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 4 5 ( 2 0 2 0 ) 8 3 1 0 e8 3 2 8

Available online at www.sciencedirect.com

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Review Article

Sustainable biohydrogen production by Chlorella

sp. microalgae: A review

Jorge Jim
enez-Llanos, Margarita Ramı́rez-Carmona*,

Leidy Rendo
n-Castrillo

n, Carlos Ocampo-Lo

pez
Centro de Estudios y de Investigaci
on en Biotecnologı́a (CIBIOT), Facultad de Ingenierı́a Quı́mica, Universidad
Pontificia Bolivariana, Cq. 1 #70-01, Bloque 11, Medellı́n, Colombia

highlights

Bio-H2 is a promising energy carrier to be considered in the sustainable energy market.

Chlorella sp. generate H2 and by-products with high interest and commercial value.

Bio-H2 production parameters should be optimized for each microalgae species.

Genetic engineering and bioreactor design will enhance Bio-H2 production.

Research is needed to make large-scale Bio-H2 production economically suitable.

article info abstract

Article history: The use of fossil fuels is causing a huge environmental impact due to the emission of air
Received 10 June 2019 pollutants, greenhouse gases, and other ground and water contaminants; also, these fuels
Received in revised form are depleting; the world is facing an energy crisis in the years to come if no preventive
27 December 2019 actions are done. Renewable energies are arising as promising technologies that will
Accepted 7 January 2020 complement and even replace conventional fuels shifting the global energy matrix to a
Available online 31 January 2020 cleaner and eco-friendly future. Microalgal biohydrogen is one of those emerging tech-
nologies that is showing positive results. This work provides an overview of the key pa-
Keywords: rameters to produce hydrogen from microalgae especially from the genus Chlorella. Current
Chlorella sp. status of chemical and biological hydrogen producing technologies is presented, along with
Biohydrogen production the main metabolic processes for this purpose in microalgae, their characteristic enzymes,
Biophotolysis several strategies to induce hydrogen production, the key operation parameters and finally
Bioreactor providing some remarks about scaling-up and industrial-scale applications.
Biorefinery © 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Hydrogen economy

Abbreviations: Bio-H2, Biohydrogen; PSI, Photosystem I; PSII, Photosystem II; Fd, Ferredoxin; PQ, Plastoquinone; NADPþ, Nicotinamide
adenine dinucleotide phosphate; NADPH, Reduced NADPþ.
* Corresponding author. Universidad Pontificia Bolivariana, Cq. 1 #70-01, Bloque 11, Oficina 317, Medellı́n, Colombia.
E-mail addresses: jorgeandres.jimenez@upb.edu.co (J. Jime
nez-Llanos), margarita.ramirez@upb.edu.co (M. Ramı́rez-Carmona), leidy.
rendon@upb.edu.co (L. Rendo
n-Castrillo

n), carlos.ocampo@upb.edu.co (C. Ocampo-Lo
pez).
https://doi.org/10.1016/j.ijhydene.2020.01.059
0360-3199/© 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
 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 4 5 ( 2 0 2 0 ) 8 3 1 0 e8 3 2 8 8311

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8311
Generalities about biohydrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8312
Biohydrogen production mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8313
Direct photolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8313
Indirect photolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8313
Dark fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8314
Enzymes for biohydrogen production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8314
Hydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8315
Uptake hydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8315
Nitrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8315
Strategies for biohydrogen production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8316
Macronutrient deprivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8316
Sparging with inert gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8317
Fed-batch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8317
Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8317
Genetic engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8317
Key parameters for biohydrogen production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8318
Microalgae species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8318
Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8319
Cell concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8319
Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8320
pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8320
Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8320
Scale-up and industrial application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8322
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8323
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8323
Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8323
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8323

