cultures [8], the changes in the initial pH of the medium [9], the cell age at the start of sulfur deprivation [10], and various light intensities after sulfur deprivation [11–13]. Moreover, the most interesting discovery is that hydrogen and oxygen can be alternatively evolved by cycling a single C. reinhardtii culture between the two stages (oxygenic photosynthesis in the presence of sulfur and hydrogen production in its absence) [2,6]. Based on this phenomenon, we investigated the effects of sulfate modulation for the development of a continuous hydrogen production process using a single C. reinhardtii culture under sulfur deprived conditions. Various concentrations of sulfate ranging from 0 to 120 mM were re-added to the cell suspensions after sulfur deprivation, and these re-additions were performed 4 times in order to bring about the repeated production of hydrogen [7]. In order to optimize this system, various parameters were also studied after sulfur deprivation, such as the change of the chlorophyll concentration, residual sulfate consumption, and control of the pH by the addition of HEPES buffer.
2.
Materials and methods
2.1.
Strain and culture conditions
The strain used in this study, C. reinhardtii UTEX 90, was obtained from UTEX, The Culture Collection of Algae at the University of Texas at Austin, in the USA. The cells were grown in 250 ml Erlenmeyer flasks which contained 120 ml of Tris–acetate–phosphate (TAP) medium [14] at pH 7.2 and 25 C with shaking at 150 rpm. The cells were subjected to alternate light (12 h) and dark (12 h) cycles using cool-white fluorescent lamps with an intensity of 60 mE m2 s1 [8]. The cell number was counted by an improved Neubauer ultraplane hemocytometer. The dry cell weight (DCW) was measured using a dried filter paper method at 80 C.
2.2. Repeated production of hydrogen by sulfate re-addition The algal cells were harvested in the late logarithmic phase after 4 h from the start of the light period for a high yield of hydrogen production (about 9.0 106 cells ml1) by centrifugation at 2000g for 5 min and washed seven times with sulfur omitted TAP medium (TAP-S medium) in order to obtain the sulfur deprived condition [8]. In the TAP-S medium, sulfate was substituted with an equivalent amount of chloride salts to satisfy the cation requirement [15]. The cells were re-suspended in the TAP-S medium with a concentration of about 1.3 107 cells ml1 (0.8 g DCW l1), and then sulfur was readded to the culture medium in the form of magnesium sulfate at different concentrations ranging from 0 to 120 mM 40 ml samples of the sulfur re-added cell suspensions were placed in 100 ml serum bottles and then argon (Ar) gas was purged to remove the oxygen in the headspace of each bottle before their
re-incubation. The re-addition of sulfate and purging with Ar were repeated up to 4 times at the end of each 140 h period of culture. The cells to which sulfate was re-added were cultivated under continuous fluorescent light intensity (200 mE m2 s1) at 25 C and 150 rpm for a total of 560 h. In order to fix the pH after sulfur deprivation, 11.92 g l1 of HEPES (2-[4(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) was only just added once with TAP-S at the beginning of the 1st cycle, and then the pH was adjusted to 7.2 using 1 N NaOH.
2.3.
Analytical methods
The light intensity at the culture surface was measured with an LI-250 quantum photometer (Li-Cor, Lincoln, NE, USA). For the chlorophyll measurements, cells were harvested by centrifugation at 3000g for 5 min, and the chlorophyll in the pellets was extracted with 95% ethanol (v/v). The cellular debris was removed by centrifugation at 15,000g for 30 s. The total chlorophyll concentration was calculated by measuring the light absorption at 649 and 665 nm, based on Spreitzer’s method [14]. A Hewlett–Packard 5890 gas chromatography system (Palo Alto, CA, USA) was used to determine the hydrogen concentration. A carboxen-1000 column (Supelco, Bellefonte, PA, USA) with Ar as the carrier gas was used to separate the H2 from the other gases in the headspace of the serum bottles. The signals were detected by a thermal conductivity detector and were calibrated with a known concentration of H2. The sulfate (SO2 4 ) concentration in the culture was measured by a DX-500 ion chromatograph (Dionex, Sunnyvale, CA, USA) with an IONPAC AS4 analytical column (Dionex). The detection limit of this instrument was 2 mg/l.
3.
Results
3.1. Effect of re-added sulfate concentration on the repeated production of hydrogen The C. reinhardtii cells were cultured photoheterotrophically on the TAP medium under synchronized illumination (light 12 h: dark 12 h) at a light intensity of 60 mE m2 s1 for 5 days. The cells were harvested at the late exponential phase after 4 h from the start of the light period for a high yield of hydrogen production and re-suspended in TAP-S medium with a concentration of about 1.3 107 cells ml1. Sulfur was readded to the culture medium in the form of magnesium sulfate a total of four times at the end of each 140 h period during sulfur deprivation at different concentrations, viz. 0, 15, 30, 60 and 120 mM. Fig. 1 shows the repeated production of hydrogen afforded by sulfate re-addition after sulfur deprivation. The addition of small quantities of sulfate (up to 30 mM MgSO4 final concentration) to the sulfur deprived cell suspensions during 4 cycles resulted in an increase in the total volume of hydrogen (112 ml H2 l1 culture upon no re-addition of MgSO4; 479 ml H2 l1 culture at re-addition of 15 mM MgSO4; 605 ml H2 l1 culture at re-addition of 30 mM MgSO4; re-addition of 0 mM MgSO4 was the control experiment). On the other hand, the total volume of hydrogen decreased in the case of the re-addition of 60 mM sulfate (204 ml H2 l1 culture) and hydrogen production
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Hydrogen production (ml/l)
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international journal of hydrogen energy 35 (2010) 13387–13391
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Fig. 1 – Repeated production of hydrogen by re-addition of sulfate at various concentrations after sulfur deprived condition. The arrows represent the time of sulfate readdition depending on the sulfate concentration. (The error bars illustrate the relative standard deviation (RSD) of three replicates).
