Download Acetylene reduction and hydrogen photoproduction by wild

FEMS Microbiology Letters 167 (1998) 13^17
Acetylene reduction and hydrogen photoproduction by wild-type
and mutant strains of Anabaena at di¡erent
CO2 and O2 concentrations
A.A. Tsygankov
b
a;b;
*, L.T. Serebryakova
a;b
, K.K. Rao a , D.O. Hall
a
a
King's College London, Campden Hill Road, London W8 7AH, UK
Institute of Soil Sciences and Photosynthesis RAS, Pushchino, Moscow Region, 142292, Russia
Received 3 August 1998 ; accepted 5 August 1998
Abstract
Hydrogen photoproduction by growing cultures of Anabaena variabilis and A. azollae did not occur under air+CO2 or
argon+CO2 atmospheres at saturating light but did take place under argon alone. It was shown that CO2 inhibited
photoproduction of H2 as a result of the photosynthetic production of O2 whereas photoreduction of C2 H2 by these
cyanobacteria was not inhibited by O2 concentrations up to 20% in the assay gas phase. In contrast to the wild type of A.
variabilis and of A. azollae, H2 photoproduction by the hydrogenase-impaired mutant A. variabilis PK84 showed only a slight
dependence on O2 concentration. Thus, in the wild-type Anabaena the decrease in the observed rate of H2 evolution at elevated
O2 concentrations could be the result of an increase in hydrogenase-mediated uptake of H2 via an oxyhydrogen
reaction. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Hydrogen metabolism ; Anabaena variabilis; Mutant PK84; CO2 in£uence on nitrogen ¢xation; O2 -dependent H2 uptake
1. Introduction
Much interest is being shown at present in developing biotechnological systems with cyanobacteria
for the conversion of light energy into H2 . However,
for optimization of the process, the in£uence of gaseous substances present during growth (e.g. O2 and
CO2 ) on H2 photoproduction must be understood.
Oxygen inactivates nitrogenase which is the main
enzyme system involved in H2 photoproduction.
* Corresponding author. Institute of Soil Sciences and
Photosynthesis RAS, Pushchino, Moscow Region 142292,
Russia. Fax: +7 (967) 790532; E-mail: [email protected]
However, cyanobacteria have developed di¡erent
mechanisms for the prevention of this inactivation
[1,2].
Carbon dioxide assimilation by heterocystous cyanobacteria occurs in vegetative cells whereas molecular nitrogen is ¢xed mainly in the heterocysts [2^4].
Nevertheless, the concentration of CO2 has a complex interaction on nitrogenase activity and on H2
production by heterocysts. Photosynthetic assimilation of CO2 by vegetative cells of ¢lamentous cyanobacteria produces the reductants which are ultimately required for nitrogen ¢xation and H2
production [3]. However, it has been found that
CO2 starvation leads to enhancement of H2 produc-
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 3 6 1 - 9
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tion and acetylene reduction (equivalent to N2 ¢xation) in Anabaena variabilis [5]. Moreover, elevated
concentrations of CO2 , much above ambient, inhibited H2 production in cyanobacteria immobilized on
hollow ¢bers [6].
The aim of the present work was a study of hydrogen photoproduction by Anabaena azollae and Anabaena variabilis in a photobioreactor under di¡erent
gas mixtures. We show here that H2 photoproduction by A. azollae and A. variabilis did not occur at
saturated light with argon+CO2 in the gas phase due
to the presence of increased O2 in the medium. This
O2 e¡ect was not, however, seen with a mutant of A.
variabilis which is de¢cient in uptake hydrogenase. It
thus appears that the decrease in H2 photoproduction at increased O2 concentrations seen with wildtype Anabaena results from enhanced H2 uptake
which occurs simultaneously with H2 evolution.
2. Materials and methods
2.1. Cyanobacteria
A. azollae originally isolated from the symbiotic
association Azolla pinnata by Hoa (National center
of Scienti¢c Research, Hanoi, Vietnam), Anabaena
variabilis ATCC 29413, and the chemically generated
mutant A. variabilis PK84 (provided by Prof. S.V.
Shestakov and Dr. L.E. Mikheeva of Moscow State
University) were used in this study. The mutant A.
variabilis PK84 is de¢cient in uptake hydrogenase
and impaired in reversible hydrogenase activity [7].
