Journal of

Photochemistry Y
and
Photobiology
B:Biolosv
ELSEVLER Journal of Photochemistry and Photobiology B: Biology 43 ( 1998) 146-I 5 I

Physiological analyses of the hydrogen gas exchange in cyanobacteria

Refat Abdel-Basset, Klaus P. Bader *
Lehrstrthlftir Zellphysiologie, Fakultiitfiir Biologic, Universitiif Bielefeld, Post&h 100131, D-33.501 Bielefeld, Germany

Received I I February 1998: accepted 14 April 1998

Abstract

Mass spectrometric analysis of the light-induced hydrogen gas exchange in the cyanobacteria Oscillatoriu chalybeu, Synechocystis PCC
6803, Synechococcus PCC 6301 (Synechococcus leopoliensis; Anacystis niduluns) and Synechococcus elongatus has been carried out by
direct detection of molecular hydrogen at m/e = 2 in the H/D collector of a delta mass spectrometer. The time curves of the signals reveal
an initial outburst of hydrogen at the onset of light, with a subsequent superimposition of a hydrogen uptake in the case of Oscillatoriu
chalybea. With in vivo cultures of Svnechocystis PCC 6803, which have not been shown to photoevolve molecular hydrogen so far, we are
able to measure very small but clearly detectable evolution and uptake signals. The principal qualitative features of the transition from
hydrogen evolution to uptake duringthe illuminationperiodin the casesof Oscillutoriu chalybea and Synechocystis PCC 6803 are about
identical. Upon addition of increasing concentrations of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), an increased
stable hydrogen photoevolution shows up with the hydrogen uptake being completely suppressed. In the case of Oscillutoria chal.vbeu this
effect is obtained with 5 J.LM CCCP, whereas with Synechocystis PCC 6803 generally higher CCCP concentrations are required to stabilize
the hydrogen evolution over time. However, in the presence of CCCP the hydrogen evolution from Synechocystis PCC 6803 (which is nearly
negligible in the controls without additions) reaches values very similar to those observed with Oscillatoria chalybea. The stimulatory effect
of CCCP is clearly distinct from an uncoupling of electron transport and photophosphorylation, as the addition of ammonium chloride inhibits
hydrogen evolution but exerts the expectedeffect of stimulationof photosynthetic oxygenevolution.However,modificationof thepH value
of the reaction buffer results in a clear dependency of the hydrogen gas exchange on the external proton concentration. Basic pH values at
about > 9 diminish the gas exchange signal as a whole. Acidic pH at about 4-5 substantially increases the evolution and decreases the uptake
part of the gas exchange signals. Thus, at a pH of 4.4, the hydrogen gas exchange signal largely corresponds to the one observed with an assay
at pH 7.5 but in the presence of CCCP. When the cyanobacterial reaction assays are flushed with pure nitrogen or with N,/CO, ( I%), the
hydrogen evolution rates are by no means decreased; in some cases the hydrogen photoevolution appears rather to be stimulated by the
presence of CO, in the flushing gas. Concomitant recording of the light-induced carbon dioxide reveals a strong initial CO2 uptake which is
substantially diminished within the first minute of illumination. Thus, mass spectrometric analysis of the light-induced carbon dioxide gas
exchange favours the CO, concentrating mechanism discussed for cyanobacteria rather than the light activation of a dark inactive Calvin
cycle. 0 1998 Elsevier Science S.A. All rights reserved.

Keywords: Photosynthesis; Mass spectrometry; Hydrogen photoevolution; Carbon dioxide; Cyanobacteria

1. Introduction dioxide in green algae [ 71. Apparently the plastoquinone

In a variety of cyanobacteria the participation of hydrog-
enase(s) in the light-induced evolution of molecularhydro-
genhasbeendemonstratedand describedwith respectto their
physiological and genetic properties [ 1,2]. An uptake
hydrogenasewhich appearsto be associatedwith the thyla-
koid membranescatalysesexclusively the oxidation of hydro-
gen, thereby consumingthe very hydrogen which hasbeen
formed during nitrogen fixation [ 3-61. This reaction might
be seen in connection with the photoreduction of carbon
* Corresponding author, Fax: +49-521-106-64-10;
biologie.uni-bielefeId.de
pool of the photosynthetic electron-transport chainisrequired
for this uptake reaction, as can be derived from the DBMIB
(2,5-dibromomethylisopropyl-p-benzoquinone) inhibition;
an implication of the ATP supply, i.e., the energy statusof
the cells, has also been discussed[ 8,9 J. A secondenzyme
that is presentin cyanobacteria is called reversible hydroge-
naseand catalysesboth the uptakeof hydrogen and the reduc-
tion of protons [ lO,l 11. Although even some genetic
approacheshave been made to characterize cyanobacterial
hydrogenases[ 12-141, there is little information to date on
the physiological properties of hydrogen-evolving and -con-
E-mail: bader8 suming cyanobacteria. In particular, the regulation of the two

101 l-1344/98/$19.00 0 1998 Elsevier Science .%A. All rights reserved.