stopped when 120 mM of sulfate was re-added. The overall hydrogen production decreased gradually and the start of hydrogen production was delayed depending on the cycle number. In the case of the re-addition of 60 mM sulfate, the start of hydrogen production was delayed for 30 h at the first and second cycles and for 80 h at the third cycle in comparison with the cases of the re-addition of 15 and 30 mM sulfate, and hydrogen production stopped completely at the fourth cycle. The changes of the chlorophyll concentration and the consumption rate of residual sulfate were monitored to demonstrate the effect of sulfate re-addition after sulfate deprivation on the hydrogen production (Figs. 2 and 3). As shown in Fig. 2, the initial chlorophyll concentration was 27.7 mg l1 at the start of the sulfur deprivation phase. The chlorophyll concentration increased rapidly in proportion to the quantity of sulfate that was re-added (0–120 mM MgSO4) during the initial 24 h after sulfur deprivation, and then decreased gradually as time passed in all cases. These patterns were observed similarly during the 4 cycles in each
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Fig. 2 – Changes in cellular total chlorophyll concentration by re-addition of sulfate at various concentrations after sulfur deprived condition. The arrows represent the time of sulfate re-addition depending on the sulfate concentration.
Fig. 3 – Changes in the concentration of residual sulfate in the culture after sulfate re-addition. There is a specific maximum sulfate concentration (2 mg lL1, i.e. 20.8 mM) below which sulfur deprivation is induced. The arrows represent the time of sulfate re-addition depending on the sulfate concentration.
case. The final chlorophyll concentrations after 4 cycles were different in all cases (5.5 mg l1 after the re-addition of 15 mM MgSO4; 10.7 mg l1 after the re-addition of 30 mM MgSO4; 32.8 mg l1 after re-addition of 60 mM MgSO4). Fig. 3 shows the consumption rate of residual sulfate after sulfur deprivation. Sulfate was gradually exhausted during every cycle after its re-addition and hydrogen production was started when the sulfate concentration decreased to a specific level, namely about 2 mg l1 (Fig. 3). The consumption rate of residual sulfate was different in each case and the residual sulfate was consumed slowly as the re-added sulfate concentration increased. In the case of the re-addition of 120 mM sulfate, the concentration of residual sulfate remained above 2 mg l1 and no hydrogen was produced.
3.2. Hydrogen production by control of pH during four cycles Generally, the C. reinhardtii cells were grown and maintained in the TAP medium at an initial pH of 7.2. However, the pH of the culture increased to 8.0–8.5 during the four cycles after sulfur deprivation in all cases of sulfate re-addition (Fig. 4 (A)). Thus, we used HEPES buffer in order to stabilize the pH value for hydrogen production in the case of 30 mM sulfate re-addition. The pH of the culture increased from 7.2 to 7.5 during the initial 24 h, and then remained at about 7.5 until the end of hydrogen production (Fig. 4(B)). The total volume of hydrogen produced was 789 ml H2 l1 culture and the amount of hydrogen produced during each cycle was similarly sustained up to the third cycle with HEPES. However, it decreased abruptly at the fourth cycle (Fig. 5). The changes of the chlorophyll concentration in the sulfur deprived culture with or without HEPES are shown in Fig. 6. In the case of the sulfur deprived culture without HEPES, the final chlorophyll concentrations were 33.3 mg l1 at the first cycle, 30.1 mg l1 at the second cycle, 20.9 mg l1 at the third cycle, and 9.7 mg l1 at the fourth cycle. However, in the case of the sulfur deprived culture with HEPES, the final chlorophyll concentrations were 36.8 mg l1 at the first cycle, 35.3 mg l1 at the second cycle,
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Fig. 4 – Changes of pH in sulfur deprived TAP-S medium (A) without HEPES buffer according to the re-addition of various sulfate concentrations and (B) with HEPES buffer according to the re-addition of 30 mM sulfate. The arrows represent the time of sulfate re-addition.
26.5 mg l1 at the third cycle, and 15.2 mg l1 at the fourth cycle. As shown in the above results, the chlorophyll concentrations remained higher in the sulfur deprived culture with HEPES than that without HEPES.
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Fig. 5 – Comparison of hydrogen production by re-addition of 30 mM sulfate in sulfur deprived culture with and without HEPES buffer. The arrows represent the time of sulfate re-addition.