heat waves that indicate climate change is happening at an

Introduction
Since ancient history, mankind had sought better fuels to
cover up the increasing energy demand, from wood to animal
fat, coal, petroleum, natural gas and more recently the trend
to migrate to alternative energies like solar, wind, hydraulic
and biomass [1,2]. During the last decade, energy consump-
tion increased exponentially due to industrialization,
modernization, prolonged life style and population growth [1];
it is expected that human population will be around 9 billion
people by 2050, so it forecasts a 50% increase of fuel demand,
but requiring an 80% decrease of CO2 emissions to maintain
climate security [3].
In 2017, world energy consumption was 13511.2 toe, 34.2%
from petroleum, 23.4% natural gas, 27.6% coal, 6.8% hydro-
electric, 4.4% nuclear and 3.6% renewable energies [4]; this
current energy matrix shows an excessive dependence on
non-renewable fuels. Conventional energy reserves are
lowering, production costs are increasing and political re-
lationships involving high-reserve oil countries are
tensioning; also, greenhouse gasses and pollutants are
causing irreversible effects in global ecosystems, like the
melting of artic ice, desertification, and over appearance of
alarming rate [2,5e8], it is clear that a change is a must,
otherwise CO2 will hit levels of 1000 ppm by the end of the
century, raising temperatures between 3.2 and 5.4  C [9].
Fossil fuel exhaustion, combined with visible environ-
mental negative effects, political tensions, economic and so-
cial crisis are pushing humanity to shift to renewable and
sustainable energy sources [10e16]. It is not realistic to expect
a dramatic change immediately, but a gradual shift to these
technologies without damaging the bionomical balance could
become a direct substitute of the traditional energy sources
[6,17,18].
Biofuels are classified in three groups. First generation
biofuels are mainly extracted from maize, sugarcane, soy-
beans, wheat, barley, sugar beet, and other food and oil crops
by mechanical, chemical and biological methods; they have
reached competent production levels [6,19]. Second genera-
tion biofuels are produced from non-edible lignocellulosic
crops and organic wastes like grasses, sawdust, sludge, live-
stock manure and basically any carbohydrate-rich residue by
using microorganisms [19], bioenergy from organic wastes is
becoming more relevant showing high potential at various
stages of development, demonstration, and commercializa-
tion [10,20]; nonetheless, production of these biofuels are in
8312 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 4 5 ( 2 0 2 0 ) 8 3 1 0 e8 3 2 8
some cases energy intensive, also technical barriers like lignin
removal and cellulose degrading prevent this process to be
cost effective [19]. A major drawback of first- and second-
generation biofuels like ethanol and biodiesel is that they
compete for fresh water, fertilizers and it takes an estimated
of 1% of arable land to provide 1% supply of the global trans-
port fuel demand [6]; also, they cannot be used in unmodified
engines above small blends and they are not suitable for the
aeronautics market [21].
Third generation biofuels are produced by microalgae
which have several advantages like the ability to grow 5e10
faster than terrestrial crops, they can be cultivated in non-
agricultural areas, they can grow in nutrient-rich waste-
water so there is not competition for fresh water and they can
fix atmospherical CO2 [19,22], major example of these fuels is
biodiesel made from lipids accumulated inside the cells, and
most recently Bio-H2 has showed great potential to become a
new and cleaner energy carrier [1,2,7,23e27].
In this review, a deeper look about Bio-H2 is presented from
the general perspective about this element, passing to through
different production technologies and discussing about some
biological process parameters using microalgae as a viable
way to produce it. Several studies have presented Chlamydo-
monas reinhardtii as the best H2 producer; however, along this
review study, Chlorella species are presented as a more
adequate microorganism to conduct a sustainable process due
to the high H2 production capacity combined with production
of other by-products with high interest and commercial value;
thus, at industrial scale, a sustainable biorefinery approach for
H2 production could be accomplished by using Chlorella
microalgae.
Generalities about biohydrogen
H2 is the most common element in the universe and scientists
are interested in domain its properties. It is a non-toxic,
odorless, colorless, tasteless and highly combustible gas [1],
it has the highest energy density (142 kJ g1) compared to any
other known fuel (Methane 56 kJ g1, natural gas 54 kJ g1,
gasoline 47 kJ g1) [2,28e30]. Energy conversion of H2 is com-
parable to electrochemical and combustion processes [31],
although the conversion of Bio-H2 in power fuel cells is two
times more efficient than burning a biofuel in combustion
engine [1,32], and it only produces water as a by-product when
burned, so it does not contribute to greenhouse gasses emis-
sions making it a clean option to replace fossil fuels and
mitigate some current environmental issues [2,26,33e36].
H2 can be stored in high-pressure gas cylinders, as liquefied
gas by cryogenic applications, by adsorption on carbon
structures, by absorption in host metals and metallic hy-
drides, in complex compounds, and by metals and complexes
together with water; however, these techniques require high
energy demand and are technically challenging [17,37]. Direct
applications are more recommended; like use it as a heat
source, as clean fuel for transportation since its combustion
value is about three times the one for gasoline, for electricity
production via fuel cells, as rocket fuel and as a raw material
for many industrial processes [1,38e40].
Globally, 500 billion m3 of H2 are produced each year with
10% growth rate [28], where 40% of the total is produced from
natural gas, 30% from heavy oil and naphtha, 18% from coal,
4% from electrolysis and 1% from biomass [26,41]. From the
produced H2, 49% is used in ammonia production for fertil-
izers, 37% petroleum refining, 8% methanol production, and
6% in other applications [1,3,26,28].
Currently, H2 is mostly obtained by chemical processes like
thermocatalytic reformation of hydrocarbons, thermal
cracking of natural gas and gasification of biomass
[6,11,15,33,38,42,43], they are the most used processes for H2
production due to their technology maturity and economic
viability [35]. However, this processes use conventional fossil
fuels as raw materials that are being depleted, their trans-
formation requires high thermal energy and greenhouse
emissions are elevated, thus a higher environmental impact is
being made [7,24,44]. In order to overcome this situation, new
technologies such as thermochemical water splitting, elec-
trolysis, photoelectrolysis and especially Bio-H2 production
techniques have demonstrated great potential because they
rely on renewable-energy sources [8,11,28,34,36,39]
Bio-H2 can be produced in several ways like photo-
fermentation, dark fermentation, direct and indirect bio-
photolysis by several kinds of microorganisms including
bacteria, cyanobacteria and green algae [40,42,44e46]. Among
all these methods, H2 production by using microalgae stands
out from the rest [24,47e53], because these processes are less
energy intensive, more economical, they use renewable
sources like sunlight, CO2 emissions are low, they can be
coupled with CO2 fixation and nutrient removal from waste-
water [54e57], they do not compete with agricultural land for
food production, and operate at ambient temperature and
pressure [3,7,29,31,49,57e63].
In addition to Bio-H2, microalgae have the capacity to
produce other by-products with high commercial and scien-
tific value like carbohydrates, proteins, lipids, pharmaceutical
compounds and other biofuels such as biodiesel and bio-
ethanol, they also can serve for cosmetics, food and fish feed
[45,64e68]. Therefore, to make Bio-H2 production a viable
process, it is important to stop thinking about it as a lone
process itself but start developing it as a biorefinery integrated
process to obtain more benefits from the same cells
[47,57e59,63,69].
Bio-H2 is a green energy carrier as result of the oxygenic
photosynthesis and a H2 production metabolism described
in several green algae genus and cyanobacteria [70]. It was
first reported by Jackson and Ellms in 1896 using cyanobac-
teria Anabaene, formerly known as blue-green algae [17].
Later in 1939, Gaffron and Rubin demonstrated that green
microalgae Scenedesmus obliquus produced H2 gas under
certain illumination conditions [14,39,71]. During 1970s,
photosynthetic process grew on research interest, never-
theless productivity was unremarkable and practical appli-
cations of those discoveries were not actively developed
[17,62]. In 2000s, Melis et al. introduced the two-stage pro-
cess concept making it an important breakthrough for large
scale Bio-H2 production; they enhanced productivity up to
140 mL L1 in 80 h by cultivating Chlamydomonas reinhardtii in
flat photobioreactors separating biomass growth from H2
 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 4 5 ( 2 0 2 0 ) 8 3 1 0 e8 3 2 8
production stage under S-limited anaerobic conditions [72].
In some cases, microalgae H2 conversion efficiency is higher
than that in cyanobacteria [40,52]. lf a real life application is
wanted, it is mandatory to keep researching until acceptable
understanding is reached about the involved enzymes,
metabolic pathways, large bioreactor designs and biotech-
nological parameters for Bio-H2 production using microalgae
[14,39,73,74].
Biohydrogen production mechanisms
Bio-H2 production in microalgae is classified into two main
categories as Light-dependent and Light-independent [75].
Among the first group, the representative mechanisms are:
Biophotolysis of water using microalgae and cyanobacteria,
photofermentation mainly from photosynthetic bacteria. In
the second group, dark fermentation highlights through
fermentation of organic compounds with anaerobic bacteria
but recently some species of microalgae have been reported to
produce H2 by this mechanism [5,15,29,35]. Normally, H2
production in cyanobacteria is related with nitrogen fixation
and green algae with reduction of Hþ electrons generated
through photosynthesis [14]; thus, biophotolysis is the most
common H2 pathway used by algae.
Photosynthesis occurs in the thylakoid membranes of the
chloroplast of green algae [76], and the electron transfer relies
on two reactions known as light and dark reactions [40]. The
light reaction occurs in the PSII where pigment molecules
absorbs solar energy and water splits into O2 and protons (Hþ).
Electrons flow from PSII to PSI through the electron transport
chain composed by the PQ pool and cytochrome b6f complex
(Cyt b6f), once in the PSI, Fd generates ATP and NADPH
[40,46,77]. In the dark reaction, the energy obtained during the
light reaction is used to fix CO2 as starch [78]. When H2 pro-
duction is active, Fd donates electrons to the enzyme hy-
drogenase and it is combined with protons, yielding H2
[46,61,77]. The general reaction is shown in Eqn (1) where
electrons flow through PSII and PSI to the key producing en-
zymes (hydrogenase or nitrogenase) using the Fd electron
carrier[17,29].
Eqn (1) General scheme of H2 production associated to
photosynthesis. Adapted from Ref. [79].
Also, this process has two categories; direct biophotolysis
that uses electrons generated by water splitting in PSII, and
indirect biophotolysis that uses electron from catabolism of
endogenous substrates [40,73]. Besides those, recent studies
confirm the presence of a third light-independent fermentative
way associated to starch degradation [9,77,80]. Microalgae with
high H2 production potential are Chlamydomonas, Chlorella and
Scenedesmus [81]. Though, compared to Anabaena sp.,
Chlorella pyrenoidosa, Chlorella vulgaris, Platymonas subcordiformis
and Spirulina platensis, C. reinhardtii is the model organism for
studying H2 metabolism in green algae [45,46]. Chlorella Fusca
8313
showed same reactions to S-limitation as C. reinhardtii, there-
fore metabolism of this two species could be similar [14].
Direct photolysis
As general concept, direct biophotolysis is the most studied
method for microalgae H2 production, it uses sun light as
energy source to split water in the PSII, the residual electrons
are transferred to PSI where hydrogenase leads to H2 pro-
duction without greenhouse gases emissions and simulta-
neous release of O2 [26,60,73,82e84].
In a more specific description, water suffers photochemical
oxidation at the thylakoid membrane by PSII (water- PQ-
oxidoreductase) [73]; then, PSII energizes electrons using
photons from sunlight transferring them to PSI via electron-
transport chain and then to Fd where Fd-oxidoreductase
produces reduced Fd [60,75], this acts as an electron donor to
[FeeFe]-hydrogenase which facilitates the reduction of Hþ to
H2 and simultaneous O2 production at PSI (Eqn (2)) [7,15,73,85].
2Hþ þ 2FD 4H2 þ 2FD (2a)
2H2 O þ lightenergy/2H2 þ O2 (2b)
Eqn (2) (a) Fd reduction in PSI. (b) Overall conversion of
water to H2 by green microalgae. Adapted from Refs. [60,79,86].
Pow and Krasna demonstrated that Scenedesmus and
Chlorella cells could produce H2 by direct biophotolysis if a
correct O2 removal is guaranteed [83].
Direct biophotolysis is considered a sustainable process
with promising future prospect since it converts solar energy
into chemical energy, main reactants are widely available
such as water, sun light and CO2; and products are H2 and O2
[7,8,12,15,42]. Nevertheless, this pathway has several tech-
nical issues that holds back its true potential. Biophotolysis
must operate near to atmospheric pressure, but this is a 1000-
fold greater than the maximum tolerated by the [FeeFe]-hy-
drogenase enzyme [44]; thus, coproduction of H2 and O2 is the
major drawback in this processes due to the high sensibility of
[FeeFe]-hydrogenase enzyme to O2 that inhibits photo-
evolution of H2 resulting in low yield of Bio-H2 [7,66,73];
therefore, it is mandatory to remove the produced O2 gas in
order to have a sustained H2 production [84]. To overcome O2
sensibility, many techniques are being investigated such as O2
absorbers, inert gas bubbling and metabolic engineering for
O2tolerant enzymes [24,84]. Due to the low energy conver-
sion efficiencies, production requires a large algae cultivation
area to collect sufficient light to capture enough free energy
(DG ¼ þ237 kJ mol1), slowing the H2 production rate [60,73].
Other challenges include photosynthetic and respiration ca-
pacity ratio, concentration and processing of cell biomass [15].
Indirect photolysis