PII SlOl l-1344(98)00097-9
 R. Ahdel-Basset, K.P. Buder/ Journal
counteracting reactions is far from understood. This might be
due to the fact that in general a reaction rate of the gas
exchange is determined and to the technical specificities to
record the reaction dynamically and continuously.
This requirement can be fulfilled by a mass spectrometric
set-up with which we were able to demonstrate the hydrogen
gas exchange in the filamentous non-heterocystous nitrogen-
fixing cyanobacterium Oscillatoria chalybea. This organism
exhibits a strong hydrogen evolution upon illumination with
short (5 ps) light flashes or with continuous light; the evo-
lution is, however, diminished by a counteracting hydrogen
uptake that is initiated a few seconds after the onset of light
and which exceeds the evolution within the first minute of
illumination [ 151. A similar transition in the hydrogen gas
exchange has been described for Chlamydomonns and inter-
preted in terms of the light activation of a dark inactive Calvin
cycle, so that during an illumination period increasingly more
electrons are used for the reduction of carbon dioxide [ 161.
In the present paper we, therefore, approached a furtherphys-
iological analysis of the cyanobacterial hydrogen gas
exchange together with a direct consideration of the light-
induced carbon dioxide uptake. Moreover, we investigated
the effect of the protonophore carbonyl cyanide m-chloro-
phenylhydrazone (CCCP) on the hydrogen gas exchange in
Oscillatoria chalyben and Synechocystis PCC 6803. This
compound is often used as an uncoupler of photophosphor-
ylation from the electron transport, but also as ADRY (accel-
eration of the deactivation reactions of the water-splitting
enzyme system Y) reagent for the fast deactivation of the
higher oxidized S-states [ 17-201. Consequently, the direct
effect of CCCP on the photosynthetic oxygen evolution usu-
ally consists of an inhibition in many cases [ 211, but inter-
estingly not in all cases when a silicomolybdate-driven
instead of a ferricyanide-driven Hill reaction was measured.
Moreover, a cyclic electron flow via cyt bss9, which might be
catalysed by CCCP, has been discussed. In this sense elec-
trons appear to cycle around photosystem II with CCCP func-
tioning as a redox component that is oxidized on the donor
side and reduced on the acceptor side [ 22,231.
2. Materials and methods
2.1. Cyanobacteria
The experiments were carried out with Oscillatoria chal-
ybea, Synechocystis PCC 6803, Synechococcus PCC 6301
(Synechococcus leopoliensis; Anacystis nidulans) and Svne-
chococcus elongatus.
Oscillatoria chalybea originated from the Algal Collection
in Giittingen (Germany) and has been cultivated for years in
our laboratory on large clay plates as porous mechanical
support in Petri dishes. The cultures were kept in aclimatized
room at 26C with a 14 h light/l0 h dark cycle at a light
intensity of approximately 12 PE m- s-. The culture
medium was medium D described by Kratz and Myers [ 241.
ojPhotochemistp and Photobiology B: Biology 43 (1998) 146-151 147
This medium contains I g I- sodium nitrate and we have
shown in a previous paper that Oscillatoria chalybea does
not fix atmospheric nitrogen under these conditions. Hence,
hydrogen gas exchange cannot be derived from nitrogenase
activity 1251. Protoplasts from Oscillatoriu chalybea were
isolated according to the procedure involving enzymic diges-
tion (glucuronidase, cellulase) essentially as described ear-
lier [26].
Synechocystis PCC 6803. Synechococcus PCC 6301 and
Synechococcus elongatus were cultivated in gas wash bottles
flushed with air containing 2% CO2 (5% CO, in the case of
Synechococcus elongatus) for two to four days. Details of
the procedure have been described by Michel et al. [ 271. The
thermophilic cyanobacterium Synechococcus elongatus was
cultivated at 50C in the culture medium outlined by Casten-
holz [28]. These cyanobacteria were directly used for the
measurements without any preparation.
2.2. Mass spectrometric analysis
This was carried out with a modified delta Stable Isotope
Ratio Mass Spectrometer from Finnigan MAT (Bremen,
Germany). The various modifications of the inlet system and
the whole set-up, leading to a substantial increase in the
sensitivity and to an improvement of the response time for
dynamic measurements in particular. have been described
earlier [29]. The calibration of the set-up for the precise
quantification of the respective gas signals (oxygen, hydro-
gen, nitrogen and carbon dioxide) has also been outlined
[ 30,25 1. Hydrogen evolution and uptake were dynamically
registered at m/e = 2 in an H/D collector and recorded on an
SE 130-03 three-channel recorder from Goertz Metrawatt.
The signals were obtained by illumination of the cyanobac-
terial assays containing about 25 kg Chl in 0.15 M Tricine/
0.3 M KC1 (pH 7.5) with a Leitz projector through a red filter
transmitting light of 701 nm or by flashing with white light
(vide infra).
2.3. Oxygenjash measurements
These measurements were performed on a large bare sur-
face electrode (three-electrode system) developed and
described in Ref. [ 3 11. Flashes were supplied by the Stro-
boslave 1539A from General Radio (Concord, MA, USA)
and triggered by a laboratory-built pulse generator. At half
intensity the flashes were 5 l~s long and spaced 300 ms apart.
