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Extended H2 photoproduction by N2-fixing cyanobacteriaimmobilized in thin alginate films
Hannu Leino a, Sergey N. Kosourov b, Lyudmila Saari c, Kaarina Sivonen c,Anatoly A. Tsygankov b, Eva-Mari Aro a,**, Yagut Allahverdiyeva a,*aDepartment of Biochemistry and Food Chemistry, Molecular Plant Biology, University of Turku, FI-20014 Turku, Finlandb Institute of Basic Biological Problems RAS, Pushchino, Moscow region 142290, RussiacDepartment of Applied Chemistry and Microbiology, University of Helsinki, P.O. Box 56, FI-00014, Finland
a r t i c l e i n f o
Article history:
Received 15 July 2011
Received in revised form
10 September 2011
Accepted 14 September 2011
Available online 15 October 2011
Keywords:
Alginate films
Cyanobacteria
Heterocystous
Immobilization
H2 photoproduction
Photosynthesis
Abbreviations: Ar, Argon; Chl, chlorophCollection; PVA, polyvinyl alcohol.* Corresponding author. Tel.: þ358 2 3338078** Corresponding author. Tel.: þ358 2 3335931
E-mail addresses: [email protected] (E.-M. Ar0360-3199/$ e see front matter Copyright ªdoi:10.1016/j.ijhydene.2011.09.088
a b s t r a c t
Screening of the University of Helsinki Culture Collection for naturally good H2 producing
cyanobacteria recently revealed several promising strains. One of the superior strains is
Calothrix 336/3, an N2-fixing heterocystous filamentous cyanobacterium. Making use of an
important feature of the Calothrix 336/3 cells to adhere to the substrate, we applied an
immobilization technique to improve H2 production capacity of this strain. We examined
the basic properties of immobilization in Ca2þ-alginate films in response to the production
of H2 of the Calothrix 336/3 strain and as reference strains we used a model organism
Anabaena PCC 7120 and its uptake hydrogenase mutant, DhupL, that allow us to compare
the responses of different strains to alginate entrapment. Immobilization of the Calothrix
336/3 and DhupL mutant cells in Ca2þ-alginate resulted in prolonged H2 production over
several cycles. Immobilization of the Calothrix 336/3 cells was most successful and
production of H2 could be measured even after 40 days after immobilization.
Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
reserved.
1. Introduction the H2 produced by nitrogenase back to cell metabolism; (iii)
Cyanobacteria exhibit a unique feature within domain of
Bacteria in harnessing solar energy and splitting water into
O2 and protons. Cyanobacteria can direct electrons from
water to the production of H2 under specific conditions. H2
can be used as an environmentally friendly energy carrier,
for its lack of CO2 on combustion [1]. Cyanobacteria have at
least three enzymes, which are directly involved in H2
metabolism: (i) the nitrogenase produces H2 as a byproduct
of nitrogen fixation [2]; (ii) the uptake hydrogenase recycles
yll; GC, gas chromatogra
; fax: þ358 2 3338076.; fax: þ358 2 3335549.o), [email protected] (Y. Alla2011, Hydrogen Energy P
bidirectional hydrogenase possibly acts as an electron valve
during darkelight transition and is proposed to play a role in
fermentation functioning as a mediator in the release of
excess reducing power under anaerobic conditions [3]. In
cyanobacteria, all the enzymes involved in H2 metabolism
are oxygen sensitive. Some filamentous N2-fixing cyanobac-
teria have evolved specialized cells, heterocysts, to deal with
this problem. Enzymes located in heterocysts are protected
from O2 due to a lack of the active oxygen evolving Photo-
system II (PSII) complex, elevated thickness of the cell
phy; UHCC, the University of Helsinki Cyanobacteria Culture
hverdiyeva).ublications, LLC. Published by Elsevier Ltd. All rights reserved.
i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 5 1e1 6 1152
envelope, and increased respiration rate in heterocysts (see
for a review [4]).
Cyanobacterial H2 production is difficult to sustain in
suspension cultures in photobioreactors. In outdoor batch and
continuous cultures cyanobacteria use not more than 15% of
light energy for H2 production and the rest of energy is rewired
mainly for production of biomass [5]. Since energy flow to
biomass is proportional to the growth rate, it is important inH2
production to keep cultures at the lowest growth rate, which
requires real-time measurements of absorbance, dilution of
the cultures and deprivation of nutrients. Utilization of
suspension cultures in a two-stage system for H2 production is
evenmore complicated and energy consuming process due to
centrifugation or sedimentation steps of cell harvesting and
themediachangesaswell asdilutionsof cell densityduring the
switch between the different phases [6]. Suspension cultures
also require intensive mixing for optimal light utilization,
which in turn causes damage to the fragile cyanobacteria fila-
ments.Thesesystemsarealsohard toscaleup. Immobilization
of cyanobacteria and the design of special photobioreactors
might solve these challenges [1,7,8]. Immobilization of
biomolecules and whole cells on various substrates and into
different gels, such as solid surfaces like porous glass, sup-
ported films, (nano-)porous materials, (nano-)fibers, foams,
inorganic and organic hydrogels, latex, nanotubes, and nano-
particles has been studied extensively (see for review [9]). The
aim of immobilization is to maintain and control the catalytic
activity of the cells while improving stability and reuse of the
cells. Immobilizing separates the cells from liquid media,
increases cell density and thus, enhances the volumetric
reactivity.
