International Journal of Hydrogen Energy 31 (2006) 1585–1590www.elsevier.com/locate/ijhydene

Enhanced hydrogen production by controlling light intensity insulfur-deprived Chlamydomonas reinhardtii culture

Jun Pyo Kima, Chang Duk Kangb, Tai Hyun Parkb, Mi Sun Kimc, Sang Jun Sima,∗aDepartment of Chemical Engineering, Sungkyunkwan University, 300 Chunchun-dong, Changan-gu, Suwon, 440-746, South Korea

bSchool of Chemical and Biological Engineering, Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul, 151-744, South KoreacBiomass Research Team, Korea Institute of Energy Research, 71-2 Jang-dong, Yuseong-gu, Daejeon, 305-343, South Korea

Available online 17 July 2006

Abstract

Chlamydomonas reinhardtii is a green alga that can use light energy to produce hydrogen from water under anaerobic conditions.This work reports the enhancement of hydrogen production by controlling the light intensity in sulfur-deprived anaerobicC. reinhardtii cultures. The overall hydrogen production was dependent on light intensity in the range of 60–200 �E m−2 s−1.Maximum hydrogen production was obtained at a light intensity of 200 �E m−2 s−1 as a result of the rapid initiation of hydrogenproduction and the greatest increase of chlorophyll during the initial 24 h after sulfur deprivation. However, the hydrogenproduction was inhibited at an intensity of 300 �E m−2 s−1 of light owing to photosystem II photodamage by excess light.The maximum hydrogen production and the maximum specific production rate of hydrogen were 225 ml H2 l−1 culture and2.01 ml H2 g−1 cells h−1, respectively. Thus, hydrogen production by sulfur-deprived C. reinhardtii cultures can be maximizedby controlling the light intensity at levels below saturation.� 2006 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

Keywords: Hydrogen production; Light intensity; Sulfur deprivation; Chlamydomonas reinhardtii; Photosynthesis

1. Introduction

Green algae have a photosynthetic system similarto that of plants and can produce hydrogen by usingcarbon dioxide, sunlight, and water as carbon source,energy source, and electron donor, respectively [1,2].Hydrogen production in green algae is primarily me-diated by hydrogenase, which receives the electronssupplied to ferredoxin by the photosynthetic electrontransport chain and efficiently donates them to protons,

∗ Corresponding author. Tel.: +82 31 290 7341;fax: +82 31 290 7272.

E-mail address: simsj@skku.edu (S.J. Sim).

0360-3199/$30.00 � 2006 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.ijhydene.2006.06.026

thereby producing hydrogen gas [3]. However, hydro-gen photoproduction is transient and cannot be sus-tained because hydrogenase is strongly deactivated bythe oxygen evolved from oxygenic photosynthesis. Thislimitation has been overcome by suppressing photo-synthetic oxygen evolution through the use of sulfur-deprived medium for hydrogen production [4].

When algal cells are cultured under sulfur-deprivedconditions, one of the most noticeable adaptive re-sponses is their ability to reduce photosystem II(PS II) activity in light [4,5]. After about 20–24 h of sul-fur deprivation, the oxygen evolved by PS II decreasesto the level of oxygen uptake by respiration. After thispoint, the environment of the algal culture is convertedfrom aerobic to anaerobic conditions. This conversion

1586 J. P. Kim et al. / International Journal of Hydrogen Energy 31 (2006) 1585–1590

activates hydrogenase and subsequently initiates hydro-gen production by the activated hydrogenase [4,6].

Based on this process, the influences of manydifferent parameters have been studied for hydro-gen production in sulfur-deprived conditions, suchas the remaining activity of PS II [5,8], the use oflight-synchronized/unsynchronized culture [9], the re-addition of sulfate to the sulfur-deprived culture, theinitial cell density, and the change of initial pH in themedium [10,11], the relationship among light intensity,photobioreactor, and cell density [12], the relationshipbetween the light intensity and acetate concentration inthe culture [13], and the cell age [14].

