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 6 ( 2 0 2 1 ) 1 4 0 9 6 e1 4 1 0 8

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Process simulation and optimization for enhanced

biophotolytic hydrogen production from green
algae using the sulfur deprivation method

Rami Bechara, Fouad Azizi, Cassia Boyadjian*

Baha & Walid Bassatne Department of Chemical Engineering and Advanced Energy, Maroun Semaan Faculty of
Engineering and Architecture, American University of Beirut, Beirut 1107 2020, Lebanon

highlights graphical abstract

First-time process simulation and

optimization for hydrogen pro-
duction from algae.

Sulfur deprivation achieved via
temporal separation of algae
growth/H2 production.

Embedded logistic kinetic model
validated with experimental
results.

Optimization of reaction variables:
reaction time, sulfate concentra-
tion and pH.

Improved hydrogen yield (54%)
and production rate (17%) by opti-
mized variables.

article info abstract

Article history: This article explores the modeling, simulation and optimization of a biophotolytic cyclic
Received 30 October 2020 process for enhanced hydrogen production from microalgae, employing the sulfur depri-
Received in revised form vation method. To achieve sulfur deprivation, each process cycle contained two temporally
29 December 2020 separated steps of sulfur-controlled algae growth and sulfur-deprived anaerobic hydrogen
Accepted 18 January 2021 production.
Available online 15 March 2021 Reaction kinetics were modeled via an empirical logistic model. Reaction times, sulfate
concentrations, and medium pH levels of each cycle were controlled to optimize the rate
Keywords: and yield of hydrogen production. Consequently, 65% and 23% improved values were ob-
Green algae tained, respectively, with a smaller total process time (11%), higher ratio of algae growth-
Hydrogen to-hydrogen production time (29% vs. 21%), buffered pH (7.8), controlled sulfate injection
Sulfur controlled and intermediary algae concentrations. Two- and 15-times higher hydrogen yields were
Simulation obtained for 2- and 12-times lower initial algae concentrations. The proposed method is a
Optimization significant tool for the design and optimization of a process for enhanced hydrogen pro-
Logistic regression duction from microalgae.

* Corresponding author.
E-mail address: cb30@aub.edu.lb (C. Boyadjian).
https://doi.org/10.1016/j.ijhydene.2021.01.115
0360-3199/© 2021 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 6 ( 2 0 2 1 ) 1 4 0 9 6 e1 4 1 0 8 14097

© 2021 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

Nomenclature

extgrowth;i , exwatsplit;i , extmet;i ðkmol =hÞ Extent of algae growth, water splitting, methionine
production in algae growth step i
extdarkferm;i , extH2;prodi extmetH2;i , extresp;H2;i ðkmol=hÞ Extent of dark fermentation, hydrogen and methionine
production, in hydrogen step i
msulfate;in , ðkmol=hÞ Sulfate input rate
nCO2;in , nCO2;out ðkmol=hÞ CO2 input and output rates
nN2;in , nN2;out ðkmol=hÞ Nitrogen input and output rates
Calggrowth;out i ðmg =lÞ Concentration of algae in stream leaving the growth step i
CH2prod;out i CalgH2prod;out i , ðml=lÞ, CsH2prod;out i ðmg =lÞ Concentration of produced hydrogen in the culture
volume, concentration of algae and sulfur in the culture
volume leaving hydrogen step i
CsH2prod;in i ðmg =lÞ Sulfate concentration entering the hydrogen step i
nO2outH2;i ðkmol =hÞ Oxygen output rates leaving the hydrogen step i
inphi () pH level in a given cycle i
Calg;∞ ðmg =lÞ Algae concentration at infinite growth time
cycl; cyclc ð  Þ Actual and critical cycle number
aalg;gr;s ; salg;gr;min ; salg;gr;max , ðmg=lÞ, ah;s ; smin;h ; smax;h ðmg =lÞ Coefficient, minimum and maximum sulfate
concentrations for algae growth and hydrogen production
steps
tc;alg;growth , tc;sloss1 (h), tc;sloss2 ; tc;alg;loss ; tc;hprod1 ðhÞ Critical times for algae growth and sulfate conversion in
algae growth step, sulfate conversion, algae loss and
hydrogen production in hydrogen production step
fpH; aph; bph; apH; ppH() pH related parameters
bgrowth ; bs2 ; balg;loss 1 ; (), balg;loss 2 ; bhalg;c0 ; bhalg;t ð  Þ Logistic exponent factors
sc;sloss 1 ; sc;sloss 2 , (mg/l) Critical sulfur concentrations in growth and hydrogen
steps
facc0 ; fc0;max ; cc;halg ; bhalg;c0 ð  Þ Logistic dependence parameters of hydrogen production
on algae concentration
Vliq ðlÞ Culture volume
VH2 ;rate ðl =hÞ Calculate hourly hydrogen production rate
VH2 ;alg ðl =gÞ Calculate hydrogen yield per input algal mass

