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Enhancement of biological hydrogen production

using green alga Chlorococcum minutum

K. Paramesh a, N. Lakshmana Reddy b, M.V. Shankar b,

T. Chandrasekhar a,*
a
Department of Environmental Science, Yogi Vemana University, Kadapa 516003, Andhra Pradesh, India
b
Nanocatalysis and Solar Fuels Research Laboratory, Department of Materials Science & Nanotechnology, Yogi
Vemana University, Kadapa 516003, Andhra Pradesh, India

article info abstract

Article history: It is estimated that the fossil fuel reserves are going to deplete continuously due to
Received 5 June 2017 extensive usage. In order to cope with this crisis, it is necessary to increase the efforts
Received in revised form towards production of biofuels such as biological hydrogen (H2). It is well-known fact that
6 September 2017 the biological hydrogen is a clean and ideal energy and liberates high amount of energy per
Accepted 11 September 2017 unit mass. Several groups are working for the large scale production of H2 chemically and
Available online xxx also using photosynthetic organisms, but output is not satisfactory. The best way to ach-
ieve enhancement of H2 is through altering the photosynthetic process by applying various
Keywords: stress conditions or by natural selection. In the process of selection, Chlorococcum minutum
Chlorococcum was found with improved H2 output when compared to model green alga Chlamydomonas
Green algae reinhardtii in a massively parallel and competitive high-throughput screen of different
Temperature green algae. Both the species belongs to class chlorophyceae of green algae and live in fresh
Light water conditions. In extent various light, pH and temperature conditions were applied and
Biological hydrogen achieved the enhancement of H2 production in this species under in vitro settings.
Enhancement Augmented hydrogenase activity was found in Chlorococcum minutum when compared to
model alga and this may be one of the reason behind improved H2 output. Hence this
species may be considered as one of the best species with respect to H2 production and also
this work may be useful for future renewable energy research.
© 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

biological hydrogen or bio-hydrogen from photosynthetic or-

Introduction
Continuous increase in world population leading to extensive
usage of fossil fuels has been a subject to contentious debate
in recent years. As per the analysis of United States energy
information administration (US-EIA), the worldwide energy
consumption may raise by 56% by the end of 2040 [1]. Hence
the focus on enhancement of renewable energy such as
ganisms has gathered momentum recently. Most of these
alternate renewable energy resources depend on solar power
in one way or another. In addition climate change may compel
the production of renewable fuels that do not contribute to
increase of carbon dioxide and other greenhouse gases [2]. On
the other hand the production of H2 through chemical and
microbial methods were not sufficient and also the

* Corresponding author.
E-mail address: tcsbiotech@gmail.com (T. Chandrasekhar).
https://doi.org/10.1016/j.ijhydene.2017.09.005
0360-3199/© 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Paramesh K, et al., Enhancement of biological hydrogen production using green alga Chlorococcum
minutum, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.005
2 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 7 ) 1 e1 0