Microalgae and cyanobacteria can produce H2 from stored
glycogen [85], this approach solves the O2 sensibility problem
by temporally separating the O2 and H2 evolution reactions
into two separate stages coupled through CO2 fixation/evolu-
tion [26,44,87]. During first stage or aerobic phase, microalgae
8314 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 4 5 ( 2 0 2 0 ) 8 3 1 0 e8 3 2 8
are allowed to grow under normal cultivation conditions
converting solar energy and fixing CO2 into chemical energy as
carbohydrates, lipids and other organic molecules, the aim is
to increase the endogenous substrate content producing O2 as
a by-product [60,87]. When O2-depletion is achieved, electron
transport chain stops functioning, inducing the second stage
or anaerobic phase [87]; here, electrons from the catabolism of
the endogenous substrates (glycolytic pathway and citric acid
cycle) are used in PSI for the non-photochemical reduction of
PQ pool by light-dependent Fd reduction, then electrons are
transferred to [FeeFe]-hydrogenases resulting in H2 produc-
tion [3,7,24,60,75,83,84]. Overall reactions are presented in Eqn
(3).
12H2 O þ 6CO2 þ light energy/C6 H12 O6 þ 6O2 þ 6H2 O
C6 H12 O6 þ 12H2 O þ light energy/12H2 þ 6CO2 þ 6H2 O
Eqn (3) (a) Overall aerobic phase reaction. (b) Overall
anaerobic phase reaction. Adapted from Ref. [26].
Stuart and Gaffron demonstrated that Ankistrodesmus and
Chlorella H2 photoproduction did not require PS II, thus indi-
rect biophotolysis is possible [88].
The main advantage of this pathway is the ability to
overcome O2 sensibility of hydrogenase enzymes, however,
production rates are incomparable to other biofuels [73]. In
cyanobacteria, heterocysts protect nitrogenase thanks to a
thick cell wall that has a low diffusivity of O2 yielding more H2,
on the contrary, green microalgae lacks of heterocysts causing
inefficient nitrogen fixation under anaerobic conditions,
resulting in low H2 generated by indirect biophotolysis [60].
This methodology could be possible if bioreactor engineering
could increase light conversion efficiency for large-scale ap-
plications [73,82] by reaction spatially in two different bio-
reactors or by alternating the photosynthetic and
fermentation periods [66].
Dark fermentation
Microalgae mostly store carbohydrates in form of starch and it
is used in anaerobic condition to produce H2, yet low pro-
duction rate is achieved due to limited concentration of
endogenous substrates. Recent studies demonstrate a worthy
increment of H2 evolution in microalgae by providing them an
exogenic carbon-rich media early in the anaerobic phase
[32,47,85].
Photofermentation is the conversion of complex organic
substrates into organic acids, alcohols, CO2 and H2 in presence
of light; while dark or heterotrophic fermentation do not
require light but only organic-rich media in anaerobic envi-
ronment [9,73,86]. Although, this processes are more related
to anaerobic fermentative bacteria (e.g., Escherichia coli, Clos-
tridium spp., Thermococcales spp, Rhodobacter spp., Rhodop-
seudomonas spp.) [89,90], similar photofermentation and dark
fermentation mechanisms have been reported in certain
microalgal species (e.g., Chlamydomonas sp., Chlorella sp., Sce-
nedesmus sp.) [42,77,91].
All H2 producing pathways includes the reduction of Fd as
electron donor to [FeeFe]-hydrogenase. Direct biophotolysis
uses water splitting employing PSII and PSI, in contrast, indirect
biophotolysis uses NADH from endogenic catabolism and
fermentation from exogenous substrates working with PSI only
[9]. Microalgae prefer dark fermentative over light-driven
reduction of Fd, the pathway associated uses the pyruvate-Fd-
oxidoreductase enzyme [9]. First, organic substrates are con-
verted into pyruvate via glycolysis, then it is oxidized to Acetyl-
CoA resulting in ATP generation and release of acetate. Fd
reduction is critical to oxidize pyruvate into Acetyl-CoA, this
reduced Fd is further oxidized by hydrogenase releasing the
necessary electrons to produce H2 [79] (Overall reactions are
presented in Eqn (4)). In C. reinhardtii, key enzymes to catalyze
pyruvate are pyruvate-formate-lyase 1 (Pfl1) and pyruvate-Fd-
oxidoreductase 1 (Pfr1). Pfl1catalyze the pyruvate-acetyl-coA
conversion in the mitochondria and chloroplasts; while Pfr1
(3a)
oxidizes not only pyruvate but two molecules of reduced Fd,
generating electrons to be used for H2 production [75].
(3b)
Pyruvate þ CoA þ 2FdðOXÞ / Acetyl  CoA þ 2FdðREDÞ þ CO2 (4a)
2Hþ þ FdðREDÞ / H2 þ FdðOXÞ (4b)
Eqn (4) (a) reaction of CO2 production from pyruvate. (b) H2
production from Fd oxidation. Adapted from Ref. [79].
Glucose is the preferred carbon source because it rapidly
converts into the primary metabolite pyruvate [75], but other
carbon sources can be used since metabolism pathways
guides them to pyruvate formation. Butyric and acetic acid are
common by-products co-generated with H2 as seen in Eqn (5).
C6 H12 O6 þ 2H2 O/2CH3 COOH þ 4H2 (5a)
C6 H12 O6 þ 2H2 O/CH3 CH2 COOH þ 2H2 (5b)
Eqn (5) (a) reaction of CO2 production from pyruvate. (b) H2
production from Fd oxidation. Adapted from Ref. [79].
Boboescu et al. obtained sustained H2 production with
Chlorella sp. using beer-brewing wastewater as substrate,
suggesting its potential for algal photoheterotrophic growth,
wastewater treatment and biofuel production [9]. Also,
Chandra and Venkata proved that increased H2 production,
biomass growth or lipid accumulation could be reached by
controlling pH and light-dark cycles using microbial dark
fermentation wastewaters that limits some inhibition factors
such as dissolved O2 [92].
Fermentation process have several advantages over the
aforeknown biophotolysis pathways [82], it does not require
large cultivation space since no light must be captured, H2 is
produced at high rate and only by using organic substrates,
opening the possibility for wastewater and industrial carbon-
rich waters usage, reducing the net production costs and
generating a positive environmental impact [73]. In addition,
valuable by-products can be obtained such as antioxidants,
fatty acids, polysaccharides, glycerol and antibiotics not to
mention a near 80% substrate conversion efficiency [93,94].
Enzymes for biohydrogen production
Enzymes that contain complex metalloclusters as active sites
are key molecules to catalyze H2 production [26]. Nowadays,
there are three known enzymes responsible of H2 production
in green microalgae that can be active simultaneously:
 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 4 5 ( 2 0 2 0 ) 8 3 1 0 e8 3 2 8
Hydrogenase, uptake hydrogenase and nitrogenase. This was
confirmed over the years by studying inorganic carbon fixa-
tion, fermentation, and vitamin biosynthesis pathways in C.
reinhardtii and other green algae [44,47].
Hydrogenase
H2 production metabolism is widely known among microor-
ganisms, where hydrogenases are the main enzymes for its
evolution or oxidation [14,35]. In green microalgae, H2 pro-
duction depends on hydrogenase activation in the stroma of
the chloroplast under strict anaerobic/anoxic conditions [6],
this due to the high inhibition of the enzyme in the presence
of either external or internal photosynthetic O2 that blocks the
transcription of the H2 production genes in the metabolism
[31,37].
Hydrogenases are classified depending on the metal
cofactor inside the active cluster, where three types of hy-
drogenases have been reported: [FeeFe]-hydrogenase,
[NieFe]-hydrogenase, and [Fe]-hydrogenase [14,86]. Whereas
[FeeFe]-hydrogenase, [NieFe]-hydrogenase are ubiquitous
among microorganisms, [Fe]-hydrogenase is a homodimer
present in some methanogenic archaea and it will not be
discussed in this review [87,95]. Characteristics as activity,
maturation and structural diversity differs upon species, but
they share a common FeeS cluster, active site, and bonds with
CO and CN (Fig. 1) [40,96].
[NieFe]-hydrogenase consists of two structures. The active
site has around 60 kDa and [NieFe] bonds, the small unit has
30 kDa with 4Fee4S or 3Fee4S cluster, cysteine radicals are
bonded to the metal cofactors with S bonds. This is the
representative enzyme in cyanobacteria, [NieFe] are more O2-
tolerant than [FeeFe]-enzymes, their expression does not
require strict anaerobic environment and inhibition is
reversible [40].
[FeeFe]-hydrogenase has a monomeric structure with
molecular weight of 45e50 kDa [40], It is formed by only a
catalytic subunit and two iron groups located in the catalytic
center (H-cluster), composed of sulfur bridges and non-
proteinous ligands CN and CO attached to both Fe atoms
[97]. It is encoded in the hydA gene and located in the chlo-
roplast after enzyme maturation [29,40]. The exceptionally
high H2 production efficiency of green microalgae is partly due
the great performance of this enzyme, which produces a 100-
fold more than other hydrogenases (104 gas molecules s1)
[3,24,95]. However, it is very sensitive to O2, thus it is difficult
to produce H2 at atmospheric conditions [40]. A study con-
ducted with Chlorella sp. NC64A demonstrated [FeeFe] cluster
Fig. 1 e Structure of the hydrogenase enzyme: (a) [NieFe]-hydrogenase, (b) [FeeFe]-hydrogenase, (c) [Fe]-hydrogenase.
Adapted from Ref. [86].
8315
hydrogenase shows both fermentative and photoproductive
H2 activity [79].
O2-sensitivity by [FeeFe]-hydrogenase has been mentioned
repeatedly as an enzyme activity inhibitor and hydrogenase
gene suppressor, even a small amount of atmospheric O2 or
one generated by PSII could cause irreversible damage in the
catalytic cluster [24,35]. This O2-sensitivity is recognized as
the principal issue for the Bio-H2 production by microalgae
[30]. Notwithstanding, some algae have shown H2 production
under aerobic conditions, possibly they have O2 tolerant
mutant enzymes or the native ones may not be fully inhibited;
further research on O2-tolerant enzymes and inhibition of O2
evolution must be done [12,80].
For instance, Fan et al. conducted in vivo experiments
demonstrating that H2 activity in C. pyrenoidosa did not have
dramatic change even when cells were treated with 21% O2.
Also, C. pyrenoidosa showed 1.6 times more tolerance to O2,
extended production time and higher final yield of H2
compared to C. reinhardtii; although, H2 producing activity was
higher in C. reinhardtii [39]. Similarly, Hwang et al. confirmed
two C. Vulgaris strains with ability to activate [FeeFe]-hy-
drogenase in presence of 21 %O2 and produce low levels of H2
at 15% atmospheric O2 [91]. Other genetic engineering pro-
cedures are being performed to overcome O2-sensivity, such
as, random mutagenesis and targeted mutagenesis of the
catalytic site to prevent O2 diffusion. For now, results are more
convincing in [NieFe]-hydrogenase rather than [FeeFe]-hy-
drogenase [3,26].
Uptake hydrogenase
Contrary to hydrogenase, this enzyme is O2 tolerant [47] and
consume produced H2 by transferring electrons from H2 to
some electron acceptors like SO2 4 and NO3 [86] when dis-
solved H2 is high enough to cause a super-saturation inside
the reactor [98], therefore, overall yield of H2 production is
reduced or in some cases equal to zero [26]. Since the partial
pressure of the produced H2 is a relevant factor in this pro-
cess, systems must be perfectly designed to reduce it, for
example by nitrogen sparging or vigorous mixing [43]. Activity
of uptake hydrogenase is not desirable for the Bio-H2
production.
Nitrogenase
This enzyme is present in the heterocysts of many cyano-
bacteria and it is the responsible of fixing atmospheric N2 into
ammonia and produce H2 as a by-product [40]. There are two
types of nitrogenase: Dinitrogenase (220e240 kDa) has a
8316 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 4 5 ( 2 0 2 0 ) 8 3 1 0 e8 3 2 8
MoeFe protein that breaks up the triple bond between the two
nitrogen atoms; and, dinitrogenase reductase (60e70 kDa)
which contains only a Fe protein that transfers electrons from
the external electron donor to the MoeFe dinitrogenase
[86,97]. The turnover rate of nitrogenase is lower compared to
hydrogenases, however it requires a large amount of elec-
trons, reductants and ATP molecules from photosynthesis or
degradation of internal substrates [40].
Strategies for biohydrogen production
Sustained H2 production under standard growth conditions is
a permanent challenge, as O2 sensitivity inhibits key enzymes
[3,80], several approaches to overrun these problems include
the use of O2 scavengers, better tolerance of metalloenzymes,
increased respiratory activity, PSII inhibitors, purging with
inert gas or exploring illumination cycles [17,26].
Recently, a two-stage process that combines direct and
indirect biophotolysis proposes the separation of photosyn-
thetic O2 evolution and anaerobic H2 production by inhibition
of PSII [17,31,71]. During first stage, biomass is cultured in
nutrient rich media upon various cultivation conditions
(Phototrophic, heterotrophic, mixotrophic or photo-
heterotrophic) allowing normal oxygenic photosynthesis and
consistent increase in cell concentration; also, other carbo-
hydrates are stored as endogenous substrates. During second
stage, culture media changes to S-limitation, this induces
anaerobiosis that activates degradation of the endogenous
substrates, therefore, hydrogenases activate and subsequent
H2 production is achieved [3,13,31,38,49,62,71,99,100].
When bioreactors are sealed, headspace O2 is rapidly
consumed by microalgae inducing an anaerobic environment,
other strategies use exogenous or endogenous substrates for
its removal [7,12]. By lowering O2 partial pressure, hydroge-
nase activity is positively affected reducing adaptation time
and H2 production start sooner [7]; However, anaerobiosis
should be complemented with another PSII inhibition treat-
ment in order to induce the pyruvate-dependent metabolic
pathway [24].
Macronutrient deprivation
Normal life cycle of Chlorella cells involves four main stages:
(1) “Growth” - cell density increase, (2) “Ripening” - various
reactions occurs in preparation for cell division, (3)
“Maturing” e full mature mother cells, (4) “Division” [101].
Macronutrients as sulfur, nitrogen and phosphorous play
crucial role in microalgae life cycle [12], lack of these nutri-
ents causes important changes in cell physiology affecting
growth and ripening, thus maturing and division do not
occur [101]. Additionally, changes in protein, lipid content,
and a 10-fold increase in carbohydrate contents were re-
ported after 24 h of nutrient limitation [24]. Interestingly, this
phenomenon draws to reversible PSII inactivation by
increasing cellular respiration that consumes available O2
creating an anoxic environment and resulting in the tem-
poral separation of O2 and H2 metabolisms, giving the
microalgae the ability to produce H2 [12,40,75]; this is a widely
known condition to produce microalgae H2 [53].
In brief, H2 production under macronutrient-deficient