carbon dioxide fixing ability, rapid growth rate, advantageous

Introduction
Hydrogen is a promising fuel of great energy potential with the
advantage of zero CO2 emissions during its conversion [1].
Amongst many applications, hydrogen is employed in fuel
cells [2], and in the production of key chemicals such as
ammonia [3], adipic acid for green plastics [4] and iron [5].
Although mainly produced via fossil fuel conversion, multiple
alternative renewable pathways are under development.
These include water splitting; i.e. electrolysis, thermolysis and
photo-electrolysis and both thermal and biological biomass
conversion techniques [6].
Among various biomass sources, microalgae have gained
considerable attention as a promising source for hydrogen [7],
due to their simple cellular system, photoautotrophic nature,
surface to volume ratio, and cultivation convenience [8].
Microalgae conversion to hydrogen occur via thermochemical
and biological processes. The former includes pyrolysis,
liquefaction, gasification, and combustion [9]. Amongst these,
thermal gasification and supercritical water gasification
(SCWG) are reported to be most efficient in terms of micro-
algae conversion rate [3,10]. In this context, SCWG has gained
specific interest as it can handle algae of high moisture con-
tent, contrary to thermal gasification which requires a costly
drying process. Consequently, SCWG has been the subject of
several process simulation and further techno-economic
studies [11,12]. SCWG is believed to have high potential to
become competitive to natural gas reforming in the near
future [11], however it remains an energy intensive process
14098 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 6 ( 2 0 2 1 ) 1 4 0 9 6 e1 4 1 0 8

that is associated with tar formation and necessitates a

Inhibition of hydrogen production

by oxygen and need for high light
hydrogen purification step [6].

Low and impure hydrogen yield

Need for pretreatment of input

Alternatively, hydrogen may be produced from microalgae

High light and high substrate

via biological pathways [13]. Biological pathways occur at

Disadvantage
ambient temperatures and pressures, thus leading to low
energy intensive processes and competitive hydrogen pro-
duction costs. Moreover, they contribute to CO2 fixation and
offset [6]. Based on this, the present work considers the bio-

requirement
logical conversion of microalgae to hydrogen. Some of the

intensity
stream
major biological processes for hydrogen production from mi-
croorganisms are summarized in Table 1 [14]. Among them,
green microalgae, C. reinhardtii, produces hydrogen via direct
photolysis. Compared to other processes, direct photolysis
brings the advantages of high solar conversion of the micro-
algae, pure hydrogen production and CO2 consumption [13].

multiple carbon sources, multiple

Although promising, this process requires further develop-

Pollutant removal, combination

presence of indirect photolysis

High solar conversion, pure H2
production, CO2 consumption,
ment to alleviate hindrances and find optimal operating

No light requirement, use of

conditions prior to industrial implementation.

Advantage

with dark fermentation

Direct photolysis produces hydrogen gas from water via

High hydrogen yield

two reaction pathways (Table 2): (1) water splitting into
hydrogen protons, electrons, and oxygen via the Photosystem
II (PSII) proteins (Equation (2)) and (2) combination of hydrogen

Table 1 e Listing and comparison of different microorganisms based hydrogen production techniques [14].
protons with electrons to form hydrogen (Equation (4)), cata-

products
lyzed by the iron-hydrogenase enzymes found in the C. rein-
hardtii [19]. The major issue of the direct photolysis remains in
the hampered hydrogen production due to the sensitivity of
the iron-hydrogenase enzyme to oxygen produced from
photosynthesis [20]. This necessitates the introduction of
anaerobiosis, i.e. suppressing oxygen presence, hence photo-
synthetic activity. Anaerobiosis is achieved through nutrient
Microorganism

Green microalgae (C.

[21], and most effectively, sulfur deprivation [22]. Melis et al.
Anabaena variabilis
[22] proposed a two-step process for temporal separation of

Cyanobacteria
acetobutylicum

hydrogen and oxygen employing the sulfur deprivation

Clostridium

reinhardtii)
method. In the first step, algal cell growth occurs in a sulfur-
replete media and then in a second subsequent, non-growth
step, hydrogen production is promoted in a sulfur deprived
media. Sulfur depletion leads to partial deactivation of the
oxygen evolving Photosystem II (PSII), hence hinders photo-
synthesis and reduces oxygen production. This in turn boosts
hydrogen production in the system, which however tends to
H2 and O2 in the presence of
substrates into H2 and CO2
smaller organic acids, and

organics conversion to O2,

cease after some time. The latter is reported to refer to

Polymers converted to H2,

Dissociation of water into

morphological changes and partial reduction of algal cells,

Conversion of organic
alcohols with no light
Mechanism

Photosynthesis then

due to both aerobic respiration (Equation (6)) and photo-

fermentation (Equation (5)) reactions which remain active in
under sunlight

light and are unaffected by sulfur deprivation [22,23]. To

H2 and CO2

circumvent this cease in hydrogen production with partial

algal cell reduction, a cyclic production approach with multi-
light

ple algae growth/hydrogen production cycles are employed.