From the above studies, we have found that it isimportant to rapidly convert from the aerobic phaseto the anaerobic phase in order to increase hydrogenproductivity in sulfur-deprived Chlamydomonas cul-tures [10]. This requires that the residual sulfate bequickly consumed in the hydrogen production stage.Here, we investigated the relationship between the hy-drogen production and consumption rates of residualsulfate by controlling the light intensity between 60and 300 �E m−2 s−1 in sulfur-deprived Chlamydomnasreinhardtii cultures. We also monitored the chlorophyllconcentration and cell number to investigate the lightinhibition of hydrogen production.

2. Materials and methods

2.1. Culture conditions of C. reinhardtii

C. reinhardtii UTEX 90 was grown in 250-mlErlenmeyer flasks containing Tris-acetate-phosphate(TAP) medium [15] at pH 7.2 and 25 ◦C with shakingat 150 rpm. Light was alternately provided betweenlight and dark phases with a 12 h interval using cool-white fluorescent lamps with 60 �E m−2 s−1 intensity.The number of cells was counted with an improvedNeubauer ultraplane hemocytometer. The dry cellweight (DCW) was measured on filter paper driedat 80 ◦C.

2.2. Hydrogen production under sulfur-deprivedconditions

The cells were harvested after 4 h from the startof the light period for a high yield of hydrogen pro-duction [9]. Algal cells were harvested at the latelogarithmic phase (about 9.0 × 106 cells ml−1) bycentrifugation at 2000 × g for 5 min and washedfive times with sulfur-free TAP medium (TAP-S

medium). In the TAP-S medium, sulfate was substi-tuted with an equivalent amount of chloride salts tosupply the cation requirement [16]. The cells werere-suspended in the TAP-S medium at a concentrationof 1.3 × 107 cells ml−1 (0.8 g DCW l−1). Then, 40 mlof the sulfur-deprived cell suspension was placed in a100-ml glass serum bottle with a 2.3-cm optical path.Oxygen in the headspace of the bottles was removed bypurging with argon gas. The algal cells were cultured un-der a continuous one-sided fluorescent light of variousintensities (60–300 �E m−2 s−1) at 25 ◦C and 150 rpm.

2.3. Analytical methods

Light intensity at the culture surface was measuredwith a LI-250 quantum photometer (Li-Cor, Lincoln,NE, USA). The measurements were made at five points(each corner and the middle) in the incubator andaveraged. We controlled the light intensity by addingcool-white fluorescent lamps. For the chlorophyll mea-surements, the cells were harvested by centrifugationat 3000 × g for 5 min, and the chlorophyll in the cellpellets was extracted with 95% ethanol (v/v). Cellulardebris was removed by centrifugation at 15, 000 × g

for 30 s. Total chlorophyll concentration was calculatedby measuring the light absorption at 649 and 665 nm,based on Spreitzer’s method [17]. A Hewlett-Packard5890 gas chromatography system (Palo Alto, CA,USA) was used to determine the hydrogen concentra-tion. A carboxen-1000 column (Supelco, Bellefonte,PA, USA) with Ar as the carrier gas was used to sepa-rate the H2 from other gases in the headspace [4] of theserum bottles. The signals were detected by a thermalconductivity detector and were calibrated with a knownconcentration of H2. The sulfate (SO2−

4 ) concentrationin the culture was measured by a DX-500 ion chro-matography (Dionex, Sunnyvale, CA, USA) with anIONPAC AS4 analytical column (Dionex). Detectionlimit of this instrument was 2 �gl−1 [18].

3. Results

3.1. Growth of the green alga C. reinhardtii undervarious light intensities

C. reinhardtii cells were heterotrophically culturedon TAP medium under synchronized illumination (light12 h:dark 12 h), and the rates of cell growth and sulfateconsumption were measured under various light intensi-ties (60–300 �E m−2 s−1). As shown in Fig. 1, the ratesof cell growth and sulfate consumption were differentwhen cells were cultivated for 7 days under various light

J. P. Kim et al. / International Journal of Hydrogen Energy 31 (2006) 1585–1590 1587

0 20 40 60 80 100 120 140 160 180

0 20 40 60 80 100 120 140 160 180

Cel

l gro

wth

(g

DC

W/l

cult

ure

)