conditions can be synthetized in 4 stages: (1) “Aerobic” e
photosynthetically cell growth and endogenous substrate
accumulation, (2) “O2 consumption” e macronutrient defi-
cient media that increases cell respiration guiding to low O2
partial pressure, (3) “Anaerobic” e gradual decrease of
endogenous substrates, pH in the medium, and accumulation
of products of fermentative metabolism such as succinate,
formate, and ethanol, (4) “H2 production” e coupled to residual
PSII activity [24,31,60,62,102].
It should be noticed that macronutrient-limitation to
create anaerobic conditions is still considered direct bio-
photolysis and not indirect biophotolysis, because PSII is not
fully inhibited but has reduced activity [60]. Also, prolonged
nutrient limitation causes a harmful effect that results in
culture death; therefore, it is relevant to regenerate PSII sys-
tem before reaching a non-return point. Strategies such as
continuous or semi-continuous regime of cultivation, re-
addition of nutrients or intensive aeration of algal suspen-
sions promote the re-oxidation of the electron transport chain
and restoring PSII for another H2 production cycle have been
proposed [24].
Sulfur is one of the six more important macronutrients
required for growth and development, mainly available as
sulfate in nature, it is necessary for synthesis of amino acids,
sulfolipids and other S-containing substances [17,31,103]. Be-
sides its relevancy, S-limitation is one of the most recent
studied methods for microalgal H2 production in the light, first
discovered by Melis et, al. using C. reinhardtii, it takes advan-
tage of the stress reactions driven by algae in order to survive
[39,49,104].
S-limited media can be achieved by changing the whole
media into a strict S-limited medium, by inoculating initial
growth media with a limited amount of sulfate or by adding
ethanesulfonate that allow growth under sulfate limitation
[14,103]. Key aspect in S-limited strategy is blocking the photo-
oxidative damage reparation cycle of PSII by stopping the D1
polypeptide chain (32 kDa) in the reaction centers, after a lag
time, residual PSII activity increments cellular respiration in
mitochondria triggering anaerobiosis in the media, proteins
breakdown and cell storages more important compounds for
survival like starch and lipids. Residual electrons from PSII
and PSI systems provides [FeeFe]-hydrogenase resulting in H2
production [31,39,60]. Zhang et, al. reported protein and starch
increase by 40 and 600% accordingly from the first 24 and 30 h
after S-limiting conditions [105], providing evidence that
sustained production could be obtained within 4 days, drop-
ping its production rate to insignificant values up to 30 days
due to endogenous substrate catabolism [17,60]. Zhang et al.
demonstrated that S-limitation was inconvenient for inducing
photoproduction of H2 in C. protothecoides [106]. While, Batyr-
ova et, al. suggested the use of seawater which is cheaper than
fresh water to lower production costs [102].
Similar to S-limitation, nitrogen is decisive for biomass
growth, N-limitation exhibits reduced photosynthetic activity
but a large increase in accumulation of carbohydrates neces-
sary for subsequent catabolism for H2 evolution [38,65,107].
One study affirms that N-limitation has more influence on H2
evolution in C. protothecoides [106]. Another investigation
shows N-deprived Chlorella sp. LSD-W2 gave the highest H2
 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 4 5 ( 2 0 2 0 ) 8 3 1 0 e8 3 2 8
production rate with 1.52 ± 0.09 mmol H2 mg chl1 h1
compared to S and P-deprived media [46]. Li et, al. found C.
pyrenoidosa could tolerate low O2 concentrations in the reactor
headspace when growth in L1-N medium, and compared to S
and P-limitation, N-limitation has less lag time to establish
anoxic conditions [99]. These studies suggest N-limitation
rather than S-limitation as more influential for H2 production
in Chlorella species. However, interesting results exposed by
Alalayah et al., affirm enhanced H2 yield by adding nitrogen
and phosphate content 10% by mole up to BBC media in
anaerobic process [38].
Phosphorus limitation has also been cited in literature,
both N and P-limitation present similar PSII inactivation
metabolism as in S-deprived media but at slower rate [87,107].
Batyrova et, al. showed similar H2 evolution by P-deprived
strategy using marine Chlorella sp. to S-deprived fresh water C.
reinhardtii [102]. However, P-limitation decreases CO2 fixation
rate due to the depletion of phosphorylated intermediates in
the pentose phosphate cycle; also, S-limitation is faster than
P-limitation [12].
Simultaneous effects of S, N and P-limitation have been
studied showing better results compared to single
macronutrient-limitation [106,108]. Pongpadung et al.,
showed that most tested Chlorella strains reached anoxic
state faster and increased H2 production when grown in
simultaneous N-limitation and S-limitation compared to
only S-deprived medium. In contrast, Chlamydomonas did
not have the same effect. C. sorokiniana KU204 showed the
highest H2-producing capacity (1.30 mL L1 h1) and accu-
mulated 89.64 mL L1 under simultaneous N-limitation and
S-limitation containing a chlorophyll content of
5e6 mg L1; also, chlorophyll content in C. sorokiniana
KU207 and Chlorella sp. KU209 decreased by 85 and 80%,
respectively, after 1 day of growth under the N-limitation.
However, in P or S-limitation chlorophyll decreased by
approximately 65e70% after 2 days, that suggest nutrient
limitation results in loss of chlorophyll content [12].
Simultaneous N and S-deprived media potentiate H2 pro-
duction rate in Chlorella sp. LSD-W2, but solely P-limitation
did not have notorious effect [46]. Even with the promising
results, this strategy is less likely to be implemented in
large-scale operations due to insufficient production rates
to be economically viable [9].
Sparging with inert gas
As mentioned, [FeeFe]-hydrogenase is strongly inhibited by
presence of O2 or H2 in the culture media, therefore it is
important to remove or displace those gases (0.1% or below
partial pressure each) in order to overcome this inhibition of
H2 production; one way to achieve it is by continuously
sparging the media with inert gases like argon, nitrogen,
carbon dioxide or methane [86,104]. It is important that gas is
of high purity with no traces of O2, also it must be considered
a proper bubbling technique to avoid shear stress of micro-
algae [14]. This technique is useful at lab scale but not
adequate for industrial scale due to the large quantity of inert
gas required to boost H2 yield that leads to extra cost for the
process [60].
8317
Fed-batch
It has been demonstrated that metabolic stress increases H2
production, however most of the best results include a
media exchange from a nutrient rich media for biomass
growth to a macronutrient deprived media to induce H2
production; this approach is viable at laboratory scale but
almost economically impossible for large scale applications
due to the cost of biomass harvesting and costly media
components [71]. Recently, the fed-batch approach emerged
as a viable large scale process. Main principle is to have a
perfectly balanced nutrient media to grow biomass and by
the time cells are ready to produce H2, macronutrient con-
centrations are low enough to simulate a deprived media;
another option is to use macronutrient microdosaging along
the process, just enough to grow biomass during the first
stage and also to recover the PSII system after the H2 pro-
duction was done in order to prepare the culture for a new
production cycle [3,71].
Inhibitors
Inhibitors are becoming more common in practice. Addition
of inhibitors occur at the end of the light phase after reaching
the targeted cellular concentration because they directly
interfere the electron flow from PSII to the PQ pool, this cuts
photosynthesis and prepares cells for the dark phase to pro-
duce H2 in anaerobic conditions [73]. Some studies show that
algae like C pyrenoidosa produced H2 photoautotrophically
without S-deprivation under intense light exposition and
addition of 3-(3,4-dichlorophenyl)- 1,1-dimethylurea (DCMU)
[47,80]; when this inhibitor is present, electrons for PSI come
from reduced endogenous substrates [75]. Similar to DCMU,
cyanide m-chlorophenylhydrazone (CCCP) inhibits PSII activ-
ity for H2 production, but it should be noticed that reduced PSII
activity decrease the amount of electrons for the [FeeFe]-hy-
drogenase, yet resulting in lower H2 yield [39]. More recent
studies explore the idea of enhancing enzyme activity rather
than inhibiting PSII activity, one example was made by shu
et al., whom showed that dimethyl sulfoxide (DMSO) in a
range of 0e1% v/v can improve Bio-H2 production efficiency of
Chlorella from 0.42% to 0.69% [30].
Another way to induce an anaerobic media is by removing
O2 chemically using compounds like sodium dithionite, con-
verting it one of the simplest methods [104]; or as more other
studies suggest, co-cultivation of algae and bacteria induced
anaerobiosis due to the high bacterial respiration that lowers
O2 partial pressure in the media [3].
Deprivation of micronutrients have been tested as well,
showing that lack of magnesium lowers the chlorophyll con-
tent in the cells leading to a smaller PSII antenna size and
further increased light utilization, respiration rate, endoge-
nous substrates accumulation and prolonged production
period [3,87,107].
Genetic engineering
Genetics have been used recently to enhance H2 production in
microalgae focusing on determinant issues to accomplish (1) a
8318 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 4 5 ( 2 0 2 0 ) 8 3 1 0 e8 3 2 8
reduced amount of chlorophyll antennaes, (2) high photo-
synthesis and respiration rates, (3) a higher carbohydrate
accumulation during the aerobic phase, (4) a higher synthesis
of xanthophyll cycle pigments, which protects PSII from
inactivation [109]; combined with tailoring strain selection,
microalgae screening would accelerate this implementation
[110].
Torzillo et al. carried a double amino acid substitution at
the D1 protein, replacing a leucine residue (L159) by isoleu-
cine, and asparagine residue (N230) by tyrosine (L159IeN230Y)
in a S-deprived C. reinhardtii strain; this mutant had a pro-
longed H2 production period, lower chlorophyll content,
higher respiration rate and photosynthethic activity
compared to the wild type [107].
Other study used Chlorella sp. HydA from the DT strain to
modify amino acid residues A105I, V265W, G113I, or V273I
around the gas tunnel to prevent O2 accessing the enzyme
active site. In vitro assay showed that the DT transgenics
produced 7-fold more H2 than the wild type in presence of 5%
O2; whereas in vivo assays showed 30-fold increase than the
wild type in presence of O2 [18]. Another technique inserts
additional copies of hydrogenase in specific conditions that all
electrons are directed to produce H2 by the genetically fused
additional hydrogenase [111].
Light effects such as differences in pigmentation, antennae
composition in the cell, losses due to reflection, scattering,
radiation outside photosynthetic active radiation region, non-
photochemical reactions and more factors from the outside
environment can affect solar-to-H2 conversion efficiency
[12,17]. Improvement of photosynthesis efficiency is hard to
achieve by conventional methods; using algal bioreactor
operation, overall efficiency can go from 5 to 15% increase;
however, by a molecular approach to engineer microalgae
10e13% efficiency is feasible [73]. Truncated chlorophyll an-
tenna size in the PS of the chloroplast could overcome the
light saturation effect related to fully pigmented cells; mutant
microalgae with less chlorophyll can be manipulated to
enhance large scale commercial applications by allowing
more light into deeper layer inside the bioreactor [82,112].
Horizontal transference of genetic material is a possible
risk; however, improvement of H2 production is mainly
focused on disruption of endogenous genes rather than
introducing new ones [82]. Genetic engineering is a powerful
tool to upgrade microalgae H2 production into a new level,
nonetheless more knowledge must be developed in order to
get more consistent results during large scale assays.
Key parameters for biohydrogen production
Bio-H2 production is a multi-parameter complex operation
that requires many optimization stages in order to reach a
low-cost and sustainable process [5,10,13,47,56,104,111]. Some
parameters to be considered include proper microalgae stain
screening, adequate macro- and micronutrients balance in
growth and H2 producing media, light intensity, photoperiods,
pH, temperature, cell concentration, type and concentration
of external substrates, aeration, agitation and bioreactor
configuration [12,13,35,38,65].
Microalgae species
H2 production depends upon the environmental conditions to
induce the specific metabolic pathways of each microalgae to
produce it [40]. Among microorganisms, most but not all green
microalgae have the genetic code to produce H2, it is diverse
and leans on several taxonomic groups [35,37]; Since the dis-
covery of H2 production by green algae in 1930s until recent
days only around 70 species from more than 30 genera have
been investigated [35,104]. Main green algae genus reviewed
for its H2 production potential include Chlamydomonas sp.
Chlorella sp. Scenedesmus sp., Anabaena sp., Platymonas sp.,
Coelastrella sp., Micractinium sp., Tetraspora sp., and Mono-
raphidium sp. [12,35,46,87,99]. Also, recent studies discovered
H2 production potential of Actinastrum, Closterium, Cosmarium,
Dictyosphaerium, Dimorphococcus, Euastrum, Pandorina, Selenas-
trum, Staurastrum, Stigeoclonium and Ulothrix [35]. C. reinhardtii
is the most common study specimen and serves as a model
organism for Bio-H2 production; engineering tools to
comprehend H2 metabolism have shown great results and
production of high-value products at laboratory scale in
closed systems made this species widely popular [100,102].
However, decisive issues regarding its sensitivity to high solar
irradiation, naturally low oil content, and its difficult growth
in outdoor ponds make this species a weak candidate for
large-scale biofuel production [113]. This is when Chlorella
species could harness this key factor and compete in the H2
economy becoming an ideal biofuel and high-value by-prod-
ucts producer.
Nowadays, production of microalgae is focused in four
genera: Chlorella, Dunaliella, Haematococcus and Tetraselmis;
where Chlorella is the most commonly used microalgae for
human nutrition and animal food [104,114,115]; therefore, it
is the most promising one for Bio-H2, considering that there
are already functioning biomass production facilities in
United States, Germany, Japan and other countries that
could be adapted for this new process [16,114]; and along