Upon sulfur re-addition, PSII proteins and algal cells are
revived and algal growth and photosynthesis are reinitiated
[23]. This is rather a simplified description of a complex dy-
namic biological system, which is well documented in the
Dark fermentation [15,16]

Photo-fermentation [17]

literature [24,25].
Indirect photolysis [18]

Direct photolysis [18]

Although sulfur-deprivation is promising and would boost

hydrogen production, the efficiency of this two-step process
and the yields of hydrogen production remain far from
Technique

commercialization [26]. Therefore, enhanced hydrogen pro-

duction using the earlier proposed sulfur deprivation process
has been the focus of both experimental and modeling works.
Experimental works of Tamburic et al. [27], Kim et al. [28] and
 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 6 ( 2 0 2 1 ) 1 4 0 9 6 e1 4 1 0 8
Table 2 e Design specifications for algae growth and hydrogen production: manipulated and target variables.
Corresponding reaction/feed
Algae Growth Oxygen Equation 1: Algae growth via photosynthesis
Production 6CO2 þ 6H2 O/C6 H12 O6 þ 6O2
Equation 2: Water splitting (direct photolysis) reaction
2H2 O/4Hþ þ O2 þ 4e
Equation 3: Methionine production reaction
MgSO4 þ 5CO2 þ 5H2 O þ 0:5N2 þ Hþ /Mg þ C5 H11 NO2 S þ 8:5O2
Sulfate feed
CO2 feed
N2 feed
Hydrogen production Equation 4: Hydrogen gas Production
algae depletion 2Hþ þ 2e /H2
Equation 5: Photo-fermentation
C6 H12 O6 þ 6H2 O/6H2 þ 6CO2
Equation 3
Equation 6: Algae cellular respiration
C6 H12 O6 þ 6O2 /6CO2 þ 6H2 O
Lehr et al. [29], for instance, studied the effect of various
process parameters on rates and yields of hydrogen produc-
tion employing the two-step cyclic process with sulfur
deprivation.
The parameters that impact the process performance are
light intensity, carbon source, temperature, pH, algae con-
centration, sulfate concentration, cycle number, and time.
Continuous illumination [30] with an optimal light intensity of
200 mE m2s1 [31] and CO2 bubbling to intermediary CO2
concentrations (5e10%) [32], as well as an ambient tempera-
ture [33] were identified as the most beneficial for optimal
hydrogen production. The use of a pH stabilizing buffer was
also found to be effective [33], with an optimal value in the
range of 7.5e7.7. Moreover, the concentration of algae
entering the hydrogen production step was identified as a key
variable, with hydrogen production yields increasing at low
algae concentrations and plateauing at high algae concen-
trations [34]. The concentration of the injected sulfate is
another important variable; for example, Kim et al. [28] re-
ported that while sulfur concentrations of 15 and 60 mM yiel-
ded improved hydrogen production rates, optimal rates were
obtained at a sulfur concentration of 30 mM. In contrast,
extreme concentrations of 0 and 120 mM led to worse results
[28]. Time is also a significant parameter; shorter time spans
kept constant hydrogen production rates for several operating
days, albeit at small values [35], whereas longer time spans
provided higher hydrogen yields that plummeted after the
fourth cycle [28,29,36,37].
This process has also been the subject of many modeling
works. Two main families of models exist: Mechanistic or ki-
netic expression models and logistic models that exhibit sig-
moid like trends [38]. First, we briefly summarize some of the
mechanistic models reported. Zhang et al. [39] modeled the
photo-autotrophic and photo-mixotrophic algae growth and
studied the impact of each of CO2, light attenuation and cell
decay, ultimately proposing an optimal reactor configuration.
Alalayah et al. [40] modeled hydrogen production by catabo-
lism of injected glucose, via a first order reaction. The most
relevant work is that of Williams et al. [26]. The authors
14099
Manipulated Variable Target Variable
extgrowth;i Calggrowth;outi
exwatsplit;i CH2prod;outi
extmet;i CsH2prod;in i
msulfate;in CsH2prod;in i
nCO2;in nCO2;out
nN2;in nN2;out
extdarkferm;i CalgH2prod;out i
extH2;prodi extH2;prodi ¼ 1
extmetH2;i CsH2prod;out i
extresp;H2;i nO2outH2;i ¼ 0
presented a mechanistic model considering the essential
physiological processes occurring within the algal cells during
sulfur deprivation. The role of sulfur in photosynthesis,
growth, and decay of cell culture and finally hydrogen pro-
duction was explicitly modeled. Moreover, computer-aided
optimization was employed, where sulfur concentrations
were controlled to optimize hydrogen production rates and
yields. The model was a further improvement of similar pre-
vious mechanistic models presented by Park and Moon [41]
and Fouchard et al. [42].
Logistic or sigmoid models were employed thanks to the