0.0

0.2

0.4

0.6

0.8

1.0

Cultivation time (h)

Su

lfat

e co

nce

ntr

atio

n (

mg

/l)

0

10

20

30

40

50

60

70

60 µE/m2s

130 µE/m2s 200 µE/m2s 300 µE/m2s

(A)

(B)

Fig. 1. Rate of cell growth (A) and sulfate consumption (B) de-pending on light intensity in the nutrient replete medium.

intensities. During the cell growth phase, the algal celldensity increased with gradually increasing light inten-sity from 60 to 300 �E m−2 s−1, reaching the maximumlevel (about 0.87 g DCW l−1 culture) at 300 �E m−2 s−1

(Fig. 1A). Moreover, both the chlorophyll and starchconcentrations showed a similar tendency to increasewith increasing light intensity (data not shown). InFig. 1B, the rate of sulfate consumption was di-rectly proportional to the cell density, with boththe maximum rate of sulfate consumption and theminimum residual sulfate concentration occurring at300 �E m−2 s−1 (7 mg l−1). However, there was littledifference between the data at 200 and 300 �E m−2 s−1.We concluded that the rates of both cell growth and sul-fate consumption saturated at 200 �E m−2 s−1 of light.

3.2. Effect of light intensity on conversion to thehydrogen production phase

To demonstrate the effect of light intensity on hy-drogen production in sulfur-deprived Chlamydomonascultures, hydrogen production was conducted at various

Time after sulfur deprivation (h)

0 20 40 60 80 100 120 140Hyd

rog

en p

rod

uct

ion

(m

l H2/

l cu

ltu

re)

0

50

100

150

200

25060 µE/m2s

130 µE/m2s200 µE/m2s300 µE/m2s

Fig. 2. Hydrogen production by sulfur-deprived culture of C. rein-hardtii under various light intensities. (Each data point representsthree analytical replicates of a single experimental run.)

Light intensity (mE/m2s)

0 50 100 150 200 250 300 350

Tim

e af

ter

sulf

ur

dep

riva

tio

n t

hat

resi

du

al s

ulf

ate

was

exh

aust

ed (

h)

20

30

40

50

60

70

Fig. 3. The time after sulfur deprivation as a function of the lightintensity that residual sulfate reaches zero in the culture.

light intensities from 60 to 300 �E m−2 s−1 (Fig. 2). Thevolume of hydrogen produced increased proportionallyto the increase in light intensity, reaching a maximumproduction of 225 ml H2 l−1 culture at 200 �E m−2 s−1

of light. Also, the initiation time for hydrogen produc-tion gradually decreased from 62 to 22 h with increasinglight intensity. Fig. 3 shows the time at which residualsulfate was exhausted after sulfur deprivation. Con-sumption rate of residual sulfate in the sulfur-deprivedculture was accelerated with increasing light intensityand saturated at 200 �E m−2 s−1 of light. We also foundthat the initiation of hydrogen production dependson the consumption rate of residual sulfate. The lagtime for hydrogen production decreased with increas-ing light intensity, thus higher light levels prolong thetime for hydrogen production under high-intensity

1588 J. P. Kim et al. / International Journal of Hydrogen Energy 31 (2006) 1585–1590

Table 1Total time, volume of hydrogen production, and specific hydrogen production rate by sulfur deprived Chlamydomonas cells depending onvarious light intensities

Light intensity Start time of H2 End time of H2 Total time of H2 Total volume of H2 Specific production(�E m−2 s−1) production (h) production (h) production (h) production after rate of hydrogen

sulfur deprivation (ml H2 g−1 cell h−1)

(ml H2 l−1 culture)

60 61 140 79 85 ± 3.6 0.76 ± 0.09130 39 140 101 158 ± 6.4 1.41 ± 0.12200 24 140 116 225 ± 10.4 2.01 ± 0.08300 22 140 118 185 ± 5.2 1.65 ± 0.11

(A)

(B) Time after sulfur deprivation (h)