with production of other by-products, a biorefinery approach
flexible enough to build a bio-economy is possible
[17,20,116].
Representative Chlorella species evaluated for H2 produc-
tion are C. pyrenoidosa, C. vulgaris var. vulgaris, C. lewinii, C.
sorokiniana, C. fusca, C. homosphaera, Parachlorella kessleri
(Formerly C. kessleri), C. protothecoides, C. salina, and C. vacuo-
lata. It has been proven that those species accumulate large
amounts of endogenous carbohydrates under nutrient limi-
tation, and have remarkable H2 production rate
[12,35,40,46,87,99,104]. Also, production of high commercial
value by-products was confirmed such as carotenoids, vita-
mins, unsaturated fatty acids, glycerol, lectins, mycosporine-
like amino acids, anti-freeze proteins, butylated hydrox-
ytoluene, specific polysaccharides, glycoproteins and gluta-
thiones [104]. Future research should be focused on
developing resistant strains to outdoor conditions, especially
light intensity and temperature shock [100].
Within the up mentioned Chlorella species, C. sorokiniana
has the higher potential for a biorefinery approach where H2
can be generated [114,117]. It has great nutrient uptake and
growth rate [104,118], It produces carotenoids, antioxidants,
 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 4 5 ( 2 0 2 0 ) 8 3 1 0 e8 3 2 8
fatty acids [119], and it is heat and irradiant stress tolerant
[120,121].
When studying H2 production with Chlorella sp., correct
knowledge of taxonomy is advised in order to prevent
misleading data. Since the discovery of genus Chlorella by
Beijerinck in 1890, more than a hundred species have been
classified. Recent discoveries indicate new taxonomy and
incorrect classification in some strain collections. Therefore,
some C. pyrenoidosa strains in collections have been renamed
as Chlorella sp., C. sorokiniana, C. vulgaris, C. protothecoides/A.
protothecoides, and C. vacuolata. In the case of C. vulgaris, strains
in some collections changed to P. kessleri, C. protothecoides/A.
protothecoides, C. saccharophila or C. sorokiniana [115].
Light
Light is one of the key parameters that regulates algae growth,
it promotes starch and ADP synthesis, and in consequence
high concentration of endogenous substrates that increase H2
production rate [13,122].
Effects of light in microalgae for biodiesel production are
well studied, but there is a lack of studies that focus on light
optimization for H2 production in both aerobic and anaerobic
stages of the process [47].
During the light stage of the process, microalgae use light
to support photosynthesis favoring accumulation of starch
and lipids [62,122]. For the second stage or dark process, the
exposure of cells to high light intensities help to establish
anaerobic conditions faster due to intense damage of the PSII
and reversibly inhibition of PSI [75,123]; It assists hydroge-
nases and other enzymes to reach an optimum activity earlier
by increasing sulfur and O2 consumption, also it degrades
stored starch and proteins [13,24]. H2 yield can be increased by
manipulating light energy during the anaerobic phase [32].
Microalgae have a theoretical maximum of 8e10% solar-to-
biomass energy conversion efficiency under low light and low
partial O2 pressures, resulting in a near 22% of light-to-H2
conversion [7,26,37]; However, in real experiments with a
best-case scenario energy conversion for green microalgae are
around 2e3% [37].
Light (L)/dark (D) cycles have an important role in Bio-H2
production as photosynthesis takes place during the light
phase producing biomass, endogenous substrates like starch
and capturing photons in the PSII system to be later consumed
anaerobically during the dark phase when H2 is obtained [104].
Different cycles like 24D, 21L/3D and 18L/6D have been studied
and results vary depending on other factors like culture media
and microalgae strains [86]. 24D experiments showed the best
H2 yield but this process is not sustainable since photosyn-
thesis is not performed in dark conditions, so eventually
biomass and endogenous substrates will decay after some
time taking the culture into a non-renewable point [124].
Considering that 48 photons are needed to fix a CO2
molecule to form glucose [17], at high photon flux densities,
the rate of photon absorption by PS pigments exceeds rate the
rate of use in photosynthesis, resulting in dissipation as
fluorescence or heat [37], this is known as a photoinhibition
effect that damages PSII affecting algae growth and even
causing death [13,42,47]. However, high levels of H2 were ob-
tained when working with a 24L/48D cycle suggesting that
8319
initial high light exposure caused photoinhibition and favor-
ing anaerobic conditions [123]. Kojima & Yamaguchi found
that the rate of H2 release increased hyperbolically with light
intensity [83].
Shading is a known effect, it occurs when in a high density
culture, cells at the surface experience photoinhibition and
prevent cells from the deeper space to absorb enough pho-
tons, decreasing its photosynthetic activity [13,37].
As light distribution is uneven inside the reactor, proper
kinetic data is difficult to acquire [71]. Although, a well-
balanced light distribution inside the reactor cause an
equilibrium of the exposition/shading effects and H2
production can be enhanced [31,73,84], therefore, hydrody-
namics of the photoreactors is a key design parameter
[69,86].
Lakatos et al., successfully used acetic acid and 50 mmol
photon m2 s1 without nutrient deprivation to inhibit PSII
and produce H2 [77]. Kojima & Lin concluded that highest rate
of H2 was achieved when more than 90% of the culture was
kept at illumination below 1.8 klx [84]. Tsygankov et al. used
25 mE m2 s1 (as low light) for stage 1 and
110e120 mE m2 s1 (as high light) in the anaerobic stage to
improve H2 yield [125]. Rashid et al. and later confirmed by
Kaushik & Sharma, stated that wavelength affects H2 yield,
750e950 nm are not suitable for H2 production, red and blue
color LEDs give more H2 than white because green algae and
cyanobacteria has a maximum absorption wavelength of
680 nm, UV is also a good resource of electromagnetic radia-
tion [47,86].
Cell concentration
Cell density has a relevant effect in the culture and H2 pro-
duction since it determines the amount of light that can go
through the cell suspension. Density of cultures depends
whereas it is autotrophic, heterotrophic or mixotrophic
microalgae fermentations and several parameters can affect it
like light penetration, CO2 concentration, and presence or
absence of inorganic or organic compounds [126].
Studies show that high initial concentration increased O2
consumption rate until fully inhibition of PSII, thus it short-
ened the transition between aerobic to anaerobic stage, it
activated hydrogenase activity quicker and H2 production
lasted longer [3,31,39,62]. However, cell density must have a
perfect balance; when cultures have high cell density, tran-
sition to anaerobiosis might occur too fast because of self-
shading, then it will only accumulate small amounts of
starch and combined to reduced light intensity inside the
vessel it can negatively affect H2 evolution rate [14,84]. In the
other hand, when algae concentration is too light, cultures
will not stablish anaerobic conditions because overall cell
respiration is not enough to consume all dissolved O2 [14].
It is important to maintain an active growth phase while
keeping microalgae as preculture; thus, it is recommended to
start a fresh medium with density around 1-2x104 cell
mL1when concentration hits 1.9  107 cell mL1 [14].
Kim et al. conducted a study to determine at which stage of
cell growth (early-exponential phase, mid-exponential phase,
late-exponential phase, and stationary phase) H2 production
would be optimal; they found that cells harvested during late-
8320 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 4 5 ( 2 0 2 0 ) 8 3 1 0 e8 3 2 8
exponential phase having high chlorophyll content produced
more H2 in anaerobic conditions [31]. Kojima & Yamaguchi
demonstrated that at low concentration photoevolution was
high and production time increased until reaching a culture
density of 0.7 kg m3; after that point, both evolution rate and
production time decreased [83]. Batyrova et al., obtained a
maximum H2 volume (20 mL L1) when working with an initial
chlorophyll content of 0.8 mg L1, a concentration above
2 mg L1 did not produce any H2 in S or P-deprived medium
[102]. Similar results were obtained by Hemschmeier et al.
who reported an optimal chlorophyll concentration of
20e25 mg mL1 [14].
Substrates
Chlorella microalgae can produce H2 using both endogenous
and exogenous substrates via heterotrophic metabolism
[5,38,126]. As for endogenous, the most important one is
starch which is formed during the light stage of the process
and is further consumed to provide electrons for the H2 pro-
duction [49]. Once all available starch is consumed, cell must
be regenerated using light and nutrients to reactivate PSII and
start a new cycle of biomass growth [37]; it should be noticed
that cell metabolism is first directed to synthesize growth
products, before accumulating starch [49]. Accumulation can
be induced by N-depletion, S-depletion, high light intensity or
high CO2 concentration; other endogenous substrates such as
lipids can be increased under heterotrophic and mixotrophic
culture conditions by P-depletion, high salt concentration
medium and high iron concentration [127].
Exogenous substrates can increase H2 production, the na-
ture of the carbon sources and their concentration determine
the economic viability of the process [13], some organic
sources are sugars like glucose, fructose, malt extract; organic
acids like acetic acid, malic acid, acetate and even organic
wastes; the yield depends on the type of carbon source, cell
concentration and microalgae species [47].
Initial glucose concentration has an important role in H2
yield, Rashid et al. concluded that optimal concentration is
5 g L1 [13]; on the other hand, Alalayah et al. found that
optimal concentration is 10 g L1 of glucose [38]; addition of
30 mM glucose was suggested by Song et al., which reduced
lag time for H2 evolution by 10 h compared to a process
without any added glucose [49]. Bala Amutha & Murugesan
showed that C. vulgaris MSU 01 strain had a H2 production
peak at 4 g L1 of corn stalk and CO2 bubbling [62], while Oli-
veira et al. claimed to get a 6-fold increase in biomass and lipid
accumulation in cultures grown in 0.01 g L1 acetate [5].
However, all authors confirm that going beyond a critical
concentration of a carbon source could modify the metabolic
pathway and get unwanted side products or H2 yield drops
due to a high demand of energy to oxidize external carbon
sources. Addition of a carbon source cannot regenerate PSII
after a production cycle is completed; therefore, it is manda-
tory to go back to autotrophic photosynthesis to synthetize
vital lipids, proteins and other molecules to maintain cell's life
[13,49].
The use of pure organic substrates has two major draw-
backs: rapid contamination with heterotrophic microorgan-
isms and expensive cost for obtaining the source [80]. Recent
studies explore the possibility of using microalgae to perform
photoheterotrophic degradation of organic acid-rich dark
fermentation effluent, as microalgae feed from organic acids
generated in the culture and bacteria can maintain a low O2
environment [9]. Use of organic wastes is also mentioned
considering availability, cost, carbohydrate content and
biodegradability, this has advantages such as giving added
value to agricultural wastes and reduction of CO2 emissions
[10]. Finally, the use of industrial carbon-high effluents is
mentioned, they are a cheap resource that come from the
dairy industry, olive mill, baker's yeast and brewery waste-
water that could increase H2 production [63,123].
pH
Green microalgae are found in many ecosystems and condi-
tions; they normally grow in a pH range of 4e10 [38,47], and a
small number of species grow at lower limits [104]. Regarding
H2, pH is a significant parameter, even smaller changes in the
medium affect both biomass growth and metabolic pathways
related to Bio-H2 evolution, it controls the activity of the H2
producing enzymes and interferes with the degradation of
endogenous substrates [6,13,42].
During photosynthesis, initial pH decrease thanks to car-
bonic acid generated after reaction of CO2 and water, as the
process goes, pH increases in the medium as algae uptake
dissolved CO2 and by-products are formed [47,63]. At low pH
values H2 is produced by hydrogenase via pyr-
uvateeFdeoxidoreductase pathway, however, acetate and
ATP are formed over H2 due to inhibition of [FeeFe]-hydrog-
enase [47].
Optimum pH varies depending on the microalgae species.
Bala Amutha and Murugesan detected a 14-fold increase in H2
production when pH of the medium changed from 5 to 8, with
a notorious abrupt gradient at pH < 8 [62]. Rashid et, al. pro-
duced more H2 using Chlorella sp. at pH 7.5e8.5 than at 9.0, yet
the drastic change was found at pH 7.0 with only 250 mL L1
obtained [13]. Song et al. explored the influence of external
substrates in pH, they employed MA medium and pH did not
change considerably under 30 mM glucose; however, at higher
concentration, pH dropped, lowering the optimal values. For
40 mM and 50 mM glucose, the final pH was 7.4 and 7.0,
respectively [49].
In general, several studies conclude that around pH 8.0
Chlorella species display the maximum Bio-H2 production rate
[6,13,38], while minimum was observed at pH 6.0 [38]. As
reference, C. reinhardtii optimum pH was 7.7 under S-deprived
conditions [6].
Temperature
Microalgae are found in different environments, for example,
Desmococcus olivaceus, Chlamydomonas sp, C. vulgaris, along
with cyanobacteria have been found in desert crusts [128],
thus the active temperature ranges for biomass growth and H2
production have a direct relationship on the algae species [86].
Thermotolerant green algae are rare [128]; nevertheless, spe-
cies like C. sorokiniana have tolerance up to 42  C [121]. Some
Chlorella species have high tolerance to dry conditions, irra-
diation and variable temperatures [104]. Heat-shock response
Table 1 e Growth and H2 production conditions of several microalgae strains from the Chlorellaceae family.
Growth H2 production Ref
Medium Other substrates/ Light Light:Dark T ( C) pH Medium Other substrates/ Light Light:Dark T ( C) pH Initial Agitation Maximum H2
Conditions intensity cycle (h) Conditions intensity cycle (h) concentration (rpm) production rate or
(mmol (mmol [Cell mL1] accumulation
m2s1) 2 1
m s ) x106
C. lewinii KU201
TAP Only 0.35 mM N4Cl 35 14:10 25 7.3 TAP-P Only 0.35 mM N4Cl 35 24:00 25 7.3 25e35 110 11.50 mL L1 [12]
TAP Only 0.7 mM N4Cl 35 14:10 25 7.3 TAP-S Only 0.7 mM N4Cl 35 24:00 25 7.3 25e35 110 13.03 mL L1 [12]
C. Protothecoides