downstream processes are expensive [3e5]. Moreover pro-
duction of bio-hydrogen from photosynthetic algae is an
interesting area of research since several years [5,6]. Several
methods were standardized for H2 production from model
green alga Chlamydomonas reinhardtii (C.r) and also from other
algal species, but still no system is developed for efficient and
continuous production [7,8].
Algae are primitive plants that can range from microscopic
to large seaweeds with various modifications in physiological,
biochemical and molecular pathways specifically with respect
to production of biofuels [9,10]. Advantage with green alga is,
its simple life span with photosynthetic capacity. Due to useful
carbohydrate, protein, lipid and significant biomolecular sub-
strate production and also the ability to grow using non-
potable water sources, algae are being explored as a potential
feedstock for biofuel production. The production of H2 from
algal source using water and solar radiation as the driving
force in the process of photosynthesis besides application of
specific conditions such as sulfur deficiency [7,11,12] or addi-
tion of copper [13] enhanced the output. Addition of any stress
such as hypoxic/anoxic or fermentation conditions are also
favorable for the production of H2 in some living organisms
including certain microorganisms, primitive and advanced
plants. Furthermore several enzymes are involved in both
photosystem I and II (PS 1 and PS II) including photolysis of
water in PSII (Fig. 1A) and oxygen tolerance process. Among all,
hydrogenases play a key role in the production of hydrogen
[14,15]. Hydrogenases are classified into iron-iron, iron-nickel
and iron groups based on active site metal content [16]. As
mentioned above, use of light as energy and electrons obtained
either from the splitting of water at PSII or from fermentative
catabolism are transferred through a series of electron transfer
cofactors to PSI and reached to ferredoxin (Fd). Most of the
electrons are routed from ferredoxin to ferredoxin-NADPþ
reductase (FNR), resulting in the production of NADPH
(Fig. 1B). If the metabolism does not require NADPH for carbon
fixation or growth conditions suppress CO2 fixation (calvin
cycle), then alternative electron sinks like hydrogen production
are activated. Under these conditions, the organism can push
the flow of electrons from Fd to the enzyme hydrogenase,
which evolves H2 via proton reduction (Fig. 1C). This is
accomplished in model alga C. reinhardtii by an [FeFe]-
hydrogenase and to date, there are two chloroplast hydroge-
nase proteins HydA1 and HydA2 identified. Since many hy-
drogenases are highly sensitive to oxygen and can be
irreversibly inactivated within seconds or minutes and anaer-
obic condition is indispensable for its function [17]. By taking
this advantage genetic engineering researchers recently
focused to insert additional copies of hydrogenase and in
specific conditions, all the electrons are routed to produce H2
by this genetically fused additional hydrogenase [18].
Rapid screening of different photosynthetic algae using
various environmental conditions is another way to find out
candidate species with respect to H2 production. Different
environmental factors belonging to both biotic and abiotic
groups play a major role in H2 production in different algae
[3,8,9]. It is well-known fact that sun or artificial light are the
main sources of energy for photoautotrophs including algae
and these organisms use light energy to convert carbon di-
oxide into organic compounds [6,10,11]. Both pH and tem-
perature are also play as key growth factors in algal cultivation
and growth. Both the factors determine the solubility,

Fig. 1 e Schematic representation of hydrogen production from an algal cell. TM-Thylakoid membrane, PSI-Photosystem I,
PSII-Photosystem II, Fd-Ferridoxin, FNR-Ferridoxin NADP reductase, PC-Plastocyanin, PQ-Plasto quinone, Cyt-Cytochrome,
NADPH- Nicotinamide adenine dinucleotide phosphate-oxidase.