sigmoid dependences which both algae growth and hydrogen
production exhibit [43]. For example, Zhang et al. [44] used an
improved logistic growth model for algae microcystis. Amutha
et al. [45] modeled the progress of cumulative hydrogen pro-
duction from the Chlorella vulgaris algae, using batch reactors
by a modified logistic model. Tamburic et al. [46] used a lo-
gistic function to model the growth kinetics of C. reinhardtii
and studied the effect of different light regimes and CO2 feeds
on algae growth and hydrogen production. Logistic models
were considered, under specific conditions of low substrate
concentrations, as a special form of mechanistic Monod ki-
netics [47], making them suitable to describe batch microbial
growth.
Although comprehensive, the previous works do not pre-
sent a global process view and do not take advantage of
computer-based process modeling, simulation, and optimi-
zation to explore the effect of process conditions. Conse-
quently, the current work addresses hydrogen production
from C. reinhardtii microalgae via direct photolysis. This pro-
cess relies on cyclic micro-algal biophotolytic hydrogen pro-
duction employing sulfur deprivation. To the authors’ best
knowledge, this is the first-time this process has been
modeled and simulated. Our approach relies on simulating
the process using ASPEN PLUS® followed by an optimization
of the essential process parameters such as time, sulfur con-
centrations and the pH of the medium. The simulation utilizes
an empirical logistic kinetic model that would allow repli-
cating the pre-existing experimental data and further
14100 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 6 ( 2 0 2 1 ) 1 4 0 9 6 e1 4 1 0 8
optimizing various process parameters to enhance hydrogen
production rates and yields. The significance of the current
work is that it can serve as a tool to help identify the best
operating conditions and optimal designs for enhanced
hydrogen production from the direct biophotolysis of micro-
algae, using the sulfur deprivation method.
Methodology
The process was simulated using Aspen Plus V11, a com-
mercial process simulator which was successfully used to
model several biochemical processes for energy production,
namely bioethanol and bioenergy from sugarcane [48], bio-
diesel from plants [49] and thermochemical conversion of
algae [3]. The applicability of this model to the process at hand
is detailed in the below section.
Process simulation model
The work presented herein considered four cycles of tempo-
rally separated two-step processes. The first step is sulfur rich,
wherein water splitting, oxygen production, and algae growth
occur. The second step is sulfur deprived, wherein anaerobic
hydrogen production and eventually algae depletion occur.
Anaerobiosis is maintained by nitrogen purge of oxygen pro-
duced from photosynthesis [28]. Sulfur rich/sulfur deprived
Fig. 1 e (a) Block flow diagram of one production cycle, (b) Block flow diagram of a sequence of production cycles.
environments are created by controlled injection of sulfate to
the photosynthesis step [28,29,36].
Green microalgae Chlamydomonas reinhardtii was selected
due to its propensity for photolytic hydrogen production and
its common use in the literature. The light intensity was
assumed constant at 200 mE m2s1, with ambient operating
temperature and pressure. Stoichiometric carbon dioxide flow
rates were used, and both carbon dioxide (nCO2;out ) and nitro-
gen (nN2;out ) flow rates leaving the different algae growth steps
were kept constant. Further, water evaporation and mass
transfer limitations were assumed to be negligible.
Fig. 1 (a) shows the block flow diagram of one process cycle.
The algae growth step had four different input streams: algae,
carbon dioxide, sulfate, and nitrogen. This step produced
sulfur-depleted, concentrated algae. Nitrogen purge removed
oxygen, creating an anaerobic environment. Hydrogen pro-
duction occurred in the second step and the resulting partially
reduced algae was used as input for the following cycle.
Fig. 1 (b) depicts the block flow diagram of the four cycles of
the process. Therein, algae stream was injected as input to the
first cycle and passed through the different growth/depletion
steps of all four cycles before exiting the fourth and last cycle.
Sulfate, nitrate and carbon dioxide were added prior to the
start of each cycle to initiate photosynthesis (Equation (2)) and
methionine production (Equation (4)). Pure hydrogen gas was
produced at the end of each cycle, excess CO2 was dissolved in
the output algae stream.
 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 6 ( 2 0 2 1 ) 1 4 0 9 6 e1 4 1 0 8 14101