The amperometric oxygen signals were processed by using
the calculation program developed by Schulder et al. for an
Atari Mega ST4 computer [ 321.
3. Results and discussion
In a previous paper we have shown the time-resolved
hydrogen gas exchange of a filamentous non-heterocystous
cyanobacterium (Oscillatoria chalybea) under strictly
148 R. Abdel-Basset. K.P. Bader/Joumal
(a)
Fig. 1. Mass spectrometric
v lmin
recording of the hydrogen gas exchange in Oscil-
Iatoria chalybea detected at m/e = 2 induced by illumination with red light
(701 nm) for 1 min. (a) Control assay containing 25 pg Chl without
additions. (b) Identical assay but in the presence of 5 (*M CCCP yielded a
net hydrogen evolution of 1.4 nM Hz.
anaerobic conditions and upon illumination with continuous
red light or with 5 ks light flashes, respectively. The initial
phase of the light-induced evolution of molecular hydrogen
lasted only for about 10 s and was significantly superimposed
by an uptake phenomenon which exceeded the evolution in
early phases of the illumination and resulted in a net uptake
signal of the overall gas exchange (see Ref. [ 151 and Fig.
1 (a) ) . The ratio between evolution and uptake could by no
means be influenced or shifted, e.g., towards a higher evo-
lution rate: addition of various substrates or pyridin nucleo-
tides had no effect (results not shown). With otherorganisms
such as the unicellular green alga Scenedesmus obliquus, the
photoproduction of hydrogen could be substantially
increased by addition of glucose, ethanol or acetate [33].
Upon addition of the protonophore CCCP, however, the
hydrogen evolution in Oscillatoria chalybea was strongly
stimulated and stabilized during the experiment. Concomi-
tantly, the above-mentioned hydrogen uptake was completely
inhibited or suppressed to zero. Thus, the overall gas
exchange consisted exclusively of an evolution signal in the
presence of 5 FM CCCP (Fig. 1(b) ) . The coccoid cyano-
bacterium Synechocystis PCC 6803, which had hitherto been
shown to photoevolve no or only minimal amounts of molec-
ular hydrogen in control assays without additions [H. Bothe,
personal communication], was in fact able to perform the
.-
Syncrhoqy.stk PCC 5803
mle=2
5 z
5 5p
ii
i-4
I Control
off
P 4- PL
2
t t
? Light on
i?
*
Fig. 2. Mass spectrometric recording of the hydrogen gas exchange in Synechoqstis
nm) for 1 min. (a) Control assay containing 25 pg Chl without additions. (b) Identical assay but in the presence of 5 pM CCCP. (c) Identical assay but in
the presence of 40 p.M CCCP. Sensitivity and all other conditions were as in Fig. 1.
of Photochemistry and PhotobiologJa B: Biology 43 (1998) 146151
same (qualitative) type of reaction but only with a very small
(b) reaction rate (about l-5% of the Oscillatoria signals) and a
generally less pronounced uptake portion of the gas exchange
(Fig. 2(a) ) . However, in this case addition of CCCP exerted
an enormous stimulatory effect on the hydrogen evolution;
in the presence of CCCP the hydrogen evolution of Synecho-
cystis qualitatively and quantitatively corresponded to the
Oscillatoria signals. Thus, it looks as if the observation that
no hydrogen appeared to be evolved by Synechocystis was
due to the counteracting uptake and evolution reactions anni-
hilating each other. Under the condition of an inhibited uptake
and/or a stimulated evolution, a substantial net evolution also
showed up with this cyanobacterium (Fig. 2(b) ) . This com-
pares with the observation made by the group of Bothe, who
described a strong photoevolution of hydrogen with Syne-
chocystis PCC 6803, but only in the presence of methylviol-
ogen/dithionite and other effecters [H. Bothe, personal
communication, and Ref. [ 3411. However, in the case of
Synechocystis, the CCCP-stimulated evolution was less sta-
ble and required higher CCCP concentrations (40 p,M or
more) for an optimal effect (Fig. 2(c) ). Under identical
conditions we observed no mass-spectrometrically measur-
able hydrogen gas exchange with either Synechococcus PCC
6301 (Synechococcus leopoliensis; Anacystis nidulans) or
the thermophilic cyanobacterium Synechococcus elongatus.
The effect of CCCP on the cyanobacteria investigated is
clearly distinct from its property of being an uncoupler of
photophosphorylation, as another well-known uncoupler,
ammonium chloride, induced a concentration-dependent
inhibition of the hydrogen evolution (Fig. 3(a) ) but exerted
the usual effect on the photosynthetic oxygen evolution,
namely a stimulation (Fig. 3 (b) ) . Under the assumption that
NH&l diminishes the trans-membrane proton gradient in
these cyanobacteria as well, it appears that a stable proton
gradient is even required for hydrogen evolution and by no
means limits the electron transport. In the case of oxygen
evolution, CCCP substantially decreased the signals as has
been repeatedly described in the literature [ 2 1,231. Fig. 4
shows the inhibitory effect of 3 p,M CCCP on the oxygen-
evolution amplitudes in Oscillatoria chalybea induced by a
train of 5 p,s flashes according to the Kok model. This effect
(b)
6 syncchoystis PCC6803 (I
CCCP
Off
1
2
e
2:
I i-4
on
, lmin , lmin
ligh fon -
PCC 6803 detected at m/e = 2 induced by illumination with red light (701
 R. Abdel-Basset. K.P. Bader/ Joumol of Photochemist? and Photobiolog? B: Biology 43 (1998) 146151 149