Please cite this article in press as: Paramesh K, et al., Enhancement of biological hydrogen production using green alga Chlorococcum
minutum, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.005
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 7 ) 1 e1 0
influence the availability of CO2 and essential nutrients and
have control on biochemical pathways including photosyn-
thesis [9,10,15,19]. All the factors mentioned above control the
biochemical pathways in turn output [19]. Modifications in
these factors such as light, pH, temperature, media composi-
tion, gaseous phase etc., have been done for improvement of
bio-hydrogen production in different algal species including
Chlamydomonas, Chlorella, Scenedesmus etc. [20e23]. However a
significant method of H2 production is not developed for in-
dustrial level. In the process of random screening Chlorococcum
minutum (C.m) was found with enhanced H2 production when
compared with model green alga C. reinhardtii. Both C. rein-
hardtii and C. minutum are belongs to class chlorophyceae of
green algae and live in fresh water habitat. Initially, dark
fermentation products in this species were compared with
that of C. reinhardtii [24]. But in the present study enhance-
ment of biological hydrogen production is focused using
various environmental and biochemical approaches.
Furthermore present work will also help us to understand the
response of C. minutum in different environmental conditions
with respect to hydrogen production.
Experimental
Algal samples such as Chlamydomonas reinhardtii and Chlor-
ococcum minutum were collected from University of Madras
and Acharya Nagarjuna University, India. Later preservation
protocols were established using liquid nitrogen depending on
the algal species for further usage.
Culture media preparation and culture conditions
Prior to preparation of medium it is prerequisite that glass-
ware (Borosil, India) must be washed using detergents/teepol
(10%) solution properly for maintaining optimal sterilized
conditions in in vitro experiments. In addition the contami-
nated vessels were decontaminated using an autoclave
(INLAB Equipment, Madras, India) at 121  C and 103,421.36 Pa
for 20 min and later washed with detergent, tap water,
distilled water and finally desiccated in an oven (Kemi, K04.3,
Ernakulam, India). Regular algal medium i.e. tris acetate
phosphate [(TAP) Fisher Scientific and Hi-Media, Mumbai,
India] and in specific conditions, tris acetate phosphate
without base (TAP-B) medium were prepared for present
investigation. In general pH of media was adjusted to 7.0 with
0.1 N HCl or 0.1 N NaOH using pH meter (Elico limited, India)
and it was made to a known volume. As it is mandatory to
provide sufficient gaseous phase for healthy algal growth,
5.0 ml of medium was dispensed into 30.0 ml serum vials and
all the vials were autoclaved for 15 min at 121  C and
103,421.36 Pa. After the completion of sterilization, all the
samples were removed from autoclave and cooled down to
room temperature and were kept in laminar air flow chamber
(Hitech products, No. 14, Chennai, India). Various set of ex-
periments which include different light and/or pH and/or
temperature conditions along with nutrient alterations were
carried out. In addition red and blue light along with light/dark
conditions were also applied to check the existence and
probable role of different photoreceptors in H2 production.
Please cite this article in press as: Paramesh K, et al., Enhancement of biological hydrogen production using green alga Chlorococcum
minutum, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.005
3
Provision of blue and red light conditions was done using
separate bulb system in the same culture room. Various pH
conditions such as 5.0, 6.0, 7.0 and 8.0 were tested in the
present investigation. Generally optimum temperature be-
tween 20 and 40  C was observed for growth of different algal
species based on the literature. In the present study, most of
the experiments were carried out at 25  C but to know the
impact of low and high temperatures on algal growth and H2
production, experiments were also carried out at 15  C and
35  C respectively.
Culture inoculation and growth maintenance
Prior to inoculation, the laminar air flow chamber was turned
on with ultra violet light for 30 min and later smeared with
ethanol. All the requirements for inoculation such as steril-
ized inoculation needles, small wood sticks, different media,
algal samples etc., were transferred inside the chamber.
Inoculation was carried out using sterilized loops and small
wood sticks near the spirit lamp. Initially hands were cleaned
with 70% ethanol and later gloves were used to minimize
contamination. In general all the cultures were kept in orbital
shaker (PELICAN equipment, ROTEK-LES, Mumbai, India) with
140 rpm speed and incubated in a culture room at 25  C. In
addition continuous photo period was maintained at a photo
flux of 107.02 cd with white fluorescent tubes. Visual obser-
vation of the cultures was carried out using hemocytometer to
know the phase of the cultures and cell count. Later the
effect of different treatments were quantified on the basis of
percentage of cultures showing response. Cultures with
contamination were removed frequently.
Growth assay
Initially growth assay of C. minutum along with model alga C.
reinhardtii was performed in light and dark conditions using
TAP agar medium. Before that the cultures were diluted with
TAP liquids to maintain equal biomass and later 10.0 mL of
each culture was spotted onto agar plates containing TAP
nutrients (pH 7.0) to know the comparative growth prolifera-
tion pattern in both the species. Plates were incubated at 25  C
in culture room with a photo flux of 107.02 cd and growth
pattern was photographed after five days.
Cell count and biomass estimation
Cell count and equal amount of biomass will give appropriate