Discussion
The repeated production of hydrogen by sulfur deprived C. reinhardtii culture depended on the re-addition of micromolar concentrations of sulfur. It was observed that an increase in the re-added sulfur concentration of up to 30 mM resulted in a gradual increase in the total hydrogen production after sulfur deprivation (Fig. 1). The chlorophyll concentration also increased as the re-added sulfur concentration was increased up to 30 mM after sulfur deprivation (Fig. 2). According to the report of Kosourov et al. [7], the study of H2 photoproduction in sulfur deprived algal cells in the presence of PSII electron transport inhibitors revealed that at least 80% of the electrons required for H2 production originate from the residual water-oxidation activity in PSII (residual PSII activity). In green algae, chlorophyll is the pigment primarily responsible for harvesting the light energy used in photosynthesis. When light is absorbed by the antenna chlorophyll in the photosynthetic membranes of C. reinhardtii, electrons are released from an oxygen-evolving complex through the watersplitting reaction [16], and then hydrogen is synthesized from the two electrons plus two protons by hydrogenase under anaerobic conditions. Therefore, more hydrogen was produced in the case of the re-addition of a higher sulfate concentration, owing to the conversion of the greater number electrons that were released at a high chlorophyll concentration [7]. On the other hand, the re-addition of sulfate at concentrations higher than 60 mM resulted in a significant decrease in the total amount of hydrogen produced, even though the increase in the chlorophyll concentration was greater than that observed after the re-addition of 30 mM sulfate. Moreover, in the case of the re-addition of 120 mM sulfate, hydrogen production was stopped altogether. These results were related to the consumption rate of residual sulfate after sulfur deprivation (Fig. 3). In fact, we observed that sulfur deprived conditions occurred below a specific level of sulfate concentration, viz. 2 mg sulfate/l culture, not at a sulfate concentration of zero. In the case of the re-addition of 60 mM sulfate, the consumption rate of residual sulfate declined gradually with increasing cycle number, which resulted in a decrease of the
international journal of hydrogen energy 35 (2010) 13387–13391
hydrogen productivity (Fig. 3). When the sulfur deprived condition was more quickly induced, hydrogenase was activated more quickly, because the culture conditions became anaerobic more rapidly, and the synthesis of hydrogen by the activated hydrogenase began sooner [7]. In the case of the readdition of 60 mM sulfate at the fourth cycle and 120 mM sulfate during first cycle, H2 production stopped because the PSII repair cycle was reactivated and the resultant O2 production inactivated hydrogenase function [7]. Therefore, a major requisite for enhancing hydrogen productivity by sulfur deprived single C. reinhardtii culture is the application of an appropriate sulfate concentration. In the current study, we expected that the level of hydrogen productivity would be maintained by the re-addition of sulfate after sulfur deprivation in every cycle. However, the amount of hydrogen produced decreased gradually whenever sulfate was re-added in the cases of the re-addition of 15–60 mM of sulfate and the pH then increased continuously in all cases of sulfate re-addition. Kosourov et al. [9] reported that an optimal pH existed for hydrogen production in sulfur deprived culture. Accordingly, we performed hydrogen production by the readdition of 30 mM sulfate in sulfur deprived culture with HEPES buffer, in order to maintain the hydrogen productivity during all 4 cycles. The total volume of hydrogen produced in the culture with HEPES buffer was 1.3 times higher than that without HEPES buffer (Fig. 5). The maintenance of the pH after sulfur deprivation played an important role in maintaining the residual PSII activity required for hydrogen production [9]. The chlorophyll concentration with HEPES buffer remained higher than that without HEPES buffer, even though the overall chlorophyll concentration decreased gradually during the culture after sulfur deprivation (Fig. 6). Therefore, the hydrogen productivity increased in the sulfur deprived culture with HEPES buffer, due to the higher residual PSII activity induced by the higher concentration of chlorophyll. However, the hydrogen production decreased sharply at the fourth cycle, because the reduction of chlorophyll occurred in the culture due to the application of a strong light intensity (200 mE/m2s) for a long period of time (560 h) [13].
5.
Conclusion
This study shows that there is a good possibility of developing a continuous process for the enhanced production of hydrogen by sulfate re-addition using a single cell culture under sulfur deprived condition. Hydrogenase activity and reduce equivalents from the residual PSII H2O-oxidation are the key factor for H2 production by C. reinhardtii. After sulfur deprivation, the readdition of a small amount of sulfate intensified the hydrogen productivity, due to the enhancement of the residual PSII activity, and it was found that there is an optimal sulfate concentration. The adjustment of the pH with HEPES buffer makes it possible to maintain the level of hydrogen productivity, due to the preservation of the residual PSII activity. Therefore, the repeated and increased production of hydrogen can be achieved by controlling the amount of re-added sulfur and adjusting the pH in the sulfur deprived culture.
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Acknowledgments This Research was performed for the Hydrogen Energy R&D Center, one of the 21st Century Frontier R&D Programs, funded by the Ministry of Science and Technology of Korea.
references
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