Journal of Biological Education

ISSN: 0021-9266 (Print) 2157-6009 (Online) Journal homepage: http://www.tandfonline.com/loi/rjbe20

Light dependent production of hydrogen gas

by green algae. The future energy carrier in the
classroom?

Rbbe Wnschiers

To cite this article: Rbbe Wnschiers (2000) Light dependent production of hydrogen gas by
green algae. The future energy carrier in the classroom?, Journal of Biological Education, 34:4,
214-217, DOI: 10.1080/00219266.2000.9655721

To link to this article: http://dx.doi.org/10.1080/00219266.2000.9655721

Published online: 13 Dec 2010.

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 Interactive Learning
Light dependent production
of hydrogen gas by green
algae. The future energy
carrier in the classroom?
Robbe Wiinschiers
Department of Physiological Botany, Uppsala University, Uppsala, Sweden
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Hydrogen gas is regarded as a potential candidate for a future energy economy. Research and development
in the field of hydrogen energy is greatly encouraged on all continents. A wide range of microorganisms are
able to produce hydrogen gas, among them photosynthetically active organisms that use light as their sole
energy source. These organisms are good candidates for the photobiological production of hydrogen gas.
Green algae are of particular interest since they are capable of splitting water during photosynthesis and of
releasing hydrogen gas under certain conditions. This article describes a small bioreactor that can be run in
the classroom and used to demonstrate the concept of photohydrogen production.
Key words: Green Algae, Bioreactor, Energy, Hydrogenases, Photosynthesis.

Introduction Developments like metal hydrides, carbon nanotubes, or glass

7 believe that water will one day be employed as fuel, that
hydrogen and oxygen which constitute it, used singly or togeth
er, will furnish an inexhaustible source of heat and light, of an
intensity of which coal is not capable. I believe then that when
the deposits of coal are exhausted, we shall heat and warm
ourselves with water. Water will be the coal of the future.'
What Jules Vernes proposed in his novel L'ile mysterieuse in
1870 is now known as one of most modern future energy con
cepts. However, it is not only the depletion of fossil fuels that
forces us to investigate new energy sources. If that were the
case, we would focus our research on improving nuclear fission
energy. Nowadays, public acceptance and environmental con
cerns push international research for alternative energy sources.
Increasingly, renewable and sustainable sources of energy (e.g.
sunlight, wind power, biomass, geothermal resources, and
hydroelectric power) are contributing to meet our energy
demand. Most of these sources are coupled to certain localities
such as sun or wind rich areas. The energy then needs to be
moved from the place of production to where it is needed.
Furthermore, it has to be stored, since most renewable energy
sources are not available continuously.
In the form of hydrogen, energy can be stored until it is need
ed and transported to where it will be used. The benefits of
hydrogen make it an ideal component of a renewable, sustain
able energy system of the future. It can be produced from water,
an abundant source, using renewable electricity (electrolysis), or
sunlight via microorganisms. Technologies for storage and trans
portation of hydrogen, generally as compressed gas or cryogenic
liquid, are commercially available and already in use.
microspheres are promising to further increase the energy con
tent per weight and volume of storage systems. Fuel cells, inter
nal combustion engines, and hydrogen burners are sophisticated
devices, which convert hydrogen's energy content into electric
ity, mechanical work, or heat. The principle by-product is water,
which can be safely returned to the environment. In fact, pro
jects in several countries already demonstrate the safe and effi
cient use of hydrogen gas to drive public buses or generate heat
and electricity in pilot power plants.
Still more than 96 per cent of world wide hydrogen produc
tion depends on fossil energy resources and consumes as much
as 2 per cent of the world's energy demand. Several interna
tional programs focus on alternative ways of generating pollu
tion-free hydrogen. As already mentioned, one such alternative
is biological production (Benemann, 1996; Zaborsky, 1998).
Several microorganisms contain enzymes, known as hydroge
nases, that either oxidize hydrogen to protons and electrons or
reduce protons and thus release molecular hydrogen. The phys
iological role and biochemical characteristics of these hydroge
nases are variable (Adams et al., 1981; Boichenko and
Hoffmann, 1994). Most biologically produced hydrogen in the
biosphere evolved in microbial fermentation processes. These
organisms decompose organic matter to carbon dioxide and
hydrogen as was shown 100 years ago by the biochemist Hoppe-
Seyler. The reduction of protons to hydrogen serves to dissipate
excess electrons within the cell and usually permits additional
energy generating steps in metabolism. The hydrogen gas pro
duced is usually taken up directly by hydrogen consumers with
in the same ecosystem. These organisms use the reducing power
of hydrogen to drive metabolic processes. Hydrogen bacteria