2.2. Growth conditions
Modi¢ed nitrogen-free Allen and Arnon medium
[8] with replacement of Mo by V [9] was used for
growth of the cyanobacteria in continuous turbidostat culture in an automated helical photobioreactor
made of PVC tubing with 10 mm inner diameter and
4.35 l volume [10]. Mo was replaced by V because Vnitrogenase containing cells showed high activity and
stability in hydrogen photoproduction [9]. The cultures were grown at 113 WE m32 s31 using daylight
£uorescent lights, pH 7.0 (controlled by automated
addition of 0.2 N NaOH), 30³C, with a gas mixture
of 2% CO2 +98% air (0.5 l min31 ). The steady state
concentration of biomass corresponded to 12 Wg Chl
a ml31 (0.8 mg dry weight ml31 ).
The rate of hydrogen photoevolution in the photobioreactor was calculated on the basis of the H2
content in the output gas (measured by gas chromatography) and the gas £ow rate.
2.3. C2 H2 photoreduction and H2 photoproduction
assays
Acetylene photoreduction and H2 photoproduction assays were carried out in glass vials (14 ml)
by incubation of the samples (2 ml) under daylight
£uorescent lamp illumination (140 WE m32 s31 ) at
30³C. The gas phase contained Ar in the case of
H2 photoproduction and 20% C2 H2 in Ar for
C2 H2 reduction. The CO2 and O2 concentrations in
the gas phase during the experiments were varied as
indicated in the text and the ¢gure legends. The gas
phase was monitored by gas chromatography (Hewlett Packard 5890) and the rates of H2 and C2 H4
photoproduction were calculated on the basis of linear kinetics for the ¢rst 30 min.
Simultaneous measurements of H2 photoproduction (or C2 H2 reduction) and dissolved O2 concentration were performed in a chamber designed for O2
photoevolution measurement (DW2/2 unit, Hansatech, UK) closed at the top by Suba Seal stoppers,
with illumination by a halogen lamp (200 WE m32
s31 ) and run at 30³C. The volume of the chamber
was adjusted to 1.7 ml; 0.2 ml of cyanobacterial
suspension was used. Simultaneously with monitoring of the gas phase for H2 , the concentration of
dissolved O2 was recorded. Before the measurements, the chamber containing the cells was £ushed
with Ar for 10 min. In order to assay C2 H2 reduction, C2 H2 was added to the gas phase at a ¢nal
concentration of 20%. The reaction was started by
switching the light on. Since the dissolved O2 content
changed during the course of the experiments, the
rates of reaction were calculated for the period
when the dissolved O2 was constant for 15 min.
Standard deviation did not exceed 10% for C2 H4
and 15% for H2 measurements.
2.4. Chlorophyll measurements
The chlorophyll a content of the cells was deter-
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15
mined spectrophotometrically at 665 nm in 90%
methanol extracts [11].
3. Results and discussion
During batch cultivation under air+2% CO2 or
argon+2% CO2 atmospheres, A. azollae and A. variabilis did not produce molecular hydrogen. Even
under the argon+2% CO2 atmosphere, the dissolved
oxygen concentration in the culture was not less than
150 WM (data not shown). After the replacement of
air+2% CO2 by argon alone, A. azollae started to
evolve hydrogen (Fig. 1). The pH increased, evidently due to CO2 photoassimilation and degassing
by argon, but stabilized after about 1 h. The dissolved O2 concentration decreased and stabilized at
50 WM after pH stabilization. Hydrogen photoproduction increased rapidly during the decrease of dissolved O2 but after stabilization of O2 it increased
slowly (Fig. 1). A similar pattern of H2 photoproduction and O2 concentration in the photobioreactor
was obtained for A. variabilis ATCC 29413 (data not
shown). It is possible that the absence of H2 photoproduction under air+2% CO2 or argon+2% CO2
was the result of increased levels of dissolved O2
due to photosynthetic CO2 assimilation.
In order to check this possibility the in£uence of
Fig. 1. H2 production by the photobioreactor incorporating A.
azollae grown in turbidostat culture. At the start (0 min) the turbidostat was switched to the uncontrolled regime (t = 30³C) and
the gas phase was changed from 98% air+2% CO2 to 100% argon (0.5 l min31 ). 1: H2 ; 2: dissolved O2 ; 3: pH.
Fig. 2. H2 production by the photobioreactor incorporating A.
azollae as a function of light intensity. Gas phase: 98% argon+2% CO2 (0.5 l min31 ). Points indicated by asterisk were
measured at the rate of argon £ow 1.0 l min31 . 1: H2 ; 2: dissolved O2 .
light intensity on H2 photoproduction by A. azollae
under an argon+2% CO2 atmosphere was studied
(Fig. 2). At an irradiance of 20 WE s31 m32 there
was 22 WM O2 in the medium and no H2 production.