At first, the simulation components were chosen. Non- production step CSH2prod;in;i mg , the algal concentration
l
biomass components included oxygen (O2), carbon dioxide

(CO2), hydrogen (H2), water (H2O), nitrogen (N2), Hþ (the entering the growth step (Calggrowth;in;i mg
l )), the governing pH
product of water splitting and the source of H2 gas), and (inpH) and the cycle number (ncycle ).
magnesium sulfate (MgSO4) as the input sulfur source.
Output: (1) the sulfur concentration entering the growth
Biomass organic components were glucose (C6H12O6) as the

step (Csgrowth;in ;i mg
l ), (2) the algal concentration leaving the
model of cellular algae, and methionine (C5H11NO2S) the


product of sulfate depletion. The choice of methionine, a key growth step (Calggrowth;out ;i mg l ) which is equal to the algal
amino acid that is a substrate for synthesis of many sulfur
concentration entering the hydrogen production step
containing metabolites [50], was motivated by Irihimovitch

et al. [21]. (CalgH ;in mg l , (3) the concentrations of sulfur
2

The MIXCISLD Stream Class was adopted, with glucose



(CSH2prod;out;i mgl ), and (4) algae (CalgH2prod;out
mg
leaving the
considered as “CISOLID” and all other components as l

“MIXED”. UNIQUAC was the chosen thermodynamic method,
since the process is polar (contains water and ions) and is
conducted at an ambient pressure of 1 bar. Henry's co-
efficients were adopted to account for the solubility of gaseous
components in water.
Second, a stoichiometric reactor was employed for the two
reaction sets (Table 2). This reactor required the definition of
the different reactions and their corresponding extents. The
stoichiometric reactions for the algae growth step were
photosynthesis, water splitting, and sulfate conversion, as
expressed in Equations (1)e(3), respectively. Their related ex-
tents, extgrowth;i , exwatsplit;i and extmet;i , were calculated in the
design specifications to reach desired target values for final
output algae concentration (Calggrowth;outi ), global hydrogen pro-
duction concentration (CH2prod;outi ), and output sulfate con-
centration (CsH2prod;ini ), where i refers to the ith cycle.
The stoichiometric reactions for the hydrogen production
step were hydrogen production, photo-fermentation, sulfate
conversion, and cellular respiration, as expressed in Equa-
tions (3) and (5)e(7), respectively. The first reaction extent
(extH2;prodi ) was equal to unity, leading to total hydrogen ion
conversion. The extents of the other reactions, extphotoferm;i ,
extmetH2;i and extresp;H2;i , were calculated in design specifications
to reach a target output algae concentration (CalgH2prod;outi ), a
target output sulfate concentration (CsH2prod;outi ), and zero oxygen
in the output stream (nO2outH2;i ¼ 0), thereby counterbalancing
the traces of oxygen produced during sulfate consumption.
Additional specified parameters were the sulfate input
rateðmsulfate;in Þ, calculated to obtain a prespecified optimal
sulfate concentration (Csgrowth;ini ), and the carbon dioxide (nCO2;in )
and nitrogen (nN2;in ) input rates, which were set to obtain fixed
output carbon dioxide (nCO2;out ) and nitrogen (nN2;out ) molar
flow rates.
The stoichiometric reactor was combined with a calculator
block and attached to each cycle to kinetically link process
inputs and outputs for a correct calculation of reaction ex-
tents. This calculator made use of a logistic empirical model,
fitted against key literature data, namely Tamburic et al., 2012
[27], Kim et al., 2010 [28] and Lehr et al., 2010 [29]. The various
input/output variables are shown below, and the employed
equations are presented in Table 3:

Input: growth time (tgrowth;i ðhÞ), hydrogen production time
(th2;prod;i ðhÞÞ, the sulfur concentration entering the hydrogen
hydrogen production step, and (5) the hydrogen produced
  
per liquid volume CH2prod;out ml :
l
Based on model equations, the cycle number (ncycle ) was an
input variable only to the output algae growth concentration,
with no direct relation to other variables. All model outputs
had sigmoid dependence on (tgrowth , th2;prod ). All variables had a
linear dependence on pH, except Csgrowth;in and CSH2prod;out which
were pH independent. A sigmoid relationship also existed
between the two sulfur outputs (Csgrowth;in and CSH2prod;out ) and the
sulfur input variable (CSH2prod;in Þ. All other variables had a para-
bolic dependence on CSH2prod;in , indicating the existence of in-
termediate optimal sulfur values. Finally, all variables
pertaining to the hydrogen production step had a sigmoid
dependence on Calggrowth;out .
For the hydrogen separation section, a Flash tank model
was chosen based on Henry's Coefficients, leading to the
production of a pure output hydrogen stream. Other gases,
such as CO2, dissolve into the partially reduced algae stream
and further pass to the next cycle.
In addition, the model evaluated the following output
variables:

For any given cycle i, the total volume of hydrogen pro-
duced (VH2 ;prod i (l)), the total amount of algae input (malg;i
(kg)), the volume of hydrogen produced per algae mass
ðVH2 ;prod : malg Þi ðl =gÞ

The process average hydrogen production rate
(VH2 ;rate ðl=hÞÞ, as defined in Equation (7).
Equation 7: Expression of hydrogen production rate
P
i VH2 ;prodi
VH2 ;rate ðl = hÞ ¼ P
i tgrowthi þ th2;prodi

The ratio of total hydrogen volume produced to mass of
algae entering the 1st cycle (VH2 ;alg ðl=gÞÞ, as in Equation (8).
Equation 8: Expression of hydrogen production rate per
input algae
  P
l VH ;prodi
VH2 ;alg ¼ i 2
g alggrowth;in1
14102 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 6 ( 2 0 2 1 ) 1 4 0 9 6 e1 4 1 0 8

Table 3 e List of model equations to calculate reaction output variables*.