214 Journal of Biological Education (2000) 34(4)

 ( ) Photobiological production of hydrogen gas Wiinschiers

(Knallgas bacteria) can even grow autotrophically with hydro
gen gas as the sole reducing agent and energy substrate. In these
bacteria, oxygen serves as the terminal electron acceptor, thus,
water is formed in a biological Knallgas reaction. Although it is
estimated that 200 million tons of hydrogen are cycled within
these ecosystems each year, the atmosphere only harbours some
0.000078 per cent (v/v) hydrogen (Belaisch et al, 1990).
Around 60 years ago Gaffron and co-workers showed that the
green alga Scenedesmus obliquus could metabolize molecular
hydrogen (Gaffron, 1939). Although the physiological signifi
cance of these results is still a matter of basic research in the case of
algae (see discussion), the process of photohydrogen production
by green algae is of interest because it generates hydrogen gas from
the most plentiful resources: light and water (Schulz, 1996).
Unfortunately, hydrogen production by this process is quite inef
fective as oxygen is simultaneously produced and inhibits the
hydrogenase enzyme. Thus, in high light conditions, hydrogen
evolution usually ceases after several minutes due to an accumula
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Anaerobic adaptation
Two different setups for the bioreactor are shown in Figure 1.
The concentrated algal suspension is placed in a bottle which is
sealed with a rubber stopper. When filled only a small gas vol
ume (max. 50 ml) should be left. Flasks used for growing tissue
cultures are the most suitable (Figure 1(b)). Anaerobic adapta
tion can be performed either by flushing the suspension with
nitrogen for 3 hours or by placing in the dark overnight. The lat
ter results in anaerobic conditions due to cell respiration. After
anaerobic adaptation, add 2 g of Na-dithionite to the culture.
This reducing agent will take up the remaining oxygen and also
the oxygen produced by photosynthesis while running the
bioreactor.
Running the bioreactor
After adding Na-dithionite, the algal suspension should be illu
minated with the bright light from a slide or an overhead pro
jector. Connecting a graduated pipette to the flask (Figure 1 (a))

tion of oxygen. Current research is trying to overcome oxygen sen allows the production of hydrogen gas to be followed directly.
sitivity by physiological, biochemical, or genetic means. Also, small gas samples can then be withdrawn and analysed by
In order to demonstrate photobiological production of hydro
gen in the classroom, the small scale bioreactor described here
was developed. Its educational value lies in the combination of
both pure and applied biology. The bioreactor can be discussed
in the context of photosynthesis and draws a bridge between
physiology, ecology, and biotechnology. Taking into account that
hydrogenases are common in many prokaryotes, rarely found in
simple eukaryotes, and not at all found in higher eukaryotes is
an aspect which might be discussed in evolutionary terms
(Vourdouw, 1992). The experiment was tested as part of bio
logical education on two classes at a German Realschule (10th
year) and Gymnasium (12th year). For undergraduate teaching
the bioreactor was set up and discussed in practical courses on
plant physiology and eukaryotic microbiology.