Natural polymers like gelatin, collagen, carrageenans,
agars, and alginates have been used for the entrapment of
whole living cells [9]. Single cyanobacteria cultures or
a combination of cultures have been immobilized in inorganic
and organic hydro-gels, like silica-gels and agar for improve-
ment of H2 production [10e13]. Photoproduction of H2 for up to
5 months with maximum rate of 0.2 ml H2/mg dry weight/h
has been observed in a specifically designed laboratory-scale
photobioreactor with hollow fiber immobilized Anabaena var-
iabilis [14,15].
Immobilization of cyanobacteria in alginate has so far been
studied mostly in bioremediation and waste water purifica-
tion [16,17]. Alginate is an anionic polysaccharide composed
of a-(1,4)-linked L-guluronic and b-(1,4)-linked D-mannuronic
acid residues. The gel formation of alginate is a very quick and
biocompatible process. The hydrogel formation occurs at
room temperature due to electrostatic interaction between
the carboxylic groups on the guluronic acid residues and
divalent ions, like Ca2þ and Ba2þ. The pH of a 4e6% alginate
solution is close to 7, and the polymerization process does not
shift the pH inside the matrix, which is important for optimal
survival of cells [17,18]. Alginate is also cheap and can be
produced from a renewable resource in large scale. However,
the mechanical stability of alginate polymers still needs
improvement, and different approaches have been utilized to
overcome this problem. Alginate has been mixed with poly-
vinyl alcohol (PVA) [19] or sodium carboxymethyl cellulose
[20], and also supporting structure inside the gel has been
applied [18,21] to increase mechanical stability. Using Ba2þ as
a polymerizing agent instead of Ca2þ can make the gel more
rigid [17], but decreases its diffusion properties. Immobiliza-
tion of Lyngbya sp., a filamentous and non-heterocystous
cyanobacterium, in Ca2þ-alginate gel resulted in 25%
increase in H2 production yield as compared to free cells
during 8 days [22], whereas immobilization ofAnabaena azollae
in polyvinyl foam and in Ca2þ-alginate beads increased H2
production by a factor of two and prolonged net H2 photo-
production till 16 h [23]. Other successful approaches to
improve H2 production include an immobilization of cyano-
bacteria Plectonema boryanumwithin alginate beads for 12 days
[13], Oscillatoria in agar matrix [24] and also entrapment of
Phormidium valderianum together with Halobacterium halobium
and Escherichia coli within PVA-alginate beads [19].
In the case of phototrophs, the immobilization of cells
within beads or onto a firm surface like glass fibers, lead to
more efficient light-utilization compared to suspension
cultures [25,26]. Importantly, the entrapment of cells into thin
films shows even better light-utilization efficiency than in
beads [26]. For instance, green alga, Chlamydomonas reinhardtii,
immobilized within thin alginate/plastic screen films per-
formed extended H2 photoproduction at high efficiency due to
an increased light-utilization efficiency, high cell density and
stability of H2 production in the presence of O2 [18,27]. Similar
effect was obtained by immobilizing non-sulfur photosyn-
thetic purple bacteria in thin latex films [28,29]. The coating-
immobilized Rhodopseudomonas palustris cells were able to
produce H2 for 4000 h.
Our recent work describing the screening of the University
of Helsinki Culture Collection (UHCC) for naturally good H2
producing cyanobacteria revealed several promising strains
[30]. One of the superior strains is Calothrix 336/3 isolated from
Lake Enajarvi, Finland. Calothrix 336/3 is a filamentous, N2-
fixing cyanobacterium with ellipsoidal heterocysts located at
the base of the filaments. Common feature of Calothrix 336/3 is
that filaments adhere to the substrate to form thin mats or
bristle-like clumps. Taking into account this feature, we
applied an immobilization technique to improve growth and
H2 production capacity of this strain. In this study, we
immobilized cyanobacteria within thin Ca2þ-alginate films. As
reference strains, we used a model organism Anabaena PCC
7120 (hereafter WT Anabaena) and its uptake hydrogenase
mutant, DhupL [31], that allow us to compare the responses of
different strains to alginate entrapment. It is known that
many factors in the initial formation of the alginate gel and
during further processing can affect the final properties of the
gel, thus also the H2 production capacity of the entrapped
cells. In this study, we examined the basic properties of
immobilization in Ca2þ-alginate films in response to the
production of H2 of the filamentous N2-fixing cyanobacteria,
Calothrix 336/3, WT Anabaena and DhupL mutant of Anabaena.
2. Materials and methods
2.1. Growth conditions
The Calothrix 336/3 cells were grown in Z8 media [32] without
combined nitrogen sources (Z8x), at pH 7.5 under illumination
of 40 mmol photons m�2s�1 and continuous bubbling with air
i n t e r n a t i o n a l j o u rn a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 5 1e1 6 1 153
at 22 �C. The Anabaena PCC 7120 and the DhupL mutant were
grown under the same conditions, but growth temperature
was 26 �C.
2.2. Immobilization of cyanobacterial cells
The cells for immobilization were harvested by centrifugation
and immobilized in Ca2þ-alginate-film strips as described
previously [18]. Cell pellets were re-suspended in fresh
medium and mixed thoroughly with sterile 4% alginate (Na-
alginate from brown algae, 71238, SigmaeAldrich, St. Louis,
MO), using the formulation ratio: 0.5e2 g wet cell weight,
0.5 ml H2O, and 1 ml 4% alginate. A template was constructed
consisting of untreated, white-polymer insect-screen, placed
over the sticky side of a strip of Scotch-type tape. The intro-
duction of the screen inside the alginate matrix significantly
increased the mechanical stability of the alginate film. The
assembled templates were washed twice with 70% ethanol
and dried. The alginate mixture with cells was then pipetted
onto the template and drawn down by hand with a sterile
glass pipette. The alginate films were polymerized by directly
spraying the unpolymerized films with a 50 mM CaCl2 solu-
tion. Finally, the templates with immobilized cells were cut
into 3 cm2 strips. The thickness of the film was about
0.2e0.5 mm.
2.3. H2 production assay and determination of H2
The alginate strips were transferred into 20-ml vials contain-
ing 5 ml of Z8x medium. The vials were sparged with Argon
(Ar) and sealed gas-tight with butyl-rubber-stopper screw-
caps. For H2 production assay in suspension cultures, 5 ml of
harvested cells re-suspended in fresh Z8x medium were
transferred into 20-ml vials sparged with Ar. After sealing,
different amounts of pure CO2 were added into vials. The vials
with immobilized cells were placed in a growth chamber at
26 �C temperature under continuous illumination from the top
(cool white fluorescent lamps (Mitsubishi/Osram); 130 mmol
photons m�2s�1). Suspension cultures were shaken at 80 rpm,
and illuminated from two sides (cool white fluorescent lamps;
total of 130 mmol photons m�2s�1). At the end of each cycle
(wonce per week), the vials were opened and the gas phase of
the headspace was changed back to anaerobic condition, and
CO2 was added to the initial level in the beginning of the
experiment.
For H2 and O2 determinations, 150 ml samples were with-
drawn from the gas phase of the vials with a gas-tight syringe
(Hamilton Co.) and injected into Gas- Chromatograph (GC,
Perkin Elmer Clarus 500) equipped with a thermal conduc-
tivity detector and a Molecular Sieve 5A column (60/80 mesh).
Calibrationwas donewith 0.5%H2 (AGA, Finland). Ar was used
as a carrier gas. H2 and O2 production rates were calculated on
the basis of the chlorophyll (Chl a) content of the cells. The Chl
contentswere determined spectrophotometrically at A665 nm
after extraction of cells with 90% methanol [33]. For cell dry
weight determination, the alginate films were destroyed with
50e100 mM Na-EDTA, cells were harvested by centrifugation,
washed four timeswithMQ-water, and incubated overnight at
60 �C.
3. Results
3.1. H2 production from the immobilized cells
The immobilization technique described in the Materials and
Methods section was used to entrap cyanobacterial cells
within thin Ca2þ-alginate films. Fig. 1A demonstrates long-
term H2 production experiment with the Calothrix 336/3 cells
entrapped in Ca2þ-alginate matrix under microaerobic
conditions. The H2 production yield in the headspace of the
vials by the entrapped Calothrix 336/3 cells in the absence of
CO2 reached the steady-state level after 3 days of incubation.
Whereas the amount of H2 in the vials supplemented with
different level of CO2 was gradually increasing about 4 days.
After 5 days of incubation, when H2 production yield slowly
reached its steady-state level or started to decline, the new
cycle was initiated: the vials were opened, the stoppers were
replaced, the gas phase was changed back to Ar atmosphere,
and appropriate amount of CO2 was added to the vials.
Maximum H2 production was observed during the second,
third and fourth cycles in the headspace of the vials with the
entrapped cells in the presence of 10%, 6% and 2% CO2,
respectively. After reaching the maximum level, a gradual
decrease in H2 production was observed in all samples. During
the 6th cycle, after 5 weeks of immobilization, the samples
supplemented with 2% CO2 were still producing H2 at the level
of the first cycle. However, the cells in the vials without CO2
supplement stopped H2 production already after the second
cycle (Fig. 1B).
Photosynthetic performance during the long-lasting
experiments was also monitored by measuring the amount
of O2 in the headspace (Fig. 1B). The highest O2 evolution of
cells was observed in the vials supplemented with 10% CO2,
however there was a significant decrease in O2 evolution after
3 cycles (Fig. 1B). O2 evolution capacity of the cells without any
CO2 supplementation dropped down to the “0” level already
after the second cycle, as expected. Importantly, even after 38
days of immobilization, the entrapped Calothrix 336/3 cells
with CO2 supplement were still able to produce O2, indicating
a long viability of this strain.
In order to investigate the long-term viability and H2
production capacity of the Calothrix 336/3 cells entrapped in
Ca2þ-alginate films, the GC vials with immobilized cells
without CO2 supplement were kept at low light conditions
(5 mmol photons m�2 s�1) and at room temperature (RT) for
extended time period. After 84 days the medium inside of the
GC vials was changed to fresh Z8x and the headspace gas
phase was changed back to Ar atmosphere with 6% of CO2
supplemented. The production of H2 in headspace was again
determined during each cycle regularly. Interestingly, already
starting from the first cycle, 12 weeks after the initial immo-
bilization, the entrapped cells from the recovered films
produced nearly similar amount of H2 as the fresh cells in the
newly made films (Fig. 1C). Moreover, the entrapped Calothrix
336/3 andAnabaena cells were viable even after 6months from
the initial immobilization (data not shown). The cells were
stored in initial medium (RT, low light conditions, 5 mmol
photonsm�2 s�1) without adding any CO2. Importantly, during
this time period the cells did not grow out of the alginate films.
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0,16
0,18
0 5 10 15 20 25 30 35 40
Ar+0% CO2Ar+2% CO2Ar+6% CO2Ar+10% CO2
mo
l H
2/
m2
Time [d]
A
0
0,1
0,2
0,3
0,4
0 5 10 15 20 25 30 35 40
Ar+0% CO2Ar+2% CO2Ar+6% CO2Ar+10% CO2
mo
l O
2/ m
2
Time [d]
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0,16
0,18
84 89 94 99 104 109 114 119 124 129
Ar+from 0% to 6%CO2
mo
l H
2/ m
2
Time [d]
C
B
Fig. 1 e H2 production from the Calothrix 336/3 entrapped in Ca2D-alginate thin film. H2 production (A) and O2 evolution (B) of
Calothrix 336/3 cells entrapped in Ca2D-alginate thin films. Cells were grown under illumination of 40 mmol photonsmL2sL1
and continuous bubbling with air in Z8x media, without combined nitrogen sources, at pH 7.5 at 22 �C. The cells were
harvested by centrifugation and immobilized in Ca2D-alginate thin film. Concentration of cells in the film was 40 mg
Chl mL2. 5 ml of fresh Z8x media was transferred to 20 ml GC vials along with the piece of film. The gas phase of the vials
was made anaerobic by Ar and CO2 was added to vials at final concentration of 0%, 2%, 6% and 10%. The vials were
incubated at 26 �C under illumination of 130 mmol photons mL2 sL1. The amount of H2 and O2 gases in headspace of the
vials was determined by GC. At the end of each cycle (indicated by downward arrows), the gas phase was changed back to
anaerobic conditions, and CO2 was added accordingly. The maximum H2 and O2 production rates correspond to 35 mmol H2
mg ChlL1 hL1 and 115 mmol O2 mg ChlL1 hL1, respectively. (C) H2 production from the Calothrix 336/3 cells after 84 days of
initial entrapment of the cells. The vials with entrapped cells without any addition of CO2, (shown in 1A) were kept at low
light conditions (5 mmol photons mL2 sL1) for 12 weeks (84 days). Then the medium in the vial was changed to a fresh Z8x
and gas phase on headspace was changed back to Ar atmosphere with 6% of CO2. H2 production at each cycle was
determined as described above. Values on the X-axis indicate the days after initial immobilization.
i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 5 1e1 6 1154
We used the WT Anabaena and its DhupLmutant as control
strains in this study. In contrast to Calothrix 336/3, no H2
production was observed in the entrapped WT Anabaena cells
in the absence of CO2 (Fig. 2A). The addition of CO2 had a clear
positive impact on H2 production. The amount of H2 in the
headspace with entrapped WT Anabaena cells reached
a steady-state after 3 and 4 days of incubation of the vials
supplemented with 2% and 6% CO2, respectively (Fig. 2A). The
entrapped WT Anabaena cells produced H2 during 3 cycles,
however H2 production yield decreased substantially (w80%)
0
0,01
0,02
0,03
0,04
0,05
0 5 10 15 20
Ar+0% CO2Ar+2% CO2Ar+6% CO2Ar+10% CO2
Time [d]
mo
l H
2/ m
2
A
0
0,1
0,2
0,3
0,4
0 5 10 15 20
Ar+0% CO2Ar+2% CO2Ar+6% CO2Ar+10% CO2
mo
l H
2/ m
2
Time [d]
B
Fig. 2 e H2 production from the Anabaena WT (A) and its DhupL mutant (B) entrapped in Ca2D-alginate thin films. Cells were
grown, immobilized in Ca2D-alginate thin films and H2 production was monitored as described in Fig. 1. Only the growth
and incubation temperatures differed from those in Fig. 1, being 26 �C and 30 �C, respectively. Concentration of the cells in
the film was 90 mg Chl mL2. Maximum H2 production rate corresponded to 9 mmol H2 mg ChlL1 hL1 in the WT and 30 mmol
H2 mg ChlL1 hL1 in the DhupL mutant cells.
i n t e r n a t i o n a l j o u rn a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 5 1e1 6 1 155
already after the first cycle. Importantly, the maximum H2
production rate of the WT Anabaena cells (9 mmol H2
mg Chl�1 h�1) was much less than that of the Calothrix 336/3
cells (35 mmol H2 mg Chl�1 h�1). Addition of 10% CO2 increased
H2 production only slightly in the entrapped WT Anabaena
cells compared to the samples with 0% CO2.
The yield of H2 production of entrappedDhupL cellswith 0%
CO2 reached a steady-state after 6 days of incubation.
Supplementation of the entrappedDhupL cells with 2% and 6%
CO2 significantly increased the H2 production yield, whereas
the cells with 10% CO2 in the headspace did not demonstrate
further improvement. The H2 yield of entrapped DhupL cells
supplemented with 2% CO2 reached the maximal level after 6
days (w0.3 mol H2 m�2), which then decreased significantly
after each cycle (Fig. 2B). It is worth to note that the entrapped
DhupL cells demonstrated higher H2 production yield during
the first cycle compared to the films with the Calothrix 336/3
cells. This difference in H2 production yieldmight be related to
higher cell concentration in the Ca2þ-alginate films with
entrapped DhupL. However, it is important to note that the
maximum specific H2 production rate by alginate-entrapped
Calothrix 336/3 cells (day 3e4, 35 mmol H2 mg Chl�1 h�1) was
slightly higher compared to that of immobilizedDhupLmutant
cells (day 1e2, 30 mmol H2 mg Chl�1 h�1) in the first cycle (Figs.
1A and 2B). Optimization of the cell concentration in the films
for best H2 production needs further investigation.
3.2. H2 production by suspension and immobilized cellsduring long-term experiments
In order to compare the capacity of H2 production by
suspension and immobilized cells during long-term experi-
ments, the H2 production assay was performed also in the
suspension cultures of Calothrix 336/3 (Fig. 3), WT Anabaena
(Fig. 4A) and DhupL (Fig. 4B). Interestingly, the addition of CO2
to the suspension cultures did not have any significant effect
on the yield of H2 production in Calothrix 336/3 during the first
cycle (Fig. 3). During the following cycles, yield of H2 produc-
tion by the cells supplemented with different amounts of CO2
decreased gradually showing higher yields at high CO2
concentrations, except 10%, whereas cultures without sup-
plemented CO2 stopped producing H2 after the first cycle
(Fig. 3). The yield of H2 production in suspension cultures of
Calothrix 336/3 (Fig. 3) was higher than that in suspension
cultures of WT Anabaena (Fig. 4A) but lower than in DhupL
toward the end of the first cycle (Fig. 4B). Different to the
alginate-entrapped cells (Fig. 2A), the suspension cultures of
WT Anabaena and DhupL cells supplemented with 2% and 6%
CO2 did not significantly enhance the H2 production (Fig. 4A
and B) and addition of 10%CO2 resulted in lowerH2 production
compared to 0% CO2 during the first cycle (Fig. 4A).
It is known that cyanobacterial enzymes involved in H2
metabolism are O2 sensitive. Despite the fact that the vials
0123456789
10
0 5 10 15 20 25
Ar+0% CO2Ar+2% CO2Ar+6% CO2Ar+10% CO2
Time [d]
mm
ol H
2/ L
Fig. 3 e H2 production from the Calothrix 336/3 in suspension culture. Cells were grown as described in Fig. 1. The cells were
harvested by centrifugation and re-suspended in fresh Z8x medium at Chl concentration of 5 mg Chl mLL1. 5 ml cell culture
was transferred to 20 ml GC vials and the gas phase of the vials was changed to Ar. CO2 was added to vials accordingly. The
vials were incubated at 26 �C under light intensity of 130 mmol photonsmL2 sL1. The amount of H2 in headspace of the vials
was determined by GC. Detected maximum H2 production rate was 25 mmol H2 mg ChlL1 hL1.
i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 5 1e1 6 1156
were sparged with Ar, the concentration of O2 inside the vials
increased gradually in the light due to the presence of func-
tional PSII (Fig. 1B). The highest O2 content of 12% was
measured in the headspace of the vials with entrapped Calo-
thrix 336/3 cells (Fig. 1B). To evaluate the impact of O2 on the
production of H2 by the Calothrix 336/3 cells, we performed H2
production assay in the presence and absence of O2 (Fig. 5).
The vials with suspension culture of Calothrix 336/3 cells were
sparged with Ar (control), Ar-air (air in which N2 was replaced
with Ar) or normal air, sealed with septa and incubated for
24 h under light intensity of 130 mmol photons m�2 s�1. In the
0
1
2
3
4
5
6
0 1 2 3
Ar+0% CO2Ar+2% CO2Ar+6% CO2Ar+10% CO2
Time [d]
mm
ol
H2
/ L
A
02468
101214161820
0 2 4 6 8Tim
mm
ol H
2/ L
B
Fig. 4 e H2 production from the Anabaena WT (A) and DhupL mu
described in Fig. 1. The cells were harvested by centrifugation a
of 5 mg Chl mLL1. The H2 production phase was monitored as i
13 mmol H2 mg ChlL1 hL1 and 30 mmol H2 mg ChlL1 hL1 in the
presence of normal air in headspace of the vials, the H2
production rate of the cells was only about 7% of that in the
control cells (Fig. 5), whereas in the presence of Ar-air 24%
decrease was observed.
Next, an experiment was performed with regular supple-
mentation of the cells with CO2 to avoid possible limitation of
H2 production by CO2 deficiency in entrapped cyanobacterial
cells. Fig. 6 demonstrates the photoproduction of H2 by the
Calothrix 336/3 cells entrapped in alginate films during 15 days
without opening and re-gassing the vials. For simplicity we
call this experiment as continuous culturing. The entrapped
4 5 6 7 8
10 12 14 16 18
Ar+0% CO2Ar+2% CO2Ar+6% CO2Ar+10% CO2
e [d]
tant (B) in suspension culture. Cells were grown at 26 �C as
nd re-suspended in fresh Z8x medium at Chl concentration
n Fig. 2. The maximum H2 production rate corresponds to
WT and DhupL mutant cells, respectively.
Argon
Argon air
Air
0
20
40
60
80
100
120
H2
pro
du
ctio
n rate (%
)
Fig. 5 e Effect of O2 on the H2 production rate of Calothrix
336/3. Cells were grown as described in Fig. 1, harvested by
centrifugation and r-suspended in fresh Z8x medium at
Chl concentration of 5 mg Chl mLL1. 5 ml cell culture was
transferred to 20 ml GC vials and the gas phase of the vials
was changed to Ar (microaerobic), air or argon air, in which
N2 is replaced with Ar. The vials were incubated at 26 �Cunder light intensity of 130 mmol photonsmL2 sL1 for 24 h.
i n t e r n a t i o n a l j o u rn a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 5 1e1 6 1 157
Calothrix 336/3 cells were incubated in the vials supplemented
with 2%, 6% and 10% CO2 by regularly injecting appropriate
amounts of CO2 into the headspace. The amount of CO2 used
by the cells in a given time was estimated approximately. The
maximum H2 yield was observed at 9th day in the vials sup-
plemented with 10% CO2 (0.2 mol of H2 m�2), which is signif-
icantly higher compared to themaximumH2 yield (0.14mol of
H2 m�2) observed during the experiments with the cycles
(third cycle, day 19, Fig. 1A). Higher maximum H2 yield from
the cells entrapped in the alginate films during continuous
culturing might be due to higher cell concentration (170 mg
Chl m�2) compared to H2 production experiments with cycles
(Fig. 1A, 40 mg Chl m�2). The H2 production yield by the cells
supplemented with 2% and 4% CO2 increased gradually but
did not reach the maximum steady-state level during 15 days.
It is also worth to note that multiple injections without
changing the septum during continuous culturing might
result in higher leakage of H2 from vials than in previous
experiments (Fig. 6). Also, the film used for continuous
culturing was thicker (w1 mm) than that used in previous
experiments (w0.2e0.5 mm), which might result in slow
0
0,05
0,1
0,15
0,2
0,25
0,3
0 2 4 6Time [d]
Ar+2% CO2Ar+6% CO2Ar+10% CO2
mo
l H
2/ m
2
Fig. 6 e Continuous H2 production from the Calothrix 336/3 entra
and H2 production analyzed as described in Fig. 1. Concentratio
w0.5 mm thick. CO2 addition to the vials indicated with arrows
diffusion of H2 from the film, thus making it easier for the
uptake hydrogenase, also present in Calothrix, to recapture of
H2. The level of O2 in the headspace of vials after 15 days of
incubationwas 34%. It is highly possible, that due to leakage of
H2, thickness of the film, and high O2 concentration, the H2
production capacity of the Calohrix 336/3 cells observed in
Ca2þ-alginate films during continuous culturing is
underestimated.
The possible effects of immobilization in alginate films on
the growth of the Calothrix 336/3 cells were also studied at
different conditions. The entrapped Calothrix 336/3 cells
incubated under aerobic and low light (20 mmol
photons m�2 s�1) conditions for 2 weeks demonstrated higher
biomass production (dry weight) compared to the control
samples measured immediately after cell entrapment in
alginate films (Fig. 7). However, the growth rate of the
entrapped Calothrix 336/3 cells was still much slower than that
in suspension cultures growing with air-bubbling (data not
shown). In contrast, the cells incubated at microaerobic, high
light (130 mmol photons m�2 s�1) conditions showed lower
biomass production (Fig. 7), which might be due to a photo-
inhibitory effect of the actinic light. Interestingly, at micro-
aerobic low light conditions no significant changes in biomass
of the entrapped Calothrix 336/3 cells was observed (Fig. 6).
3.3. Morphology of the immobilized cells
The microscope images of the Calothrix 336/3 cells are shown
in Fig. 8. The image of the cells from suspension cultures was
taken 9 days after inoculation at normal growth conditions
(Fig. 8A) and immobilized cells 6 weeks after the initial
entrapment in alginate films (Fig. 8B). In suspension cultures,
the Calothrix 336/3 cells are tapering with well-defined ellip-
soidal heterocyst at the end of the filaments. It is worth to
mention that the cell morphology was similar also after 6
weeks of H2 production in the suspension cultures (in gas-
tight vial under Ar atmosphere) (data not shown).