Ch

loro

ph

yll c

on

cen

trat

ion

(m

g/l)

15

20

25

30

35

40

60 µE/m2s 130 µE/m2s 200 µE/m2s 300 µE/m2s

0 20 40 60 80 100 120 140

0 20 40 60 80 100 120 140

Cel

l nu

mb

er (

x106

cells

/ml)

8

10

12

14

16

18

Fig. 4. Changes of cell number (A) and chlorophyll concentration(B) under various light intensities during hydrogen production bysulfur-deprived C. reinhardtii culture.

illumination and consequently increasing hydrogenproduction (Table 1).

3.3. Optimizing light intensity for hydrogen productionin sulfur-deprived C. reinhardtii cultures

The changes in cell number and chlorophyll concen-tration were monitored under light intensities from 60

to 300 �Em−2 s−1 during hydrogen production. In allcases, both the cell number and chlorophyll concentra-tion increased sharply during the initial 24 h and thendecreased gradually (Fig. 4). The increases in both cellnumber and chlorophyll concentration were saturated atthe highest levels at a light intensity of 200 �E m−2 s−1

during the initial 24 h. As shown in Fig. 4, the cellnumber and chlorophyll concentration decreased moresharply after 70 h under illumination at 300 �E m−2 s−1

of light than under any other light conditions. TheChlamydomonas cells cultured at 300 �E m−2 s−1 oflight seemed to be inhibited by excessive light. Accord-ingly, the hydrogen production at 300 �E m−2 s−1 waslower than that at 200 �E m−2 s−1. Although the timefor hydrogen production was longer at 300 �E m−2 s−1,the chlorophyll concentration was greater during theinitial 24 h under 200 �E m−2 s−1 of light. Therefore,the specific production rate of hydrogen was maximalat 200 �E m−2 s−1; the maximum volume of hydrogenproduced was 2.01 ml H2 g−1 cell h−1 (Table 1).

4. Discussion

Light is critical for photosynthetic microorganismssuch as green algae and photosynthetic bacteria. We ob-served that the rate of cell growth and sulfate consump-tion were dependent on light intensity in C. reinhardtiiculture (Fig. 1) and were saturated at 200 �E m−2 s−1 oflight intensity. Wykoff et al. also reported that the rate ofoxygen evolution by C. reinhardtii increased dependingon light intensity and that oxygen evolution became sat-urated at light intensities above 200 �E m−2 s−1 [12,13].Based on those results, we hypothesized that light inten-sity might affect hydrogen production in C. reinhardtiicultures because the conversion to the sulfur-deprivedcondition was dependent on the light intensity.

We found that hydrogen production in sulfur-deprivedChlamydomonas cultures was dependent on light inten-

J. P. Kim et al. / International Journal of Hydrogen Energy 31 (2006) 1585–1590 1589

sity. The maximum hydrogen production was obtainedat 200 �E m−2 s−1 of light, and the maximum hydrogenproduction was 2.6-fold that at 60 �E m−2 s−1 of light.There are two reasons for this hydrogen production de-pendence on light intensity in sulfur-deprived culturesat up to 200 �E m−2 s−1 of light. The first is that theconversion to the hydrogen production stage was accel-erated by high light intensity, resulting in prolonged hy-drogen production time (Table 1). The result that starttime of hydrogen production depend on light intensityhas also been reported previously [13]. However, we dis-cussed this phenomenon with rate of sulfate consump-tion after sulfur deprivation. As shown in Fig. 3 andTable 1, consumption rate of residual sulfate accordingto light intensity brought about the lag time of hydro-gen production in sulfur-deprived culture. Accordingly,the sulfate consumption rate was important to hydrogenproduction after sulfur was removed from the culturemedium. Residual sulfate in the culture was responsi-ble for the lag time of hydrogen production. The sulfur-deprived condition was more quickly induced in cul-tures at higher light intensities; hydrogenase was thenactivated more quickly because the culture conditionmore rapidly became anaerobic; and consequently, hy-drogen synthesis by the activated hydrogenase begansooner. Thus, the period of hydrogen production wasprolonged in proportion to the light intensity, resultingin greater hydrogen production.

