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b i o m a s s a n d b i o e n e r g y 6 3 ( 2 0 1 4 ) 5 4 e5 7

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Enhanced hydrogen production by immobilized

cyanobacterium Lyngbya perelegans under varying
anaerobic conditions

Kamra Anjana a,*, Anubha Kaushik b,**

a
Department of Environmental Science and Engineering, Guru Jambheshwar University of Science & Technology,
Hisar 125001, India
b
University School of Environment Management GGS Indraprastha University, New Delhi, 110075

article info abstract

Article history: Hydrogen (H2) production was studied using a non-heterocystous, filamentous cyanobac-
Received 18 March 2012 terium, Lyngbya perelegans in free and immobilized forms, where cells were incubated
Received in revised form under O2 free anaerobic environment with alternate light and dark period (21 h light/3 h
24 July 2013 dark). For immobilization, agar and alginate systems were taken and it was revealed that
Accepted 20 January 2014 hydrogen production was more sustained in alginate trapped cells as compared to agar
Available online 24 February 2014 trapped cells and free cells. Cell density and size of bound cell system were also important
parameters influencing H2 production in immobilized systems. The type of gas mixtures
Keywords: sparged for creating anoxic environment in the headspace had a significant influence on H2
Lyngbya perelegans production showing methane (CH4) to be most conducive component of the gaseous
Non-heterocystous mixture.
Immobilized cells ª 2014 Elsevier Ltd. All rights reserved.
H2 production
Cyanobacterium

environment [3]. It is therefore, necessary to design an oper-

1. Introduction
Photobiological hydrogen production using readily available
substrates like water and carbondioxide achieved by different
renewable technologies can provide a means of reducing our
dependence on fossil fuels for energy [1]. Renewable and non-
polluting features of photoproduction of H2 makes it ideal,
clean and sustainable energy source in contrast to chemical
process [2]. Due to extreme sensitivity of the hydrogenase
enzyme to molecular oxygen, which is produced by the cya-
nobacteria by photosystem II in the presence of light, pro-
duction of H2 is usually for a very short period in oxygenic
ating system that would ensure stable and long term
hydrogen production. For dealing with the problem of oxygen
inactivation of H2 evolving system and obligatory production
of oxygen during photosynthesis, partially inactivated O2
evolution system of algae was done by S-deprivation of the
medium [4]. Another approach for increased H2 production
involves cell immobilization in some suitable matrix and
creation of anoxic environment, especially in case of fila-
mentous cyanobacteria, in which breakage of filaments and
structural degeneration; normally occur due to agitation,
resulting in decreased in hydrogen production. Cell immobi-
lization provides protection to enzymes mediating metabolic