values of H2 at the end of the experiments. Hemocytometer
was used for cell count and also to know the growth condition
of both the algal cultures. Before that culture vials were taken
off from the orbital shaker and 90 mL of each algal sample was
mixed with 10 mL of glycerol and covered with coverslip and
finally transferred onto hemocytometer. Hemocytometer was
inserted under the microscope and cells were counted in all 5
square boxes and values were noted (cells/ml). On reaching the
mid-log phase, all the cultures were used for estimation of
biomass i.e. chlorophyll content. In certain cases the algal
cultures before observing in the hemocytometer was treated
with 0.1 ml Lugol's iodine to 0.9 ml of culture for starch grain
observation and photograph purpose. Simultaneously
4 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 7 ) 1 e1 0
chlorophyll contents were also estimated using regular spec-
trophotometer (Shimadzu, UV-1800, Mumbai, India) by dis-
solving 100 ml culture with 900 ml of 80% acetone and kept in
dark conditions for 2 min. Later centrifuge 2 min at 25,000  g
and supernatant was used for estimating the O.D values at 663
(chl-a) and 645 (chl-b) for chlorophyll experiment. Based on
chlorophyll data all the samples were diluted with the help of
liquid TAP medium to get uniform amount of biomass and
later moved for subculture. In the subculture primary algal
inoculum was used maximum of 100 mL to 4.9 ml TAP medium
in 30.0 ml serum vial bottles.
Hydrogen estimation using gas chromatography-thermal
conductivity detector (GC-TCD)
After secondary inoculation, the serum vial bottles were
sealed with the help of rubber stopper and aluminum cap for
creating hypoxia condition and kept in orbital shaker at
140 rpm up to 24 h in continuous light condition. After
reaching optimal growth (mid-log phase) and as mentioned
previously chlorophyll amount were also measured using
other replicates and proceed for purging with nitrogen to
remove oxygen to create anoxic conditions. Later depending
on the set of experiments, cultures were incubated in different
light and/or pH and/or temperature and/or nutrients with
continuous shaking at 140 rpm. After 10 min of purging with
nitrogen, all the samples were removed and estimated for
hydrogen using GC-TCD (Shimadzu, GC-2014, Japan) at
different time intervals i.e. 6, 12, 24 and 48 h according to the
procedure of Lakshmana Reddy et al. [25]. Nitrogen was used
as a carrier gas at a flow rate of 15 ml/min. The packed column
was maintained at 40  C and the temperature for the detector
Fig. 2 e Growth assay of Chlorococcum minutum and
Chlamydomonas reinhardtii in light and dark conditions. All
the cultures were grown in TAP with pH 7.0 along with
25  C. Cultures were maintained in 107.02 cd light
conditions. Inoculum contains equal biomass and
photograph was taken after five days.
Please cite this article in press as: Paramesh K, et al., Enhancement of biological hydrogen production using green alga Chlorococcum
minutum, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.005
and injector was set at 100 and 80  C respectively. The total H2
and O2 volumes were calculated according to the peak area,
which was pre-calibrated by injecting known concentrations
of standard gases. Specifically H2 contents were estimated
using minimum of three replicates of peak area values. The
statistical work has been carried out using excel program of
computer for different parameters.
Estimation of hydrogenase enzyme activity
Before going to estimate hydrogenase activity, all the cells
must be grown until early to mid-log growth phase. Here
culture conditions for algal growth was normal i.e. continuous
light, pH 7.0 along with 25  C temperature. For anaerobic in-
duction, cultures were spun down at 3500  g for 10 min at
25  C and resuspended in AIB (anaerobic induction buffer) so
as to get 30 mg/ml cell suspension. Later cultures were purged
with nitrogen gas up to 30 min then were continued to incu-
bate in orbital shaker with 140 rpm for 90 min in the dark. In
in vitro hydrogenase enzyme activity experiment, add 1.0 ml
methyl viologen (MV) reaction buffer and mix with 200 mL of
100 mM sodium dithionite (SDT) in 30 mM NaOH in the glove
box. After that it was purged with N2 gas for 20 min and later
add 100 ml of the anaerobically induced cells to the MV reac-
tion buffer plus SDT. Samples were incubated for 10 min and
then 250 ml of the head space was injected into GC-TCD for
determining the activity.
Results and discussion
In the present investigation an attempt has been made to
know the production of biological hydrogen from Chlorococcum
minutum species. Initially growth assay was performed to
standardize the growth conditions for C. minutum and then
compared with model alga C. reinhardtii. Later different envi-
ronmental factors such as light, pH, temperature including
altered nutrient levels were tested for the growth of C. minu-
tum under in vitro conditions. Finally the H2 produced from the
above mentioned stresses were estimated through GC-TCD
and all the results were documented below.
Growth pattern of Chlorococcum minutum in light and
dark conditions
The growth pattern of C. minutum in light and dark conditions
was compared with that of C. reinhardtii using agar media
plates that contained TAP nutrients. Maximum growth of C.
minutum was noticed in the presence of light similar to model
alga (Fig. 2). Interestingly C. minutum species has shown sig-
nificant growth even in darkness within five days. But in C.
reinhardtii after one week the growth was still slow and mild.
This experiment helped us to establish the growth conditions
for C. minutum and also to know the H2 production levels.
Enhancement of hydrogen production in various light
conditions
After reaching the mid-log phase, all the cultures were purged
with nitrogen to remove oxygen and later serum vial bottles
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 7 ) 1 e1 0 5