The second reason is that the cell number and chloro-phyll concentration increased with increasing lightintensity during the initial 24 h of the hydrogen pro-duction stage (Fig. 4). When light is absorbed by theantenna chlorophyll in the photosynthetic membranesof C. reinhardtii, electrons are released from an oxygen-evolving complex through the water-splitting reaction,and then hydrogen is synthesized from the two elec-trons plus two protons by hydrogenase under anaerobicconditions. Therefore, more hydrogen was producedat higher light intensity owing to the conversion ofmore electrons being released at high chlorophyll con-centration.

However, hydrogen production decreased at 300�E m−2 s−1 of light even though the hydrogen produc-tion time was longer and the energy available for elec-tron production was greater than at 200 �E m−2 s−1.This was because the cell number and chlorophyllconcentration were reduced sharply at 300 �E m−2 s−1

compared to the values at 200 �E m−2 s−1 after 24 hof sulfur deprivation (Fig. 4). According to studies byWykoff et al. [7], Melis et al. [4], and Ghirardi et al.[6], the oxygen evolution capacity decreased to approx-imately 30% of the initial value within 24 h after sulfate

was removed from the culture medium. The decline inphotosynthetic oxygen evolution resulted from the con-version of PS II centers from the QB-reducing to theQB-nonreducing form, which is an intermediate in thePS II repair cycle [19]. The QB-nonreducing form ac-cumulated because the photodamaged D1 could not beefficiently repaired due to the lack of sulfurated aminoacids such as methionine and cysteine. Accordingly,the destruction of PS II is accelerated with increasinglight intensity under sulfur-deprived conditions, result-ing in the rapid reduction of chlorophyll and cell lysis(Fig. 4). Therefore, the low hydrogen yield at a highlight intensity under sulfur-deprived conditions wasattributable to the accelerated photodamage of PS II.

Recently, Melis’s group worked to lower the rateof photodamage by genetically truncating the chloro-phyll antenna size of the photosystems; this improvedlight utilization efficiency of the alga and can apply inalgal hydrogen production under mass culture condi-tions [20,21].

In conclusion, light intensity was an important fac-tor for the rapid conversion to anaerobic conditions byresidual sulfate consumption in C. reinhardtii cultures.Hydrogen production increased proportionally to lightintensity up to 200 �E m−2 s−1 in this system; increasedhydrogen production was attributable to rapid conver-sion to the hydrogen production stage and increasedchlorophyll concentration during the initial 24 h aftersulfur deprivation. However, hydrogen production de-creased at the highest light intensity (300 �E m−2 s−1)

owing to rapid chlorophyll decomposition and cell lysis.Therefore, to enhance hydrogen production in sulfur-deprived C. reinhardtii cultures, it will be necessary tocontrol the light intensity as also reported by previousauthors [12,13] in order to minimize photodamage ofthe cells and maintain the hydrogen production yield.Our results may be useful in designing a photoreactorfor algal hydrogen production by increasing the lightintensity range without light inhibition.

Acknowledgment

This research was performed for the HydrogenEnergy R&D Center, one of the 21st Century FrontierR&D Program, funded by the Ministry of Science andTechnology of Korea.

References

[1] Das D, Veziroglu TN. Hydrogen production by biologicalprocesses: a survey of literature. Int J Hydrogen Energy2001;26:13–28.

1590 J. P. Kim et al. / International Journal of Hydrogen Energy 31 (2006) 1585–1590

[2] Gaffron H, Rubin J. Fermentative and photochemical productionof hydrogen in algae. J Gen Physiol 1942;26:219–40.

[3] Appel J, Schulz R. Hydrogen metabolism in organisms withoxygenic photosynthesis: hydrogenases as important regulatorydevices for a proper redox poising? J Photochem Photobiol1998;47:1–11.

[4] Melis A, Zhang L, Forestier M, Ghirardi ML, SeibertM. Sustained photobiological hydrogen gas production uponreversible inactivation of oxygen evolution in the green algaChlamydomonas reinhardtii. Plant Physiol 2000;122:127–35.