* Corresponding author.
** Corresponding author.
E-mail addresses: anjanakamra@yahoo.co.in (K. Anjana), anubhakaushikes@gmail.com (A. Kaushik).
0961-9534/$ e see front matter ª 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biombioe.2014.01.019
 Author's personal copy
b i o m a s s a n d b i o e n e r g y 6 3 ( 2 0 1 4 ) 5 4 e5 7
activities against environmental disturbances, thus enabling
the cells to be used for longer duration [5,6]. Besides this,
immobilization effectively separates the cells from the liquid
phase, significantly increases the cell density and as a
consequence, allows for more efficient light utilization on a
per unit area basis [7]. Effect of immobilization on facilitated
hydrogen production has been reported mostly in unicellular
microalgae [8,9].
The present study was aimed at evaluating H2 photopro-
duction by a hitherto unexplored filamentous cyanobacte-
rium Lyngbya perelegans in immobilized form using agar and
alginate as the carriers for immobilization as there are very
few reports on such aspects in filamentous cyanobacteria.
These immobilization matrices with several advantages like
natural origin, non-toxic nature and low cost are suitable for
industrial use [10]. Also, the polymerization process does not
shift the pH inside the matrix and the pH of the alginate
solution used is near neutral [7], which is optimal for cya-
nobacterial growth. Further, the polymerization process
takes place at room temperature involving non-toxic calcium
ions.
The study further analyzes important variables of the
bound cell system like size of the immobilized bead/cube and
concentration of cyanobacterial inoculum on H2 production.
Photoproduction of H2 by the cyanobacterial system in sus-
pension and bound form was also studied under different
anoxic environments created by sparging with gaseous mix-
tures of different types.
2. Materials and methods
2.1. Organism and growth conditions
L. perelegans, a non-heterocystous, filamentous cyanobacte-
rium was isolated from sub-tropical fresh water environment
of Faridabad district, Haryana, India, where it occurs as a
predominant species. Pure culture of this strain was obtained
by streaking on basal agar medium (BG-11) at pH 8.5 (buffered
with 0.1 M tris amminomethane) using standard isolation and
culturing techniques [11]. The cyanobacterium was grown in
BG-11 medium for 7 days under fluorescent light
(56.85mmol m2 sec
1) with 12/12 h light and dark photoperiod
at 28  3  C temperature. Nitrogen supplement was given in
the form of NaNO3 (1.5 g/L) in the medium for growth. Initially
nitrogen free medium was used for growth as some species of
Lyngbya are reported to possess nitrogenase. However, the
cells failed to grow after 2e3 days suggesting lack of nitroge-
nase in this species, which was later confirmed by performing
acetylene reduction assay using gas chromatograph (Hardy
et al., 1973).
2.2. Immobilization
Cells from 7 d old culture were harvested by centrifugation
at 3000 rpm for 2 min and washed with distilled water prior
to immobilization. Two types of gels, agar and alginate,
were used for binding the cyanobacterial cells. For agar
cubes, 4% agar was dissolved in 10 ml of distilled water at
100  C and cooled to 50  C. 10 ml of cyanobacterial
55
suspension was then added to the flask, mixed and cooled
to 30  C on a cold glass plate. The gels of immobilized
cyanobacterium were cut into small cubes of 1 mm3. For
alginate beads, cyanobacterial suspension and 4% sodium
alginate (1:1) were mixed and the suspension was dropped
into 2% CaCl2 solution through a small syringe making
small beads of 1 mm diameter.
For studying the effect of size of the immobilization matrix
agar cubes of different sizes viz. (0.5)3, (1.0)3 and (2.0)3 mm and
alginate beads of 0.5, 1.0 and 2.0 mm diameter were used for
hydrogen production experiments.
2.3. Measurement of hydrogen evolution
Hydrogen production at different time intervals (according to
the experimental design) was estimated by withdrawing 1 ml
aliquot of headspace gas phase with a gastight syringe and
injecting it in a calibrated gas chromatograph (Agilent 6890N)
equipped with a 13X molecular sieve column, thermal con-
ductivity detector (TCD) and nitrogen as carrier gas. Temper-
ature of the column was 75  C while the injector and detector
temperatures were maintained at 75  C and 150  C,
respectively.
All the H2 production assay experiments were performed
in sealed vials (15 ml) in triplicates. For experiments with
free cells, 2 ml (containing 0.03 g dry wt.) of 7 d old cya-
nobacterial culture in mid log phase was suspended in 1 ml
of BG-11 medium and for experiments with bound cell
system, 3.5 g of immobilized alginate beads/agar cubes
(containing same amount of cyanobacterial cells by dry
weight as in free cells) were added to the vials containing
the medium.
Experiments were also performed by varying cyanobacte-
rial dose (0.5, 1.0, 2.0 g) for determining the effect of cyano-
bacterial density per bead/cube on H2 production. Anoxic
conditions were maintained in H2 assay by replacing the air in
the vials with argon, an inert gas. For determining the effect of
different anoxic environments on hydrogen production by L.
perelegans, gas mixtures in different ratios (v/v) viz. only Ar
(13), Ar:CO2 (12:1), CO2:N2 (6:7), N2:Ar (4:9), CO2:Ar (2:11) and
CH4:Ar (11:2) were used.
Sealed vials containing immobilized or suspension culture
were kept in orbital shaker (Orbitek LT) with continuous stir-
ring at 100 rpm at 25  C with alternate light and dark condi-
tions (21 h light of 56.85 mmol m2 sec
1/3 h dark). These
conditions were previously optimized in free culture of L.
perelegans by changing the system variables [12].
3. Results and discussion
Table 1 shows the effect of size of both immobilized systems
(agar and alginate) on the rate of H2 production. Results show
that 1.0 mm3 agar cubes and or 1.0 mm diameter alginate
beads are optimum for maximum H2 production as compared
to other two sizes of immobilized systems. Hence, 1.0 mm3
agar cubes and or 1 mm diameter alginate beads were used
further in all other experiments.
Hydrogen production by free and immobilized (agar and
alginate) cyanobacterium showed marked variations (Fig. 1).
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56 b i o m a s s a n d b i o e n e r g y 6 3 ( 2 0 1 4 ) 5 4 e5 7

25
Table 1 e Effect of the size of immobilized cell system of L.

(nmol/h/mg dry wt.)

perelegans on hydrogen Production. 20

H2 production
Size of immobilized Hydrogen production 15
cell system (nmol/h/mg dry wt.)
10
Agar cube Alginate bead
(0.5)3a 18.60  0.61 19.68  0.39 5
(0.5)3b 0
(1.0)3a 19.79  0.71 21.49  0.14 2 4 8 20 24
(1.0)3b Time (h)
(2.0)3a 19.04  0.54 20.45  0.25
0.5g 1.0g 2.0g
(2.0)3b
a
mm diameter in alginate bead. Fig. 2 e Effect of cyanobacterial cell density on hydrogen
b
mm3 in case of agar cube. production by immobilized (agar) cells of L. perelegans.