0 1
Calggrowth;out ¼ CalgH
2 ;in
B Calg;s ðsÞ  Calg;∞ C
B C
Calggrowth;in þ BCalg;∞ þ   þ fpH *inphC*gðtÞ
@ cycl bcycl A
1þ
cyclc
Calg;s ¼ aalg;gr;s *ðCSH2prod;in  salg;gr;min Þ*ðCSH2prod;in  salg;gr;max Þ
fpH ¼ aph*cycl þ bph
!bgrowth !
tgrowth
gðtÞ ¼ 1  1 = 1 þ
tc;alg;growth
Csgrowth;in asloss2
asloss1 *Calggrowth;out   bs2
CSH2prod;in
1þ
sc;sloss1
CSH2prod;in þ  bb1 *Calggrowth;out
tgrowth
1þ
tc;sloss1
CSH2prod;out CSH2prod;in  sout;∞
CsH2prod;out ¼ sout;∞ þ  bs2
time
1þ
tc;sloss2
asloss3
ss ¼ !b
Calggrowth;out
1þ
sc;sloss2
CalgH2prod;out Calggrowth;out  f ðs; pHÞ
CalgH2prod;out ¼ f ðs; pHÞ þ !balg;loss1
time
1þ
tc;alg;loss
f ðs; pHÞ ¼ aalg;s ðCSH2prod;in  smin;c ÞðCSH2prod;in  smax;c Þ*ð1 þ apH*INPHÞ
tc;alg;lossmax
tc;alg;loss ¼ !b
Calggrowth;out alg;loss2
1þ
cc;algloss
CH2prod;out CH2prod;out ¼ facc0*fact*facs*facph
!bhalg;c0 !
Calggrowth;out
facc0 ¼ fc0;max  fc0;max = 1 þ
cc;halg
!bhalg;t
time
fact ¼ 1  1=1 þ
tc;hprod1 þ btc;hprod2 *Calggrowth;out
facs ¼ ah;s ðCSH2prod;in  smin;h ÞðCSH2prod;in  smax;h Þ
facph ¼ 1 þ ðapH*C2alggrowth;out þ bpH*Calggrowth;out Þ*inpH
*
The first line for each variable represents the said function whereas the second live gives the dependences of different parameters on model
input variables.

Process optimization these variables, based on literature experience, as well as trial

An optimization block was added to the model with the goal of
identifying optimal operating conditions. The manipulated
variables were the cycle growth time (tgrowth i ðhÞÞ, the hydrogen
production timeðth2;prod i ðhÞÞ, the sulfate concentration
entering the hydrogen production step (CsH2prod;in i ðmg∕lÞÞ and
the pH (inpH). Table 4 details the upper and lower limits of
and error. The input algae concentration to the first cycle was
considered an independent variable and therefore, not
included amongst these variables.
To optimize the global process performance, the chosen
objective function (Equation (9)) was calculated based on the
hydrogen production rate VH2 ;rate (l/h) and the nominal
hydrogen production volume per g algae VH2 ;alg (l/g). Although

Table 4 e Defining lower and upper bounds for optimization variables.

Variable Lower limit Upper limit Variable Lower limit Upper limit
tgrowth 1 ðhÞ 1 100 tgrowth3 ðhÞ 1 100
th2;prod 1 ðhÞ 30 120 th2;prod3 ðhÞ 30 120

mg
mg
sH2prod;in 1 0.5 5 sH2prod;in3 0.5 5
l l
tgrowth 2 ðhÞ 1 100 tgrowth4 ðhÞ 1 100
th2;prod 2 ðhÞ 30 120 th2;prod4 ðhÞ 30 120

mg
mg
sH2prod;in 2 0.5 5 sH2prod;in4 0.5 5
l l
inpH 0 1
 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 6 ( 2 0 2 1 ) 1 4 0 9 6 e1 4 1 0 8
each of these parameters should have different optimal con-
ditions, the objective function attempted to balance this by
using a combined optimal value [26]. As a result, the objective
function, Equation (9), was an average of the normalized
values of VH2 ;rate (l/h) and VH2 ;alg (l/g), each obtained by dividing
the actual value by its maximum.
Equation 9: Definition of objective function
VH2 ;rate VH2 ;alg
Maximize obj ¼ 0:5* þ 0:5*
VH2 ;rate;max VH2 ;algmax
Results and discussion
The model shown in Table 3 was tested and fitted against
literature results [27e29], as indicated in Fig. 2. This figure
provides a parity plot for the different process variables: (a)
Calggrowth;out , (b) Csgrowth;in , (c) CSH2prod;out , (d) CalgH2prod;out , and (e) CH2prod;out .
The obtained R2 values of these variables were 0.9742, 0.9943,
0.975, 0.977, and 0.9562, respectively. These values were
Fig. 2 e Parity plot line experimental vs. model results (a) Calggrowth;out , (b) Csgrowth;in , (c) CSH2prod;out , (d) CalgH2prod;out , and (e) CH2prod;out
14103
considered to be in line with other research works (R2 ¼ 0.95
[27], 0.98 [51], and 0.99 [52]), and thus, deemed acceptable. The
average slope and normalized intercepts were on the other
hand calculated at 95.6% and 4.34%, respectively, with results
approximately 4% away from the identity (y ¼ x) line.
The fitting of the parity plot line was realized by manipu-
lating the values of key desired parameters. The first of these
parameters were the upper sulfur limits; the two variables,
smax;c and smax;h linked to the hydrogen production step had
mg
smaller values 5.43 and 5.01 l , respectively, than
mg
salg;gr;max ¼ 7.02 l , related to the algae growth step. This reit-
erated the need for a lower sulfate concentration in the
hydrogen production step. Second, the time dependence of
the various sub-steps was studied. Algae loss was deemed as
least critical because of its high characteristic time tc;alg;loss ¼
60564h, which rendered algae loss to be slow. Sulfur depletion
had the smallest characteristic times, tc;sloss1 ¼ 45:74 h and
tc;sloss2 ¼ 54:12 h which rendered depletion to be fast. These
values indicate that both slow algae loss and fast sulfate
14104 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 6 ( 2 0 2 1 ) 1 4 0 9 6 e1 4 1 0 8