[5] Antal TK, Krendeleva TE, Laurinavichene TV, Makarova VV,Tsygankov AA, Seibert M. et al. The relationship betweenthe photosystem 2 activity and hydrogen production in sulfurdeprived Chlamydomonas reinhardtii cells. Biochem BiophysMol Biol 2001;381:371–4.

[6] Ghirardi ML, Zhang L, Lee JW, Flynn T, Seibert M, GreenbaumE. et al. Microalgae: a green source of renewable H2. TrendsBiotechnol 2000;18:506–11.

[7] Wykoff DD, Davies JP, Melis A, Grossman AR. The regulationof photosynthetic electron transport during nutrient deprivationin Chlamydomonas reinhardtii. Plant Physiol 1998;117:129–39.

[8] Antal TK, Krendeleva TE, Laurinavichene TV, Makarova VV,Ghirardi ML, Rubin AB. et al. The dependence of algal H2production on Photosystem II and O2 consumption activities insulfur-deprived Chlamydomonas reinhardtii cells. Biochimicaet Biophysica 2003;1607:153–60.

[9] Tsygankov A, Kosourov S, Seibert M, Ghirardi ML. Hydrogenphotoproduction under continuous illumination by sulfur-deprived, synchronous Chlamydomonas reinhardtii cultures. IntJ Hydrogen Energy 2002;27:1239–44.

[10] Kosourov S, Tsygankov A, Seibert M, Ghirardi ML.Sustained hydrogen photoproduction by Chlamydomonasreinhardtii: effects of culture parameters. Biotechnol andBioeng 2002;78:731–40.

[11] Kosourov S, Seibert M, Ghirardi ML. Effect of extracellularpH on the metabolic pathways in sulfur-deprived, H2-producing Chlamydomonas reinhardtii cultures. Plant CellPhysiol 2003;44:146–55.

[12] Hahn JJ, Ghirardi ML, Jacoby WA. Effect of process variableon photosynthetic algal hydrogen production. Biotechnol Prog2004;20:989–91.

[13] Laurinavichene T, Tolstygina I, Tsygankov A. The effectof light intensity on hydrogen production by sulfur-deprivedChlamydomonas reinhardtii. J Biotech 2004;114:143–51.

[14] Kim JP, Kang CD, Sim SJ, Kim MS, Park TH, Lee DH. et al.Cell age optimization for hydrogen production induced by sulfurdeprivation using a green alga Chlamydomonas reinhardtiiUTEX 90. J Microbiol Biotechnol 2005;15:131–5.

[15] Gorman DS, Levine RP. Cytochrom f and plastocyanin: theirsequence in the photosynthetic electron transport chain ofChlamydomonas reinhardtii. Proc Natl Acad Sci 1965;54:1665–9.

[16] Davies JP, Yildiz FH, Grossman AR. Sac1, a putative regulatorthat is critical for survival of Chlamydomonas reinhardtii duringsulfur deprivation. EMBO J 1996;15:2150–9.

[17] Harris EH. The Chlamydomonas sourcebook: a comprehensiveguide to biology and laboratory use. San Diego: AcademicPress; 1989.

[18] DIONEX Corporation, Determination of low concentrationsof perchlorate in drinking and groundwaters using ionchromatography, Application note 134, 2004. Availablefrom: 〈http://www1.dionex.com/en-us/by_technique/ic/anion/lp5008.html〉, accessed 16 January 2006.

[19] Melis A. Photosystem-II damage and repair cycle inchloroplasts: what modulates the rate of photodamage in vivo?Trends Plant Sci 1999;4:130–5.

[20] Melis A. Green alga hydrogen production: progress, challengesand prospects. Int J Hydrogen Energy 2002;27:1217–28.

[21] Polle JEW, Kanakagiri S, Jin ES, Masuda T, Melis A.Truncated chlorophyll antenna size of the photosystems—apractical method to improve microalgal productivity andhydrogen production in mass culture. Int J Hydrogen Energy2002;27:1257–64.