The hydrogen production of the cyanobacterium L. perelegans As hydrogen production in immobilized cyanobacteria was
immobilized in alginate and agar was approximately 2e4 found to increase several folds in the 24 h experiments, it
times higher than that by free cells during 24 h and the pro- prompted us to estimate continuous hydrogen production by
duction was slightly higher in alginate matrix. The main the cyanobacterium without any further addition of nutrient
advantage of immobilized cells is that it does not form clumps medium or argon sparging. Continuous hydrogen production
and filament breakage is prevented. Also, it does not inhibit upto 120 h (5 d) by free and immobilized cells of L. perelegans is
the growth of the cyanobacterium [8]. Alginate entrapped cells shown in Fig. 4. Hydrogen evolution rate was high during the
of Chlamydomonas could produce H2 even in the presence of first 24e48 h, both in free and bound cells, and slowed down
air, though the final H2 yields were lower than that in argon thereafter. The decline was sharp in case of free cells which
atmosphere [7]. showed 57% decline at 72 h, 82% at 96 h and ceased completely
Further investigations were carried out on the H2 produc- at 120 h. The immobilized cells on the other hand, showed
tion efficiency of the immobilized system as a function of cell stabilized H2 production in alginate entrapped cells upto 120 h
density, with the inoculum size varying from 0.5 to 2.0 g (Figs.
2 and 3). It was found that 1.0 g inoculum was most favorable
producing 22.06 and 24.0 nmol of H2/h/mg dry wt. using agar
cubes and alginate beads, respectively. This was around 10%
more than that of 0.5 g inoculum. It declined by 21e23% when
the inoculum was increased to 2.0 g. This significant decline in
the rate of hydrogen production suggests that this much cell
density (2.0 g) is high enough to decrease the mechanical
stability of immobilization system. This may further lead to
increased porosity that allows for easier diffusion of O2 into
the matrix. Oxygen diffusion inhibits the oxygen sensitive
hydrogenase enzyme system [13] and decreases hydrogen
production in both types of matrices. Faster consumption of
acetate from the medium by high density immobilized cells Fig. 3 e Effect of cyanobacterial cell density on hydrogen
may be responsible for limiting cellular respiration during production by immobilized (alginate) cells of L. perelegans.
hydrogen photoproduction, possibly increasing the intracel-
lular level of O2 and inhibiting hydrogen production [7].

Fig. 1 e Time course variation in hydrogen production in Fig. 4 e Continuous hydrogen production by free and
free and immobilized cells of L. perelegans. immobilized cells of L. perelegans.
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b i o m a s s a n d b i o e n e r g y 6 3 ( 2 0 1 4 ) 5 4 e5 7 57

with anaerobic gas mixture may solve the problem. Immobi-

Table 2 e Hydrogen production (nmol/h/mg dry wt.) by
lization protects the enzyme system and prevents the dete-
free and immobilized cells of L. perelegans sparged with
different gaseous mixtures in 24 h assay (21/3 h L/D). rioration of cells, leading to cell dilution. In comparison to free
cells, immobilized cells produced more hydrogen and the
Anaerobic gas Free cells Agar Alginate
alginate entrapped L. perelegans not only showed enhanced
mixture (v/v)
hydrogen production, but also the process was stabilized after
Ar (13) 11.11  0.61 19.79  0.71 21.49  0.14
48 h producing H2 continuously for the observed duration of
Ar:CO2 (12:1) 11.47  0.58 12.84  0.35 12.75  0.22
CO2:N2 (6:7) 17.16  0.64 22.95  0.87 36.44  0.43 120 h. Sparging the cyanobacterial H2 producing assays with
N2:Ar (4:9) 28.15  0.66 35.02  0.33 38.85  0.34 mixtures of anoxic gases like CH4 and CO2 proved particularly
CO2:Ar (2:11) 33.47  1.25 46.25  0.87 49.16  0.51 useful in increasing H2 production several times. Thus
CH4:Ar (11:2) 81.38  1.06 82.25  1.08 87.14  0.51 enhanced H2 production by immobilized cells of L. perelegans
show the potential for further research to make significant
of observation period, while agar bound cells showed a improvements in the rate and efficiency of H2 production in
gradual decline at 96 h upto 120 h. scaled-up bioreactors.
A specific experiment was also performed to examine the
effect of mixture of different anoxic gases on hydrogen pro-
duction using free and immobilized cells. Based on our earlier references
observations of increased hydrogen production by free cells
under the influence of different combinations (as mentioned
in Section 2.3) of various anoxic gas mixtures (publication [1] Esper B, Badura A, Rogner M. Photosynthesis as a power
under process), combinations resulting in maximum supply for (bio-) hydrogen production. Trends Plant Sci
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mixed with CO2 (11:2 v/v), resulted in about three fold increase Greenbaum E, et al. Microalgae: a green source of renewable
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