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Repeated production of hydrogen by sulfate re-addition insulfur 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 Koreab Polar BioCenter, Korea Polar Research Institute, KORDI, Incheon 406-840, Republic of Koreac Biomass Research Team, Korea Institute of Energy Research, Daejeon 305-343, Republic of Korea

a r t i c l e i n f o

Article history:

Received 9 November 2009

Accepted 18 November 2009

Available online 24 December 2009

Keywords:

Repeated hydrogen production

Green algae

Chlamydomonas reinhardtii

Sulfur deprivation

Sulfate re-addition

pH adjustment

* Corresponding author. Tel.: þ82 31 290 734E-mail address: simsj@skku.edu (S.J. Sim

0360-3199/$ – see front matter ª 2009 Profesdoi:10.1016/j.ijhydene.2009.11.113

a b s t r a c t

Biological hydrogen production by the green alga, Chlamydomonas reinhardtii can be induced

in conditions of sulfur deprivation. In this study, we investigated the repeated and

enhanced hydrogen production afforded by the re-addition of sulfate with monitoring of

pH and concentration of chlorophyll and sulfate. Without adjustment of the pH, the

optimal concentration of re-added sulfate was 30 mM for the hydrogen production. By the

re-addition of 30 mM of sulfate and the adjustment of the pH during 4 cycles of repeated

production, we obtained the maximum amount of 789 ml H2 l�1 culture, which is 3.4 times

higher than that of one batch production without adjustment of pH, 236 ml H2 l�1 culture.

This means that the enhancement of the hydrogen production can be achieved by the

careful control of the sulfate re-addition and pH adjustment in the sulfur deprived culture.

ª 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

1. Introduction Green algae can generate hydrogen from light and water,

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 bio-

logical method of producing hydrogen using green algae in

order to reduce this dependence [1–3].

1; fax: þ82 31 290 7272.).sor T. Nejat Veziroglu. Pu

both of which are plentiful resources in nature [4]. In green

algae, hydrogen photoproduction is catalyzed by hydroge-

nase, 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 photo-

synthetic 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 photosyn-

thesis 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 sepa-

ration of the photosynthetic O2 and anaerobic H2 evolution

activities in the green alga, Chlamydomonas reinhardtii (two

blished by Elsevier Ltd. All rights reserved.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 3 3 8 7 – 1 3 3 9 113388

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 concentra-

tions of sulfate ranging from 0 to 120 mM were re-added to the

cell suspensions after sulfur deprivation, and these re-addi-

tions 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 concen-

tration, 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 m�2 s�1 [8]. The cell number

was counted by an improved Neubauer ultraplane hemocy-

tometer. The dry cell weight (DCW) was measured using

a dried filter paper method at 80 �C.

2.2. Repeated production of hydrogen by sulfatere-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 ml�1) by centrifu-

gation 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-sus-

pended in the TAP-S medium with a concentration of about

1.3 � 107 cells ml�1 (0.8 g DCW l�1), and then sulfur was re-

added 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 culti-

vated under continuous fluorescent light intensity

(200 mE m�2 s�1) at 25 �C and 150 rpm for a total of 560 h. In order

to fix the pH after sulfur deprivation, 11.92 g l�1 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 centrifu-

gation 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 chlo-

rophyll 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 cali-

brated with a known concentration of H2. The sulfate (SO42�)

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 therepeated 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 m�2 s�1 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 ml�1. Sulfur was re-

added 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 concen-

tration) to the sulfur deprived cell suspensions during 4 cycles

resulted in an increase in the total volume of hydrogen (112 ml

H2 l�1 culture upon no re-addition of MgSO4; 479 ml H2 l�1

culture at re-addition of 15 mM MgSO4; 605 ml H2 l�1 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 l�1 culture) and hydrogen production

Time after sulfur deprivation (h)

0 100 200 300 400 500 600

Hy

dro

ge

n p

ro

du

ctio

n (m

l/l)

0

50

100

150

200

250 sulfate 0 µMsulfate 15 µMsulfate 30 µMsulfate 60 µMsulfate 120 µM

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 re-

addition depending on the sulfate concentration. (The error

bars illustrate the relative standard deviation (RSD) of three

replicates).

Time after sulfur deprivation (h)

0 100 200 300 400 500

Re

sid

ua

l s

ulfa

te

c

on

ce

ntra

tio

n (m

g/l)

0

2

4

6

8

10

12

14 Sulfate 0 µMSulfate 15 µMSulfate 30 µMSulfate 60 µMSulfate 120 µM

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.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 3 3 8 7 – 1 3 3 9 1 13389

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 l�1 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

Time after sulfur deprivation (h)

0 100 200 300 400 500 600

Ch

lo

ro

ph

yll co

ncen

tratio

n (m

g/l)

0

10

20

30

40

50

60

70sulfate 0 µM sulfate 15 µM sulfate 30 µM sulfate 60 µM sulfate 120 µM

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.

case. The final chlorophyll concentrations after 4 cycles were

different in all cases (5.5 mg l�1 after the re-addition of 15 mM

MgSO4; 10.7 mg l�1 after the re-addition of 30 mM MgSO4;

32.8 mg l�1 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 l�1 (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 l�1 and

no hydrogen was produced.

3.2. Hydrogen production by control of pH duringfour 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-addi-

tion. 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 l�1 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 chlo-

rophyll 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 l�1 at the first cycle, 30.1 mg l�1 at

the second cycle, 20.9 mg l�1 at the third cycle, and 9.7 mg l�1

at the fourth cycle. However, in the case of the sulfur deprived

culture with HEPES, the final chlorophyll concentrations were

36.8 mg l�1 at the first cycle, 35.3 mg l�1 at the second cycle,

Hp

7.0

7.2

7.4

7.6

7.8

8.0

8.2

8.4

8.6

8.8

sulfate 0 µM sulfate 15 µM sulfate 30 µM sulfate 60 µM sulfate 120 µM

Time after sulfur deprivation (h)

0 100 200 300 400 500

0 100 200 300 400 500

Hp

6.0

6.5

7.0

7.5

8.0

8.5

9.0

A

B

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.

Time after sulfur deprivation (h)

0 100 200 300 400 500 600

Ch

lo

ro

ph

yll co

ncen

tratio

n (m

g/l)

0

10

20

30

40

50

60

70

Without HEPES bufferWith HEPES buffer

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.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 3 3 8 7 – 1 3 3 9 113390

26.5 mg l�1 at the third cycle, and 15.2 mg l�1 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.

Time after sulfur deprivation (h)

0 100 200 300 400 500

Hyd

ro

gen

p

ro

du

ctio

n (m

l/l)

0

50

100

150

200

250

300

With HEPES buffer Without HEPES buffer

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.

4. Discussion

The repeated production of hydrogen by sulfur deprived

C. reinhardtii culture depended on the re-addition of micro-

molar 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 respon-

sible 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 water-

splitting 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 concen-

trations 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 concen-

tration, viz. 2 mg sulfate/l culture, not at a sulfate concentra-

tion 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

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 3 3 8 7 – 1 3 3 9 1 13391

hydrogen productivity (Fig. 3). When the sulfur deprived

condition was more quickly induced, hydrogenase was acti-

vated 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 re-

addition 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 re-

addition 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 equiva-

lents from the residual PSII H2O-oxidation are the key factor for

H2 production by C. reinhardtii. After sulfur deprivation, the re-

addition 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 produc-

tivity, 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.

Acknowledgments

This Research was performed for the Hydrogen Energy R&D

Center, one of the 21st Century Frontier R&D Programs, fun-

ded by the Ministry of Science and Technology of Korea.

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