(a) @I m/e = 32
de=2

Control 4 10 Flashes

L
1mM NHbCl Control

1OmM NH&
i R

T t t10 Flashes 10 Flashes

lmin
10 Flashes

-L--- 1 min

Fig. 3. Effect of ammonium chloride on the flash-induced hydrogen evolution detected at m/e = 2 (a) and on the photosynthetic oxygen evolution measured
at m/e = 32 (b). The signals represent the cumulated net evolution amplitudes obtained by a series of ten 5 ps flashes spaced 300 ms apart.

can be correlated with the property of CCCP as being an
ADRY reagent, which means that the higher oxidized redox
states SZ and S3 are deactivated at a higher rate during the
dark time between flashes [ 17-l 91. Consequently, at a given
flash frequency lower oxygen yield amplitudes are obtained
in relation to the controls. Thus, the inhibitory effect of CCCP
in the case of hydrogen uptake and the stimulation of hydro-
gen photoevolution must be explained by a different mech-
anism. From reports in the literature, it has been concluded
that CCCP inhibits the photosynthetic electron transport at
the level of the cytochrome Wfcomplex and/or in the vicin-
ity of the oxygen-evolving complex [35]. Later, it was
reported and discussed in more detail that CCCP catalyses a
cyclic electron flow by the oxidation of cyt bss9 [ 221 or the
membrane pool of plastoquinone [36]. In this sense, the
chemical might function as a redox component that is oxi-
dized and reduced by the respective components of the elec-
tron transport chain.
As it has also been suggestedthat CCCP might supply
protons for photosynthetic reactions [ 231, we investigated
the effect of the pH of the reaction medium on the light-
induced hydrogen evolution in cyanobacteria. Fig. 5 shows
that in fact an increase in the proton concentration of the
respective assayby lowering the pH resultedin a net increase
in the hydrogen evolution and concomitantly in the complete
abolition of the uptake signal. At pH 5.8 the hydrogen-evo-
lution signal was maximally enhanced,whereasat pH 4.5 no
uptake signal at all could be detected and the recording asa
whole strongly resembledthe gas exchange in the presence
of 5 PM CCCP, although the steady-statehydrogen evolution
appearedlessstablein the caseof acidic pH. Our observations
show that the light-induced evolution of molecularhydrogen
can be maximized by carrying out the reaction in the presence
of CCCP, which might beof interest underthe appliedaspects
of photobioreactors where the counteracting oxidation of
hydrogen would be highly unfavourable. Further technical
and principle problemsrelevant to the concept of an efficient
use and application of photobioreactors have recently been
outlined [ 371. The observation that aninduction of hydrogen
evolution in the caseof an inactive S~~choc~stis PCC 6803
control assay can be achieved by CCCP addition (vide