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 4 5 ( 2 0 2 0 ) 8 3 1 0 e8 3 2 8
TAP 25e35 14:10 25 7.3 TAP-S Only 0.35 mM N4Cl 40e50 25 25e35 5.533 mL L1 h1 [48]
TAP 25e35 14:10 25 7.3 TAP-S Only 0.7 mM N4Cl 40e50 25 25e35 4.004 mL L1 h1 [48]
TAP Only 0.35 mM N4Cl 30e35 14:10 25 7.3 TAP-N 30e35 24:00 25 25e35 59.5 mL L1 [106]
TAP Only 0.35 mM N4Cl 30e35 14:10 25 7.3 TAP-NS 30e35 24:00 25 25e35 82.5 mL L1 [106]
C. Pyrenoidosa
TAP 2.0 g L1 sodium 180 24:00 28 7 TAP-S 180 24 dark, 28 20 mg Chl L1 23.12 mL L1 [80]
acetate then 24:00
TCP 10 mM NaCO3 180 24:00 28 7 TCP DCMU at 9 180 24 dark, 28 20 mg Chl L1 93.86 mL L1 [80]
then 24:00
C. Salina Mt
TAP Potoeterotropically 120 24:00 30 7.2 TAP-S 120 24:00 30 7.2 5 0.5 mL L1 h1 [117]
C. Sorokiniana Ce
TAP Potoeterotropically 120 24:00 30 7.2 TAP-S 120 24:00 30 7.2 5 1.35 mL L1 h1 [117]
C. sorokiniana KU204
TAP Only 0.35 mM N4Cl 35 14:10 25 7.3 TAP-P Only 0.35 mM N4Cl 35 24:00 25 7.3 25e35 110 69.00 mL L1 [12]
TAP Only 0.7 mM N4Cl 35 14:10 25 7.3 TAP-S Only 0.7 mM N4Cl 35 24:00 25 7.3 25e35 110 89.64 mL L1 [12]
C. sorokiniana KU208
TAP Only 0.35 mM N4Cl 35 14:10 25 7.3 TAP-P Only 0.35 mM N4Cl 35 24:00 25 7.3 25e35 110 17.51 mL L1 [12]
TAP Only 0.7 mM N4Cl 35 14:10 25 7.3 TAP-S Only 0.7 mM N4Cl 35 24:00 25 7.3 25e35 110 30.18 mL L1 [12]
C. Vulgaris
BBM 200 mL min1 Air 160 W 24:00 30 6.9 BBM-S 10 g L1 glucose þ 10% 8 2.85 mol mol1 [38]
(0.04% CO2) N. P salts glucose
C. Vulgaris MSU01
Modified 112 24:00 Modified 4 g L1 corn stalk 112 30 7.4 60 1.34 mL L1 h1 [62]
BG-11 BG-11
C. Vulgaris MSU-AGM 14
ACM 15 24:00 ACM-S 20% seaweed extract 15 30 6.7 1.215 OD 2.0  103 g L1 h1 [6]
C. Vulgaris NIER-10003
MA 0.3 vvm 5.0% CO2 120 24:00 25 8.6 MA-S 30 mM glucoseb 120 24:00 40 150 238 mL L1 h1 [49]
MA 100 mL min1 Air 120 24:00 20e22 8.6 MA-S 5.0 g L1 fructoseb 120 20e22 8 150 24 mL L1 h1 [13]
(5.0% CO2)
Chlorella sp. AARL G014
JM Autotropically 30.8 24:00 25 JM-S 54 24:00 25 -a 0.46 mmol mg Chl [35]
a1 h1
TAP Mixotropically 30.8 24:00 25 TAP-S 54 24:00 25 -a 0.49 mmol mg Chl [35]
a1 h1
Chlorella sp. KU209