depletion were beneficial for the process. Ultimately, the two The simulation results are detailed in Table 6, along with
most limiting steps of this two-step process were algae growth the experimental data shown in brackets. Some differences
and hydrogen production, characterized by their high char- existed between calculated and experimental measurements
acteristic times of tc;alg;growth ¼ 65:11 h andtc;hprod1 ¼ 88 h, for the rate of hydrogen production. Simulation results were
respectively. In fact, these values indicate that these pro- higher for the third cycle and lower for the fourth. This was
cesses were both slower than sulfur depletion. also true for algae concentrations, with the greatest difference
The resulting flowsheet was first solved for a Base Case observed for the fourth cycle. This can be related to error
chosen from the literature [28], to test the validity of the accumulation with increasing the cycle number. Nonetheless,
overall simulation model. The values of the variables, deduced the obtained results seemed to grasp the evolution of the
from literature [28] and provided in Table 5, were used as input different variables, namely the decrease in hydrogen pro-
variables for the Aspen Plus® flowsheet. This model consid- duction rates and algae concentrations. Moreover, the average
ered only the algae concentration to the first algae cycle as hydrogen concentration was equal for both cases. This good
input, whereas algae concentrations entering the remaining approximation supports the use of the proposed model. Table
cycles were calculated as outputs of the previous cycle 6 also shows the extents of the two hydrogen production re-
(Calggrowth;in i ¼ CalgH2prod;out i1 ; i > 1Þ. actions, water splitting and photo-fermentation. The results
indicate the predominance of water splitting over photo-
fermentation for hydrogen production. This can be attrib-
uted to the low algae concentrations along with slow
fermentation kinetics [53].
Table 5 e Chosen Values for different input variables [28]. Further, the following were observed: (1) the liquid volume
Variable Cycle 1 Cycle 2 Cycle 3 Cycle 4 decreased negligibly with every cycle, namely due to water

mg splitting, (2) hydrogen production ; VH2 ;prod i (l), decreased from
CsH2prod;ini 2.27 2.09 2.12 2.62
l one cycle to the next, with a drastic drop in the fourth, (3)
tgrowth i ðhÞ 24.50 33.80 32.90 30 algae mass (malg;i ) entering the cycle reached a maximum in
th2;prod i ðhÞ 115.96 107.33 107 109.51
the second cycle and slightly decreased in later cycles,
inpH 1 1 1 1

mg reaching a final value comparable to that of the initial value.
Calggrowth;in 28
i l Consequently, the hydrogen production per algae input
Vliq i ðlÞ 25000 ðVH2 ;prod : malg Þi followed a decreasing pattern, with sharp
drops observed in the second and fourth cycles (30% and 26%
respectively). The drop in the fourth cycle can be attributed to
the decreased hydrogen production rate, whereas that in the
Table 6 e Results of simulation of the Aspen Plus® model. second cycle can be attributed to the related peak in algae
Variable Cycle 1 Cycle 2 Cycle 3 Cycle 4 concentration. Finally, the Base Case values of the two vari-
ables to be optimized, VH2 ;rate and VH2 ;alg , were found to be
CH2 prod;i (ml/l) 244 (244) 223 (234) 186 (212) 122 (84)
(Experimental) 34.5 l/h and 27.272 l/g, respectively.
CalgH2prod;out (mg/l) 37.50 (38.6) 35.30 (36) 29.03 (29) 23.60 (17) Several optimization runs were then performed for three
i

extwatsplit;i (kmol/h) 0.121 0.106 0.087 0.056 different initial algae concentrations, namely, 2.5 mg/l, 15 mg/
H2watsplit;i (kmo/hl) 0.484 0.424 0.348 0.224 l, and 30 mg/l. The optimal values are highlighted in Table 7
extphotoferm;i (kmol/h) 0.0012 0.0011 0.00105 0.00086 along with values of the Base Case (Calggrowth;in 1 ¼ 28 mg=lÞ. It
H2darkferm;i (kmol/h) 0.0288 0.0264 0.0252 0.02064 should be noted that the Base Case values were based on data
Vliq i ðlÞ 24995 24991 24988 from literature [28].
VH2 ;prod i (l) 6301 5804 4901 3314 The three optimized runs exhibited a general increase in
malg;i (kg) 0.750 0.975 0.917 0.795 the total process time, along with a decrease in algae con-
ðVH2 ;prod : malg Þi (l/g) 8.40 5.90 5.30 4.16 centrations. These results illustrate that an increase in pro-
VH2 ;rate ðl∕hÞ 34.50 cess time was required to provide sufficient time for algae
VH2 ;alg ðl∕gÞ 27.272 growth and hydrogen production and to compensate for the