were kept in orbital shaker for further growth. High levels of
photo-production of H2 in C. minutum was noticed when
compared to C. reihardtii in light condition (Fig. 3). In some
cases, certain dark period was also necessary for conversion
of sugars into alcohol and other metabolite formation
including hydrogen. Generally certain species and strains of
Chlamydomonas, Chlorella, Nannochloropsis etc. tolerate light
fluctuations and exhibits the maximum growth rate including
yield [19]. Depending on the availability of light, photosyn-
thetic efficiency is determined and after crossing the satura-
tion limit it causes photoinhibition or photoblast which leads
to variation in cell volume and number [26,27]. Algae including
other advanced plants overcome light limitation to some
extent by desaturation of chloroplast membranes and adap-
tation can occur through multiple mechanisms such as
changes in pigments, growth rate, dark respiration rate and
the availability of essential fatty acids [28,29].
Red light favors the growth and hydrogen production in C.
minutum
Parallel experiments were conducted to know the possible
role of photoreceptors which involved in growth and H2 pro-
duction. Interestingly C. minutum exhibited abundant growth
and H2 production in red light condition (Fig. 4). This data
reveals that the existence of phytochrome or like photore-
ceptor in this species and also supports the works of Rockwell
and Lagarias [30]. In contrast C. reinhardtii prefer blue light in
agreement with Voigt et al. [31] where blue light was shown to
be essential for the division of cells. Number of reports
confirmed that primitive plants including algae also possesses
blue light-mediated cryptochrome receptors and their func-
tion [32e34]. In extent Miyachi et al. [35] studied the starch
formation in Chlorella vulgaris under blue and red light con-
ditions and confirmed that carbon pathway in photosynthesis
is regulated by blue light even under low intensity. In contrast
blue light negatively impacts in resetting the circadian clock of
C. reinhardtii strain CC124 [34,36]. Interestingly both red and
blue light does not show any impact on photosynthetic effi-
ciency at low level of carbon dioxide in C. reinhardtii [37].
Several researchers worked on the effect of ultraviolet (UV)
light and emphasized the damage caused by UV-B than UV-A
in different algal species including Chlamydomonas, Tetraspora
etc. [38e40]. Overall enhanced biological hydrogen was
observed in C. minutum in red light and this data demonstrated
that this species is unique among green algae and is an
essential candidate species for industrial scale. Probably this
may be due to efficient hydrogenase activity or modified PSI
and II electron transfer systems as showed in different algal
species [41,42].
Alteration in pH also favors the hydrogen production in C.
minutum
It is known that pH is another important growth factor in algal
cultivation and determines the solubility, availability of CO2
and essential nutrients. In the present investigation, various
pH conditions were maintained in media to know the growth
pattern and H2 production in both the species. Neutral pH (7.0)
was favorable for both the species in terms of growth as well
H2 output, but C. minutum produced more hydrogen (Fig. 5).
Generally algae can grow in a pH range of 4e10, but most of
the algae prefer neutral pH with few exceptions such as acid-
tolerant Chlorella saccharophila, Chlamydomonas acidophila,
Euglena mutabilis, Dunaliella acidophila etc. and alkali-tolerant
Spirulina platensis, Dunaliella parva, Anabaena species etc.
[15,19,43,44]. In addition C. minutum exhibited moderate
growth at pH 6.0 and also generated more H2 when compared
to model alga (Fig. 5). Generally acidic condition alters the
morphology of algal cells and limits the motility of Chlamy-
domonas applanata and Euglena mutabilis [45]. Moreover Chla-
mydomonas applanata did not show growth in acidic condition
and exhibited maximum growth at pH 7.4 [46]. Low pH con-
dition limits the nutrient uptake which leads to the reduction

Fig. 3 e Effect of light and dark on hydrogen production from Chlorococcum minutum and Chlamydomonas reinhardtii. All the
cultures were grown in TAP with pH 7.0 along with 25  C. Cultures were maintained in 107.02 cd light conditions. Inoculum
contains equal biomass (total 5.0 ml culture).

Please cite this article in press as: Paramesh K, et al., Enhancement of biological hydrogen production using green alga Chlorococcum
minutum, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.005
6 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 7 ) 1 e1 0

Fig. 4 e Effect of red and blue light on hydrogen production from both Chlorococcum minutum and Chlamydomonas reinhardtii.
All the cultures were grown in TAP with pH 7.0 along with 25  C. Inoculum contains equal biomass (total 5.0 ml culture). (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of photosynthetic ability thereby decreasing the accumulation
of total carbon and evolution of oxygen [47]. Acidic conditions
also induce metal toxicity which in turn affects the growth
and development of various algae including Chlamydomonas
acidophila and Chlorella species [19]. In acidic conditions (pH4.4)
hydrogen ions denature certain proteolytic enzymes such as
V-lysin which facilitates releasing of daughter cells from
within the parental wall [19,48]. Similarly C. vulgaris exhibits
growth in extreme acidic environments such as pH of 3.0 with
altered biochemical modifications [49]. The energy required to
maintain internal pH in these acid-tolerant algae is conserved
and maintained through cellular metabolism. This is one of
the reason that algal growth is not drastically affected under
acidic conditions [50]. Dunaliella acidophila adapts to low pH
conditions in growth media by accumulating glycerol to pre-
vent the osmotic imbalance [51]. In extent certain algal species
accumulate more saturated fatty acids and storage lipids such
as triacylglycerides (TG) to tolerate acidic conditions [52].
Similarly at pH 8.0, C. minutum displayed moderate growth
and produced more H2 when compared to C. reinhardtii (Fig. 5).
Guan et al. [53] reported that optimal hydrogen was produced
at pH 8.0 in Platymonas subcordiformis. Alkaline conditions also
limit the availability of nutrients, specifically carbon from CO2
and suppresses the process of photosynthesis [43,54]. On the
other hand in higher pH, carbon is available in the form of
bicarbonates. Hence affinity of algae towards free CO2 de-
creases under high pH conditions [54,55]. Alkaline condition
also modifies the plasma membrane moiety that suppresses

Fig. 5 e Effect of different pH on hydrogen production from Chlorococcum minutum and Chlamydomonas reinhardtii. All the
cultures were grown in TAP along with 25  C and 107.02 cd light conditions. Inoculum contains equal biomass (total 5.0 ml
culture).