8321
TAP Only 0.35 mM N4Cl 35 14:10 25 7.3 TAP-P Only 0.35 mM N4Cl 35 24:00 25 7.3 25e35 110 10.27 mL L1 [12]
(continued on next page)
 8322 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 4 5 ( 2 0 2 0 ) 8 3 1 0 e8 3 2 8

in Chlorella is related to cytochrome f in a programed cell death

[117]

[102]

[102]
Ref

[12]

0.75 mL mg Chl1 h1 [48]

1.52  103 mmol mg [46]

0.9  103 mmol mg [46]

process [129].
At normal conditions, generated electrons in PSII flows
(rpm) production rate or
accumulation

through Fd in the Calvin cycle; in contrast, under low light

Maximum H2

intensity (1e3 W m2) or low temperatures (~0  C), Calvin

0.8 mL L1 h1
12.67 mL L1

cycle and the partitioning ratio being controllable to a certain

22 mL L1

38 mL L1

Chl1 h1

Chl1 h1
extent pathways compete for those electrons, reducing H2
production [84]. Similar conclusion was obtained by Mun ~ oz

and Guieysse, showing optimal temperature 25e28 C, below
Agitation

optimal conditions growth rates are reduced whereas high

110

150

100

100 rates are increased by higher temperatures [130].