Table 7 e Results of the three optimization runs with 30-15 and 2.5 mg/l input algae concentration vs. the default case.
Case 1 Case 2 Case 3 Base Case Case 1 Case 2 Case 3 Base Case
Calggrowth, in1 (mg/l) 30 15 2.5 28 Calggrowth, in1 (mg/l) 30 15 2.5 28
tgrowth1 (h) 34.10 43.10 45.30 24.50 tgrowth2 (h) 27.7 38.7 45.1 34
th2, prod1 (h) 86.60 79.50 118.10 116 th2, prod2 (h) 94.50 88.50 112.50 107
sH2prod, in1 (mg∕l) 1.40 1.24 1.53 2.20 sH2prod, in2 (mg∕l) 2.36 1.77 1.30 2.10
tgrowth3 (h) 52.90 41.40 54.67 33 tgrowth4 (h) 4.45 5.90 23.98 30
th2, prod3 (h) 69.50 88.50 107.70 107 th2, prod4 (h) 77.90 90.87 114.90 110
sH2prod, in3 (mg/l) 2.17 2.37 2.24 2.10 sH2prod, in4 (mg/l) 2.46 1.70 2.01 2.60
inpH 1 1 1 1
VH2, rate (lh) 53.40 50.90 39.30 32.20 VH2, alg lg 31.90 64.68 390.80 25.88
malg1,2 (kg) 1.26, 1.15 1.2, 1.23 0.98, 0.98 0.98, 0.91 malg3,4 (kg) 1.28, 1.06 1.2, 0.97 1.01, 0.77 0.8, 0.63
 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 6 ( 2 0 2 1 ) 1 4 0 9 6 e1 4 1 0 8
low initial algae concentration. Algae growth times of the
optimized cases were greater than those of the default case
(except for the 4th cycle), while hydrogen production times
were always smaller. For all optimal cases, this resulted in
overall process time splits of 27% and 73% for algae growth
and hydrogen production, respectively, vs. a ratio of 21%e79%
for the default case. These results emphasize the importance
of increasing the algae growth time, which rendered higher
hydrogen production rates. The smaller hydrogen production
times of the optimized cases may also indicate that sulfur
addition occurred before hydrogen production was
completed. The optimized sulfur concentrations were lower
for the optimal cases than the Base Case, which indicate and
confirm the need to operate at low sulfur concentrations
(sulfur deprivation) to enhance hydrogen production rates. In
addition, the later cycles were found to have higher sulfate
concentrations than the earlier ones. This can be attributed to
the fact that a higher sulfur supply was crucial to enhance
algal cell growth and possibly reduce cell death in the later
cycles. This is in line with literature findings [29] where sulfate
concentration was precisely calculated and controlled to yield
a specific algae concentration. For pH, no changes were
observed in the obtained values. A pH value of 1 was
confirmed to be optimal for all three different optimization
runs, in line with [28,37].
As a result, larger values were obtained for the two
parameters,VH2 ;rate and VH2 ;alg , as compared to the Base Case
values [28]. Also, the total hydrogen production volume, ob-
tained by multiplying VH2 ;alg by Calggrowth;in 1 , was equal to 23,927,
24,255 and 24,424 l for the optimized cases (Case 1, 2, and 3,
Fig. 3 e Evolution of volume of hydrogen produced per
culture volume.
Table 8 e Process carbon and hydrogen balance for Base case and Case 1.
Carbon balance Base Case Case 1 Difference
Input CO2 0.221 0.238 8%
ðkmol=hÞ
Output CO2 0.207 0.202 2%
ðkmol=hÞ
Converted to algae 0.004 0.011 375%
ðkmol=hÞ
Converted to methionine 0.015 0.019 27%
ðkmol=hÞ
Error 1.4% 1.3%
14105
respectively) versus 18,115 l for the default cases. Comparable
overall hydrogen production volumes were observed for all
optimized cases. This further indicates the importance of
properly designing the process to maximize hydrogen pro-
duction, regardless of the initial algae concentration. This was
further emphasized when considering the volumetric
hydrogen yield that was calculated to be 2.14, 2.04 and 1.57 ml$
l1 $h1 for the three optimized cases (Case 1, 2 and 3 respec-
tively), while a value of 1.28 ml$l1 $h1 was reported for the
Base Case [28], and 1.52 ml$l1 $h1 and 0.5 ml$l1 $h1 were
obtained in investigations reported by Tamburic [36] and Lehr
et al. [29], respectively. Concerning VH2 ;alg , Case 3 yielded a
higher specific hydrogen production value (390.78 l$g1 )
compared to previous literature works [28,29], which reported
lower hydrogen yields (59.9 and 34.7 l$g1 respectively) for
similar algae concentrations, thereby stressing the signifi-
cance of proper optimal design of the process.
Concomitantly, the final algae mass of the optimal cases
were greater than that of the default case: i.e. 1.07 and 0.97 kg
for 30 (Case 1) and 15 mg/l (Case 2) respective input algae
concentrations, vs. 0.79 kg for the default case. These final
algae mass values were lower than the maximum value
observed at the second or third cycle.
Fig. 3 showcases the evolution of the hydrogen production
volume per culture volume. An overall downward trend is
observed for this ratio with increasing cycle numbers, for
almost all cases. