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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: evaaro@utu.fi (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), allahve@utu.fi (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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