Mixotrophic or heterotrophic algae growth is also affected
by temperature [63]; for instance, heterotrophic growth of C.
concentration

0.8 mg Chl L1

2.5 mg Chl L1

protothecoides is higher at 25  C [131], while C. sorokiniana on a

[Cell mL1]
Initial

mixture of acetate and butyrate (1:1 ratio) is higher at 30  C

x106

[132]. In Chlorella, temperature increase conduced to fewer lag

25e35

adaption time enhancing H2 evolution rate at 40  C, yet lag

5

time became greater and H2 evolution rate reduced below

Light:Dark T ( C) pH

7.3

7.2

7.2

7.2
H2 production

25  C [49,99].
To illustrate and compare the combined effects of the


25

30

25

25

25

30

30

mentioned parameters for growth and H2 production, an

extensive research was conducted and information was
intensity cycle (h)

organized in Table 1 considering only strains from the Chlor-

24:00

24:00

24:00

24:00

24:00

ellaceae family (Full length table is presented in Annex 1).

m2s1)
(mmol

60e65
Light

Scale-up and industrial application

120
35

45

45

30

30

Laboratory results have been reported but pilot plant data is

1.0 mL L1 Acetic acid
Light:Dark T ( C) pH Medium Other substrates/

hardly found in literature, and no evidence of a functional

Made wit sea water
Only 0.7 mM N4Cl
Conditions

commercial scale plant has been reported due to technical

barriers like light environment, climate and land space,
30 g L1 NaCl

reactor construction materials, the mechanism of culture

mixing, reactor maintenance, and long-term operational sta-
bility with maximal gas production [7,44,60,73]; yet, there have
been attempts to implement industrial-scale processes
combining organic rich wastewater treatment and algal-oil
TAP-N
7.3 TAP-S

7.2 TAP-S

TAP-S
7.2 TAP-P

7.2 TAP-P
7.8 L1-N

extraction [9].
Benemann estimated the cost of H2 at $10 USD GJ1
considering a two-stage process, cultivated in large open
ponds (140 ha), a high carbohydrate content in the biomass


25

30

25

28

28

30

30

and tubular photobioreactors (14 ha). Operating at a capacity

of 90%, the system was expected to yield 1.2 million GJ yr1. At
intensity cycle (h)

14:10

24:00

14:10

24:00

24:00

24:00

24:00

25% annual capital charge, the capital costs charged to almost

90% of total investment where algal ponds cost was estimated
at $6 USD m2 and the photobioreactors $100 USD m2 [133].
Tredici & Zittelli evaluated a near-horizontal tubular
m2s1)
(mmol

25e30
Light

reactor system using single-stage algal biophotolysis; based

120
35

25

25

30

30

on 10% sunlight conversion efficiency and 17% yearly capital

charge, the tubular photobioreactor costs were estimated at
30 g L1 NaCl/Air (2%
Other substrates/

Marine C. Pyrenoidosa IOAC707S

Potoeterotropically

Potoeterotropically

Potoeterotropically

$50 USD m2. Overall H2 production was estimated at $15 USD

Only 0.7 mM N4Cl

1.0 mL L1 Acetic

Conditions

Cells immobilized in agar.

Marine Chlorella sp. LSD-W2

GJ1 [134]
Made with sea
Table 1 e (continued )

Melis & Happe predicted that Bio-H2 would cost about $2.80
Manually twice a day.
(2.0% CO2)
water/Air

USD kg1, considering that all photosynthesis is directed to H2

production, 50% plant capacity and an estimated production
CO2)
acid
Chlorella sp. Pt6

rate of 80 kg H2 acre1 day1; authors affirm that at his price,

H2 could be competitive to gasoline assuming that 1 kg of H2 is
Medium
Growth

equivalent to a gallon of gasoline [135].

L1-N
TAP

TAP

TAP

TAP

TAP

TAP

James et al. had an estimated production cost of $1.38 USD

b
a

kg1 without storage costs using O2-tolerant microalgae. Cost

 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 4 5 ( 2 0 2 0 ) 8 3 1 0 e8 3 2 8
can be summarized as subcomponents like PBRs (42%), H2
collection assembly (34%), microalgae feed assembly (4%),
recycle and pumping systems (13%), and other costs like
control systems, consumables (7%). A direct biophotolysis
with 13.4% conversion efficiency and 9.4% biomass production
will help to reach an affordable production price [136].
Acar & Dincer estimated for direct biophotolysis a H2 cost
of $2.13 USD kg1 and a capital cost of $50 USD m2; whereas
for indirect biophotolysis an estimated H2 cost of $1.42 USD
kg1 and a capital cost of $135 USD m2 [85].
Romagnoli et al. determined that H2 production yield from
C. reinhardtii would be 109.5 tonne year1 or 15549 GJ year1
assuming the higher heat capacity of this element, but they
did not determine if that process is energy positive because
intense energy consumption was detected especially in the
cultivation and biomass processing steps. They do determine
that process should have at least 7% efficiency under outdoor
conditions to be commercially viable and compete to fossil
fuels [137]. Some studies indicate that in order to have a
positive energy balance, a 50% reduction in cost must be done
by acquiring CO2, nutrients and water at lower costs as this
items represents 80e85% of the total cost [9,138]
Bio-H2 production at laboratory scale has improved
rapidly and showed promising results [45,139], but a proper
mix of scientific knowledge with the industrial necessities is
needed in order to properly optimize and scale-up to get
realistic applications [17]. Compared to H2 obtained by
chemical processes, microalgal Bio-H2 has major challenges
to overcome [73], such as: Maintain a monoculture
throughout the cultivation process [114], low yield and en-
ergy conversion efficiency [5], O2 inhibition [39], microalgae
sensitivity to external factors that prevents them to guar-
antee a stable gas generation [60], design of an appropriate
photoreactor system (considering light, mixing, tempera-
ture, monitoring devices, size, trapping and removal of H2,
low capital costs) [17,45,54,60,73,86], flocculation, harvest-
ing techniques [71], gas purification techniques [23],
adequate systems for storage, transportation, delivery
[42,109], lack of a well-defined production technology for
commercialization [100], process management [36], costs,
and reliability [139].
All authors agree that considering the up mentioned
technical, social and political barriers, combined with a final
product price greater than other commercial fuels, H2 pro-
duction using microalgae is still not economically viable
[109], efforts should be focused on designing cheap photo-
bioreactors and the optimization of the photosynthetic
process; however, it deserves attention since it is a prom-
ising energy technology that will cause a positive environ-
mental and economical effect in a nearby future
[138,140,141].
Conclusions
Bio-H2 is a promising energy carrier to be considered in
the sustainable energy market, it can be used as heat
source, rocket fuel, precursor of chemical reactions, in
fuel cells and its combustion only produces water as by-
product, making it a versatile element. H2 is naturally
8323
produced by several microorganisms; among them, green
microalgae highlights for its production efficiency when
metabolism stress is induced. C. reinhardtii is the most
studied microalgae for H2 production; however, it is not
suitable for large-scale applications due to its sensitivity
to environmental factors. In contrast, Chlorella species are
more tolerant to environmental conditions, plus they
generate valuable by-products that could drive the entire
biorefinery process to be economically suitable. C. sor-
okiniana have shown the best results for industrial-scale
applications with optimal parameters around pH 8.0 and
30  C in anaerobic media.
There are many technical and economic barriers that are
holding back a real Bio-H2 industrial-scale application;
nevertheless, advances in genetic engineering, bioreactor
design, and deep understanding of key production parameters
are overcoming current limitations to develop and improve
Bio-H2 production processes that could complement the use
of fossil fuels in a short-term and replace them in some long-
term scenarios.
Acknowledgments
Authors would like to thank every member of the Centro de

n en Biotecnologı́a (CIBIOT) from the
Estudios y de Investigacio
Universidad Pontificia Bolivariana. This work was supported
by the especial cooperation agreement No. FP44842-113-2017
between the university and the colombian science, technol-
ogy and innovation department (COLCIENCIAS).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ijhydene.2020.01.059.
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