Increasing the irradiance resulted in an increase of
H2 photoproduction and dissolved O2 in the medium. Increasing the light higher than 70 WE s31
m32 resulted in a decrease of H2 production and
an increase in dissolved oxygen. After increasing
the gas £ow rate (2% CO2 unchanged) from 0.5 to
1.0 l min31 at 140 WE s31 m32 the O2 concentration
decreased due to increased dilution by the gas mixture (Fig. 2, point with asterisk) and the hydrogen
production increased. This indicates that hydrogen
photoproduction by A. azollae was inhibited in the
presence of CO2 due to photosynthetically produced
O2 but not by CO2 itself.
In contrast to H2 photoproduction, the rate of
C2 H2 reduction by A. variabilis ATCC 29413 and
A. azollae, grown under nitrogen-¢xing conditions
with V, was unchanged under atmospheres with 0^
20% CO2 (data not shown).
The rate of C2 H2 reduction by A. azollae was constant with increasing O2 concentration up to 280 WM
(Fig. 3). However, the rate of H2 photoproduction
decreased when dissolved O2 concentrations were
higher than 30 WM and at 280 WM it was only 18%
of the initial level.
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The measured rate of H2 photoproduction by cyanobacteria is the net result of H2 formation by the
nitrogenase system and H2 consumption by hydrogenase. The inhibition of H2 production by O2 is
probably due either to inhibition of nitrogenase-catalyzed H2 production or to an increase of H2 uptake
activity catalyzed by hydrogenase. To distinguish between these two possibilities we compared the in£uence of dissolved O2 on H2 photoproduction of A.
variabilis ATCC 29413 and of the hydrogenase-de¢cient mutant of this cyanobacterium PK84 (Fig. 4).
Hydrogen photoproduction rates by A. variabilis
wild type started to decrease at O2 concentrations
higher than 40 WM. At 315 WM O2 the rate of H2
photoproduction was 7% of the control (no added
O2 ). Similar data have been reported by other authors for wild-type Anabaena [12]. However, in contrast, A. variabilis mutant PK84 showed H2 photoproduction rates not less than 75% of the control
even at 315 WM dissolved O2 (Fig. 4).
It is important to note that the rate of C2 H2 reduction by cells of both A. variabilis wild type and
mutant PK84 did not depend on the dissolved O2
concentration up to at least 315 WM (Fig. 4), thus
supporting the conclusion that H2 uptake does not
protect nitrogenase from inactivation by O2 in Anabaena [13].
Fig. 4. The rate of H2 photoproduction (1, 2) and C2 H2 reduction (3, 4) by A. variabilis ATCC 29413 (1, 3) and the mutant
PK84 (2, 4) as a function of dissolved O2 concentration. 100%
activity corresponded in the case of H2 photoproduction: ATCC
29413, 12.0 ml h31 l31 suspension (39.4 nmol h31 Wg31 chlorophyll a); PK84, 10.5 ml h31 l31 suspension (32.3 nmol h31 Wg31
chlorophyll a) ; and for C2 H2 reduction: ATCC 29413, 10.8 ml
h31 l31 suspension (35.6 nmol h31 Wg31 chlorophyll a) ; PK84,
8.3 ml h31 l31 suspension (25.4 nmol h31 Wg31 chlorophyll a).
To conclude, our results show that the apparent
decrease of H2 photoproduction by wild-type A. variabilis (and most probably also by A. azollae) in response to increased O2 concentrations (in photobioreactor and vials) is the result of increased H2 uptake
via hydrogenase in an oxyhydrogen reaction [1,2].
Evidently, at low O2 concentrations the rate of H2
uptake by cyanobacteria with hydrogenase is limited
by O2 availability. The O2 concentration may be increased by direct addition of O2 (Figs. 3 and 4) or by
illumination of the cyanobacteria with CO2 (as in the
photobioreactor). Our data provide an explanation
for the light-stimulated H2 uptake activity found in
cyanobacteria [14,15].
Acknowledgments
Fig. 3. The in£uence of dissolved O2 on photoproduction of
C2 H4 (1) and H2 (2) by A. azollae. The suspension contained
12 Wg chlorophyll a ml31 in each case. 100% activity corresponded to 10.3 ml h31 l31 suspension (38.5 nmol h31 Wg31
chlorophyll a) for H2 photoproduction and 35 nmol h31 Wg31
chlorophyll a for C2 H2 reduction.
This work was supported by RITE (Japan), INTAS (Brussels) and Royal Society (UK). The authors wish to thank Prof. S.V. Shestakov and Dr.
L.E. Mikheeva for the kind gift of A. variabilis
PK84.
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