Materials and Methods

Cell growth and harvesting
The results described here were obtained with the green alga
Scenedesmus obliquus, but a range of other species such as
Chlamydomonas are also suitable. S. obliquus can be grown eas
ily either photoautotrophically in the light, or heterotrophically
in the dark, at 30C. Heterotrophical growth is favourable
because the cultures need only be shaken and cells grow within
a few days using the following medium: 8 mM KN0 3 , 8 mM
NaCl, 1 mM Na 2 HP0 4 , 3 mM NaH 2 P0 4 , l mM CaCl2, 550 pM
Na-citrate, 100 pM MgS0 4 , 45 pM H3BO. 7.5 pM Fe 2 (S0 4 ) 3 ,
0.7 pM ZnS0 4 , 0.3 pM CuS0 4 , 0.1 pM Mo0 3 , 0.08 pM MnCl 2 ,
0.5 per cent (w/v) glucose, 0.25 per cent (w/v) yeast extract.
For photoautotrophic growth both glucose and yeast extract are
omitted. It is recommended to prepare stock solutions. If no
autoclave and shaker are present boil 1000 ml of medium in a
3000 ml Erlenmeyer flask, allow to cool to room temperature,
inoculate with algae, and place on a magnetic stirrer. Stirring or
shaking is needed to aerate the cell suspension. For the bioreac Figure 1 (a) Simple setup of the bioreactor. The algal suspension in the
round flask was stirred to increase the number of illuminated cells. A grad
tor about 15 g (wet weight) of algae are needed which corre uated pipette with a water droplet was connected (on the left] to follow the
sponds to 3000 ml culture grown for 7 days. Cells can be har gas production volumetrically. Additionally, a Clark-type electrode was con
vested by centrifugation or by allowing them to settle overnight nected (on the right) to specifically measure hydrogen in the atmosphere of
the bioreactor. (b) A more developed bioreactor where the cells settle down
and decanting. The final cell suspension should be concentrated in a cell growth flask. Stirring was not necessary. The bioreactor was con
to 15 g (wet weight) in 150 ml medium. nected to a fuel cell, which drives a small electric motor.

Journal of Biological Education (2000) 34(4) 215

 f j Photobiological production of hydrogen gas
gas chromatography (see below) or a small scale Knallgas reac
tion. Figure 1 (b) shows a fuel cell connected to the bioreactor.
Usually the gas pressure is insufficient to supply the fuel cell
with hydrogen. In this case, the atmospheric pressure in the
bioreactor can be increased by adding nitrogen gas with a
syringe.
Measuring hydrogen gas
To measure hydrogen gas, both qualitatively and quantitatively,
two prominent techniques are available: Clark-type electrodes
and gas chromatography. The Clark-type electrode is a sensitive
instrument for studying hydrogen metabolism, where it is possi
ble to detect both low levels and small differences of hydrogen
concentrations. Moreover, the electrode allows measurements in
the gas phase as well as in aqueous solution. It consists of a Pt- and
a reference Ag/AgCl-electrode covered by a film of half-saturated
KC1 electrolyte enclosed within a Teflon membrane. Originally
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developed for measuring oxygen gas, it is only a matter of polari
ty, whether the electrode senses hydrogen or oxygen gas.
However, using a Clark-type electrode needs skill and experience.
Another highly sensitive method for measuring hydrogen gas
is gas chromatography. The gas chromatograph should be
equipped with a Molecular Sieve 5A column (40 - 60 mesh]
and a thermal conductivity detector. With a 1800 x 6.4 mm col
umn, an oven temperature of 100C, and nitrogen as a carrier
gas at 30 ml min"1, retention times of approximately 1 min can
be expected. A detailed discussion of how to use both polaro-
graphic and gas chromatographic hydrogen measurements can
be found in Hanus et al. (1980).
Results and Discussion
The small-scale bioreactor described here is an easy way to
demonstrate the light dependent production of molecular
hydrogen by green algae and has been successfully setup by
pupils. If you are not equipped to work sterile, it is suggested to
grow the cultures autotrophically to minimise the danger of
contaminating the culture with bacteria. The algae might also
be grown at room temperature, although growing rates are sig
nificantly slower than at 30C.
To get significant hydrogen production the anaerobic adapta
tion of the algae is prerequisite. This adaptation process should
be performed in the dark to avoid generation of oxygen by pho
tosynthesis. It has been shown that during anaerobic adaptation
the activity of the hydrogenase enzyme is induced. The exact
mechanism for this regulation is not well understood
(Wunschiers et al., 1999). The fastest procedure would be to
flush the algae with nitrogen or another inert gas like argon. In
this case, the gas used replaces all other gases. Creating anaero
bic conditions by respiration has the disadvantage of taking
longer (over night). It is not possible to induce hydrogenase
activity by addition of Na-dithionite!
When the algae are illuminated, hydrogen gas is produced as
shown in Figure 2. The actual rates and volumes may vary since
they are influenced by the age of the algae or light quality. Gas
chromatographic analyses of the produced gas showed it to be
pure hydrogen.
The physiological background
In general, green algae are able either to take up or produce
hydrogen in the light, depending on physiological and environ-
216
Wunschiers
10 -i 1
3 6- /
0 60 120 180
Minutes
Figure 2 Photohydrogen production of Scenedesmus obliquus as measured
volumetrically. The setup was as in Figure 1 (b). Ten grams (wet weight) of
algae were used and illuminated by the sun.
mental conditions (Boichenko and Hoffmann, 1994; Schulz,
1996). The uptake of hydrogen is called 'photoreduction of car
bon dioxide' since hydrogen donates the electrons for carbon
dioxide reduction (Figure 3(a)). This reaction takes place in the
presence of high partial pressures of both hydrogen and carbon
dioxide and both gases are taken up in a 2:1 ratio. This reaction
probably plays no significant role in nature. Although algae are
likely to encounter anaerobic conditions in their habitat, it is
very unlikely that they face high partial pressures of hydrogen. It
is believed that photoreduction of carbon dioxide can be divided
into different reactions (Figure 3(a)). One reaction is the light
independent reduction of NADP by hydrogen via a hydrogenase
(H 2 ase), its natural redox partner ferredoxin (Fd), and the
enzyme ferredoxin NADP-reductase (FNR). A second, light
dependent reaction provides ATP via cyclic electron transport
around photosystem I (PS I). In this process ferredoxin (Fd), the
proton gradient generating cytochrome b ( f-complex (Cyt b6f)
and plastocyanin (PC) are involved. Both, NADPH and ATP
supply the Calvin cycle with reductive power and energy for car
bon dioxide fixation.
The production of hydrogen is called 'photohydrogen pro
duction' and takes place in the absence of high amounts of
atmospheric hydrogen gas and carbon dioxide. Both the water
splitting photosystem II (PS II) and the ferredoxin (Fd) reduc
ing photosystem I (PS I) were shown to be involved in this
process (Figure 3(b)). Due to the absence of carbon dioxide, the
Calvin cycle can not take over electrons from the photosynthet-
ic light reactions via NADPH. Thus, it is believed that a hydro
genase (H2ase) serves as a valve by oxidising ferredoxin (Fd) and
releasing hydrogen. This process enables the algae to generate a
proton gradient for ATP production. Furthermore, the genera
tion of reactive oxygen species due to a queue of electrons in the
photosystems is prevented. On the long term, photosynthesis is
down regulated by structural changes like the dissociation of
light harvesting complexes from the photosystems. This down
regulation of photosynthesis together with the inactivation of
the hydrogenase by the produced oxygen will result in a
Journal of Biological Education (2000) 34(4)
 Q Photobiological production of hydrogen gas Wunschiers

info@h-tec.com; w e b site: www.h-tec.com

H, Warsitz Enterprises, Inc., P O Box 3 5 5 5 , San Jose, Califor
^fEbase] -* Fd -* [ F N R ] ^ nia 9 5 1 5 6 , U S A . E-mail: h 2 m a n @ s l i p . n e t ; w e b site:
NADPH + H
www.warsitz.com

Chemicals and laboratory equipment

ATP Sigma-Aldrich Techware, P O Box 14508, St. Louis, Mis
souri 6 3 1 7 8 , U S A . E-mail: sigma@sial.com; w e b site:
Fd
www.sigma-aldrich.com
/
/ \
Acknowledgement
Cyt b 6 /f ATPase
M T h e a u t h o r wishes to thank Professor L e h m a n n (University of
Stralsund, G e r m a n y ) for providing t h e fuel cell. For scholarships
PC-> PS I I thank t h e Studienstiftung des D e u t s c h e n Volkes ( G e r m a n y )
and t h e Angpanneforeningens Forskningsstiftelse (Sweden).