Filaments of entrapped cells were strongly curved and
vegetative cells were significantly larger than those in the
suspension cultures. Heterocysts in entrapped cultures were
hard to identify. This resembles the situation with symbiotic
cyanobacteria, which live in a kind of natural immobilization
system [34]. Cyanobiont microcolonies contain heterocysts
8 10 12 14 16
pped in Ca2D-alginate films. Cells were grown, immobilized
n of the cells in the film was 170 mg Chl mL2. The film was
(2% 6% 10%).
80859095
100105110115120
control air microaerobic
low light
microaerobic
high light
Bio
mass (%
)
Fig. 7 e Cell dry weight of Calothrix 336/3 entrapped in
Ca2D-alginate films. The cells were grown and
immobilized as described Fig. 1. The film of the
immobilized cells was cut into 14 cm2 pieces. 35 ml of fresh
Z8x media was transferred to 160 ml GC vials along with
the piece of film. Gas phase in the headspace of the vials
was changed to Ar or air and 6% of CO2 was added to vials.
The vials were incubated at 26 �C under illumination with
light intensity of 130 mmol photons mL2 sL1 (high light) or
20 mmol photons mL2 sL1 (low light). Dry weight of the
control samples was measured immediately after
entrapment in alginate. Alginate was removed with 50 mM
Na-EDTA. Dry weights of other samples were measured
after 2 weeks of immobilization.
i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 5 1e1 6 1158
with typical ultrastructure but may also contain altered
heterocysts with reduced cell walls, which might dominate in
all regions of the coralloid roots [35].
4. Discussion
4.1. Immobilization prolongs the H2 production ofCalothrix 336/3
All N2-fixing cyanobacteria, so far examined, possess an
uptake hydrogenase [2]. It is well known that the uptake
hydrogenase is the main obstacle for H2 production by N2-
fixing cyanobacteria. Recently released genome sequence of
Calothrix sp. SC01 revealed the presence of an uptake
hydrogenase in this particular organism (J. Craig Venter
Institute, USA). In line with this, the Southern hybridization
results confirmed that the Calothrix 336/3 strain also possesses
Fig. 8 e Microscopic images of Calothrix 336/3. The Calothrix 33
alginate films for 6 weeks under Ar and 6% of CO2 atmospher
an uptake hydrogenase (unpublished data from our lab).
Despite this fact, the Calothrix 336/3 cells demonstrate even
higher H2 production rate than the Anabaena DhupL mutant
cells, which lack the uptake hydrogenase.We should also note
that in our experiments thin alginate films were used and
thicker alginate films (�1 mm) more likely would reduce H2
diffusion from the film, and therefore increase the
consumption of H2 by the uptake hydrogenase.
Themaximum specific rate of H2 production, over the time
intervals examined, was consistently comparable between
the suspension cultures and alginate-entrapped cells in the
Calothrix 336/3, WT Anabaena and also in the DhupL mutant.
WT Anabaena produced significantly less H2 than the DhupL
mutant or Calothrix 336/3, whereas the entrapped cells and
suspension cultures of the DhupL (30 and 30 mmol H2
mg Chl�1 h�1, respectively) and Calothrix 336/3 (35 and 25 mmol
H2mgChl�1 h�1, respectively) had nearly similarmaximumH2
production rates under the conditions of this study. An
interesting feature of the entrapped cells was that they
produced substantially more H2 at much longer time course.
Noteworthy, in suspension cultures the maximum H2
production yield of Calothrix 336/3 (7 mmol H2 L�1) was lower
than that in DhupL mutant of Anabaena (14 mmol H2 L�1)
probably due to the long agitation process. Calothrix 336/3 does
not grow well during agitation and, it naturally prefers to
adhere to different surfaces for fast growth.
In suspension cultures, the H2 production reduced signifi-
cantly after the first cycle. Calothrix 336/3, in WT Anabaena did
not produce any H2 on the second cycle, whereas DhupL
decreased H2 production substantially, and stopped it
completely after second cycle (Figs. 3 and 4). On the contrary,
entrapment of the Calothrix 336/3 and the DhupL mutant in
alginate resulted in prolonged H2 production over several
cycles (Figs. 1A and 2B). Immobilization of the Calothrix 336/3
cells was most successful and production of H2 could be
measured even after 40 days after immobilization.
4.2. CO2 is an important determinant for efficient H2
production
The elevated carbon sources is likely to increase the photo-
synthetic rate and N2 demand of cyanobacterial cells, which
then function as a signal for enhanced nitrogen fixation and
subsequently also for prolonged H2 production [36,37]. In our
long-term experiments the dependency of H2 production by
6/3 cells from suspension cultures (A) and entrapped in
e (B). Alginate was removed with 100 mM Na-EDTA.
i n t e r n a t i o n a l j o u rn a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 5 1e1 6 1 159
cyanobacterial cells on the CO2 level in the headspace of the
vials was clearly demonstrated. The immobilized Anabaena
cells, particularly the DhupL mutant, responded more posi-
tively to the addition of CO2 compared to the Calothrix cells. It
is known that immobilization limits gas diffusion and there-
fore is likely to significantly change the whole cell metabo-
lism. We postulate that Anabaena and Calothrix 336/3 differ
from each other in their properties of CO2 concentration
mechanisms, and this becomes more obvious in immobilized
systems than in suspension cultures. Without CO2 supple-
mentation both the entrapped cells and suspension cultures
of Anabaena and Calothrix stopped the H2 production after the
first cycle, mainly due to a shortage in the supply of reducing
power for the function of the nitrogenase and concomitant H2
production. Intriguingly, the entrapped Calothrix 336/3 cells
supplemented with 2% and 6% CO2 demonstrated longer H2
production capacity over several cycles. This indicates that
CO2 is one of the limiting factors in H2 production [38,39]. In
line with this, regular supplementation of the entrapped Cal-
othrix 336/3 cells with CO2 during the continuous H2 produc-
tion experiments, demonstrated gradually increasing H2 level,
which did not reach the steady-state level even during the 2
weeks duration of the experiment.