Please cite this article in press as: Paramesh K, et al., Enhancement of biological hydrogen production using green alga Chlorococcum
minutum, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.005
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 7 ) 1 e1 0 7

the algal growth. Moreover alkaline pH prevents the rupture of
cell wall of mother cells and inhibits autospore release, thus
prolongs the time for cell cycle completion [19,56]. Overall this
data concludes that both pH 6.0 and 8.0 are also favorable for
growth as well for H2 production apart from productive
neutral pH condition in C. minutum.
Effect of temperature on hydrogen production in C.
minutum
Generally optimum temperature range observed was be-
tween 20 and 40  C for growth of different algal species based
on the literature with some exceptions. In the present study,
algal cultures were stirred under continuous light at tem-
peratures of 15  C, 25  C and 35  C simultaneously and
observed the growth and development of C. minutum and C.
reinhardtii. The most suitable temperature for growth of both
the species was 25  C but H2 production in C. minutum was
high when compared to C. reinhardtii (Fig. 6). Temperature
optima for H2 output has been found to vary significantly
depending on alga. Chlorella vulgaris exhibits more growth
above 30  C [57]. For Scenedesmus and Nostoc muscorum the
optimum temperature is around 40  C, whereas for Spirulina
it is between 20 and 40  C. Chlostridium and Platymonas sub-
cordiformis exhibits better growth at 37  C and 27  C respec-
tively [19,58e60]. At 15  C temperature normal growth was
not found in agreement with Vonshak and Torzillo [61]. It has
been suggested that changes in cytoplasmic viscosity under
low temperature condition is responsible for less efficient
carbon utilization and reduced electron transport at a given
photon flux rate [62]. At low temperature, Dunaliella salina
exhibited increase in fatty acid unsaturation, altered fluidity
of cell membrane and considerable impact on efficiency of
photosynthetic machinery [63].
Moreover at 35  C temperature, there was significant
decrease in growth and H2 output was noticed in both the
species (Fig. 6). Though overall growth was diminished at
35  C, comparatively C. minutum proved best with respect to
H2 production. Depending on the species, the growth rate
alters with increasing temperature i.e. above critical level
[64]. In addition certain enzymes may works in ambient
temperature, but there is high possibility of modification in
activity at high temperature. Higher temperatures reduce
protein synthesis, accumulate more free amino acids and
consequently decreased growth rate was observed in
Phaeodactylum tricornutum and Ulva pertusa [19]. Moreover
temperature also exhibits influence on degradation of
starch content in Chlorella vulgaris [65]. Two fold increase in
total carotenoid content was observed in Chlorococcum sp.,
by raising the temperature from 20 to 35  C under nitrogen
deprivation conditions [66]. Though equal amount of
biomass was maintained in each set of experiment, varia-
tion in data was found among different sets of experiments
because each set was performed at different time periods.
For example 24 h data of C. minutum in light condition
(Fig. 3), pH7.0 (Fig. 5) and 25  C temperature (Fig. 6) is not
same due to variation in initial algal inoculum. For instance,
inoculation of algal colonies with toothpick was not same
every time and another valid point is dilution factor vary
every time in each set of experiment. In the present inves-
tigation 1.5e2.5 folds of biomass variation was found in
between different sets.
In extent growth pattern and H2 content were estimated
using TAP and TAP-B (without tris base) media in both the
species. As expected low levels of H2 was found in cultures
grown on TAP-B medium when compared to TAP medium in
agreement with several reports [9,19]. Tris base is useful for
maintaining the pH of media and is compulsory to the cul-
tures for optimal growth. This preliminary data also supports
the advantage of C. minutum and future works may have more
answers with respect to H2 production in nutrient alteration
conditions.