References
Adams, M.W.W., Mortenson, L.E., and Chen, J.-S. (1981) Hydrogenase.
Downloaded by [Orta Dogu Teknik Universitesi] at 12:37 04 March 2016

II, Biochimica and Biophysica Ada, 594, 105 - 176.

J^H2ase Belaich, J.-P., Bruschi, M. and Garcia, J.-L. (1990) Microbiology and bio
chemistry of strict anaerobes involved in interspecies hydrogen transfer.
X New York: Plenum Press.
Fd
Benemann, J.R. (1996) Hydrogen biotechnology: progress and
V
/ \ / '\i 5
prospects. Nature Biotechnology, 14, 1101 - 1103.
Boichenko, V.A. and Hoffmann, P. (1994) Photosynthetic hydrogen
production in prokaryotes and eukaryotes: Occurrence, mechanism
Q _ * Cytb6/f and functions. Photosynthetica, 30, 527 - 552.
P
M Gaffron, H. (1939) Reduction of carbon dioxide with molecular hydro
/ \ gen in green algae. American Journal of Botany, 27, 273 - 283.
PS II PC PS I I Hanus, EX, Carter, K.R. and Evans, H.J. (1980) Techniques for mea
J surement of hydrogen evolution by nodules. Methods in Enzymology,
69,731 - 7 3 9 .
H,0 Vi O, + 2 H + Schulz, R. (1996) Hydrogenases and hydrogen production in eukaryot-
ic organisms and cyanobacteria. Journal of Marine Biotechnology, 4, 16
Figure 3 Light dependent hydrogen metabolism in the green alga -22.
Scenedesmus obliquus: (a) Photoreduction of carbon dioxide, (b) Voordouw, G (1992) Evolution of hydrogenase genes. Advances in
Photohydrogen production. For detailed information, please see text. Inorganic Chemistry, 38, 397 422.
Abbreviations used: S = chloroplast stroma, M = thylakoid membrane, L = Wunschiers, R., Heide, H., Follmann, H., Senger, H. and Schulz, R.
thylakoid lumen, H2ase = hydrogenase, Fd = ferredoxin, FNR = ferredoxin
(1999) Redox control of hydrogenase activity in the green alga
NADP-reductase, Cyt b/f = cytochrome bj-complex, PC = plastocyanin,
PQ = plastoquinone, PS I = photosystem I, PS II = photosystem II, ATPase Scenedesmus obliquus by thioredoxin and other thiols. FEBS Letters,
= ATP synthase, dotted lines = proton translocation, straight lines = electron 455, 1 6 2 - 164.
transfer. Zaborsky, O.R. (1998) Biohydrogen. New York: Plenum Press.

decrease of hydrogen evolution after some time. This time can Web sites
be extensively prolonged by addition of oxygen scavengers like T h e internet site www.photohydrogen.com provides informa
Na-dithionite. It seems t h a t t h e production of hydrogen u n d e r tion and links on this topic.
anaerobic conditions is a regulative device for photosynthesis.

Suppliers Robbe Wunschiers works in the Department of Physiological Botany,

Fuel cells Uppsala University, Villavdgen 6, 75236 Uppsala, Sweden. Tel. +46
18 471 2814; Fax. +46 18 471 2826, E-mail: wuenschi@yahoo.com
H-TEC, Lindenstrasse 48a, 2 3 5 5 8 Liibeck, Germany. E-mail:

Journal of Biological Education (2000) 34(4) 217