There was, however, an upper threshold level for
improvement of H2 production by CO2. The entrapped Calo-
thrix 336/3 cells supplemented with 10% CO2 demonstrated
decrease in H2 production already after the second cycle. In
this case, the decrease in H2 production might also be due to
a drop in pH, since the Z8-media is not buffered adequately.
However, our efforts so far to use altered, more buffered Z8-
media, have not yielded positive results regarding H2
production with as high as 10% CO2-level (data not shown).
It is also possible that increased O2 level in the headspace
of the samples, due to the activity of water-splitting PSII
centers, is yet another reason for down regulation of H2
production over the time. However, our results demonstrate
that the presence of 21% O2 in the headspace decreases H2
production rate in the Calothrix 336/3 cells maximum by 24%
compared to that in Ar atmosphere. This suggests that an
uptake hydrogenase in this cyanobacterium is not connected
to the respiratory chain, in contrast to A. azollae and A. varia-
bilis [5]. On the other hand, many other heterocystous cya-
nobacterial strains with uncoupled H2 uptake and respiration
exist [39].
It is important to note that after reaching the maximum
level of H2 production the yield gradually decreased during the
following cycles. Such decline most likely resulted from
a deficiency in nutrients andN2. This assumption is supported
by the fact that after H2 production experiment and further
storage of the cells, the addition of fresh nutrient medium
resulted in even higher production of H2 than in the original
immobilization experiment.
4.3. Benefit of cell immobilization for H2 production
Immobilization has a positive effect also on cell viability. In
entrapped films all three strains, the Calothrix 336/3, WT
Anabaena and DhupL cells, were viable for over 10 months in
the initial nutrient media without addition of CO2. Long-term
viability of entrapped cells is a very important issue for
economical use of cyanobacterial systems in H2 production.
Although we have not tested the viability of entrapped cya-
nobacterial cells in drying alginate films, there is a report
demonstrating that soil bacteria entrapped in dry alginate
beads remain viable for about 13 years [40].
The entrapment in Ca2þ-alginate films also reduces the
growth of cyanobacterial cells. This is promising, considering
the problems of controlling the cells between growth/recovery
and H2 production phases. It is possible that the reduced
growth results from the inability of filaments to reproduce
with spreading of hormogonia. Entrapping the filaments
inside the film hinders the dispersion of hormogonia and
small fragments of filaments that would be beneficial for
growth in culture. Thus restriction of growth by entrapment
would rewire more energy toward the production of H2,
instead of biomass production. The immobilization of cya-
nobacterial cells would be especially useful during the
switches between H2 production and recovery phase, without
the need to harvest the cells between these two phases.
Another positive point in immobilization is that nutrients can
be added continuously without harvesting the cells or use of
extra energy to mix the cells. Recently obtained Anabaena PCC
7120 nitrogenase mutants, which favor protons as a substrate
instead of N2 [41,42] could be an elegant model organism for
immobilization system operating in air atmosphere that
switches between growth and H2 production phases.
In addition, entrapment of cells into alginate films also
reduces the possibility of cell contamination. This is particu-
larly important for the long-termH2 production process, when
cells are hard to keep axenic. Also, storing the cells in rough
conditions is useful for the economical usage of the whole
system.
As a summary, we suggest that an extended H2 production
in alginate-entrapped cells might be due to several reasons: (i)
immobilization in films, in general, significantly stabilizes the
cells [6,7], whereas prolonged agitation of the cells in
suspension cultures might cause mechanical stress to fila-
mentous cells; (ii) light in the thin alginate film ismore equally
distributed [6,7], whereas in suspension cultures, constantly
changing, unequally distributed light might cause metabolic
stress in cells; (iii) alginate polymer could function as a pro-
tecting envelope from the changes of pH in Z8 medium; (iv)
cultures immobilized in alginate films are usually less
susceptible to contamination than suspension cultures.
Continuous H2 production from immobilized cells is a more
promising H2 production system in larger scale than the
production from suspension cultures.
Acknowledgments
This work was supported by the Academy of Finland mobility
grant (project # 139258), Center of Excellence program
2008e2013 (project # 118637), the EU/FP7project Solar-H2 (#
212508), and the Finnish Cultural Foundation, Varsinais-
Suomi Regional Fund, Nordic Energy Research Program
(Nordic BioH2). The DhupL mutant of Anabaena PCC 7120 was
kindly provided by Prof. H. Sakurai.
i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 5 1e1 6 1160
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