Fig. 6 e Effect of temperature on hydrogen production from Chlorococcum minutum and Chlamydomonas reinhardtii. All the
cultures were grown in TAP with pH 7.0 along with 107.02 cd light conditions. Inoculum contains equal biomass (total 5.0 ml
culture).

Please cite this article in press as: Paramesh K, et al., Enhancement of biological hydrogen production using green alga Chlorococcum
minutum, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.005
8 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 7 ) 1 e1 0
Fig. 7 e Estimation of hydrogenase activity in Chlorococcum
minutum and Chlamydomonas reinhardtii. All the cultures
were grown in TAP with pH 7.0 along with 25  C and 107.02
cd light conditions. Numbers indicates hydrogen production
in micromoles. Inoculum contains equal biomass.
Hyper hydrogenase activity in C. minutum
In order to know the reason for improved production of H2 in
C. minutum, hydrogenase activity was performed using the
algal cultures grown on normal conditions i.e. continuous
light, pH7.0 along with 25  C temperature. Generally native
hydrogenase expressed in chloroplast and later exhibits the
function depends on anoxic and/or any stress conditions. To
measure the H2 evolution, reduced methyl viologen was used
as an electron donor in detergent-permeabilized cells. Detec-
tion of its activity mainly via determining the reduction of
methyl viologen or measuring the volume of H2 evolved using
gas chromatography. In the present study, hydrogenase ac-
tivity was high in C. minutum than C. reinhardtii (Fig. 7). In
growth assay, greenness of C. minutum is almost equal to
model alga in light conditions but in case of H2 production the
variation is high which supports the probable role of hydrog-
enase including some other factors (Fig. 2). Here the enzyme
serves to recapture the electrons, oxidizing the H2 and
reducing ferredoxin. This data indicates the hyperactive
function of hydrogenase in C. minutum. Similar robust activity
was found in Chlorella and Tetraselmis hydrogenases [42,67,68].
Hydrogenase activity was performed at pH 7.0 and 25  C
temperature, but both pH 6.0 and 35  C also favors the
considerable quantity of H2 production in this species when
compared to C. reinhardtii. This data also supports the robust
activity of hydrogenase in this species. Number of copies of
hydrogenase gene may be more or there must be natural ge-
netic modification are also possible reasons for augmented H2
production in this species. This hyperactive hydrogenase can
be helpful for future genetic engineering experiments with
other algal species for enhanced H2 production.
Conclusions
Biological hydrogen is considered to be the ideal fuel for the
future because of its high energy content. Hence the entire
Please cite this article in press as: Paramesh K, et al., Enhancement of biological hydrogen production using green alga Chlorococcum
minutum, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.09.005
globe is desperately working for its efficient production from
various living organisms including algae through modern
biotechnological methods. But in the present investigation, an
attractive candidate alga C. minutum was screened and
enhanced H2 production was achieved normally when
compared to model alga C. reinhardtii besides application of
various stress conditions. Improved H2 yield was found in
continuous light specifically in red light conditions in this
species. Both pH 6.0 and 8.0 also favors the H2 output in C.
minutum apart from productive neutral pH 7.0 condition.
Though overall H2 production was high at 25  C temperature
in C. minutum, considerable quantity was produced even at
35  C when compared to model alga. In conclusion, this work
may be useful for large scale production of H2 without any
expense. Moreover hyperactive hydrogenase of C. minutum
may be useful for future research particularly for biological
hydrogen production in other algal species.
Acknowledgements
The authors are thankful to the Department of Science and
Technology (DST), Govt. of India funding in the form of
INSPIRE fellowship to KP. Authors also thankful to Prof. Kevin
Redding and Mr. Andrey Kanygin, Arizona State University,
Tempe, AZ, USA for their scientific suggestions and help.
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