FL
off
i

Flash Number on
Fig. 4. Effect of the protonophore CCCP on the flash-induced oxygen evo- , lmin
lution pattern in the frame of the coherent Kok model analysed on the flash
electrode system developed by Bader et al. [ 291. A sequence of 15 flashes
was given after a dark adaptation time of 20 min on a control assay and an Fig. 5. Mass spectrometric recording of the light-induced hydrogen gas
identical assay containing 3 FM CCCP. Flashes were 5 ks long and spaced exchange in Oscillatoria chalybea in reaction media of different pH values.
300 ms apart. Note that none of the assays contained CCCP. Conditions as in Fig. 1 (a).
150 R. A&de/-Basset, K.P. Bader/Journal qfPhotochemistry and Photobiology B: Biology 43 (1998) 146-151

supra), but in this case not with a corresponding shift in the continuouslight. However, the direct time-dependentrecord-
pH value, remains to be elucidated. ing of the carbon dioxide uptake clearly showsthat absolutely
The transition from a strong proton reduction to an increas- no light-induced activation of the CO? fixation has to be
ingly significant oxidation of hydrogen has been tentatively considered.On the contrary, it wasalways observedthat after
explained by the assumption that during early phases of an a short period of strong initial uptake, the reaction was
illumination after a sufficiently long dark adaptation, the Cal- decreasedduring the illumination so that in extreme cases
vin cycle might be inactive so that relatively few electrons even a net surplus in the carbon dioxide concentration was
can be used for carbon dioxide assimilation; in this case attained. Thus, our observationsclearly contradict the theory
protons tend to be reduced. After the onset of light, however, of light activation developed by Greenbaumsgroup for so-
the Calvin cycle is activated so that increasingly more elec- called photosystem I free mutants of Chlamydomonas
trons are funnelled from proton reduction into this metabolic [ 38,161 which, however, turned out to be photosystem I
pathway and hydrogen is even used as a substrate [ 38,161. contaminated following experiments in our laboratory
In order to investigate this interpretation, we analysed the [39,40]. However, our results perfectly match the time
effect of carbon dioxide on the photoevolution of hydrogen coursesof carbon dioxide gasexchangedescribedby Kaplan
and compared the light-induced hydrogen gas exchange at [41] and explained with an internal CO,-concentrating
m/e = 2 with the carbon dioxide assimilationdetectable at mechanismin cyanobacteria with the possibleparticipation
mle=44. of carboxysomes. By meansof a so-called carbonic anhy-
When the cyanobacterial thylakoids wereflushedwith 99% drase-like entity, CO, from the outside might be converted
N7/1% CO2 instead of pure nitrogen, the hydrogen back- to HCO,- so that a constant passive influx takes place in
ground signalwas enhanced,which suggestsa stimulation of accordancewith the permanentdiffusion gradient [ 42,431.
the dark evolution of hydrogen. Moreover, we observedabso-
lutely no inhibition of the light-induced hydrogen evolution
and also the superimposedhydrogen uptake appearedto be Acknowledgements
comparableunderboth conditions; in somecaseseven a stim-
ulation of the hydrogen evolution might be discussedinstead The generousfinancial support provided by the Alexander
( resultsnot shown). The transition betweenthe initial hydro- von Humboldt-Stiftung (R.A.-B.) and by the DeutscheFor-
gen evolution andthe subsequenthydrogen uptake. however, schungsgemeinschaft(K.P.B.) is gratefully acknowledged.
appearedto be clearly distinct from any interaction or inter- We thank Dr K.P. Michel, Mr D.P. Stephanand Mr P. Exss-
relationship betweenproton reduction and/or hydrogen oxi- Sonnefor the cultures of SynechocystisPCC 6803, Synecho-
dation on the one hand and the light activation of the Calvin coccusPCC 6301 and Synechococcuselongutus.
cycle on the other hand. Fig. 6 depictsthe direct recording of
the light-induced carbon dioxide uptake which was mass-
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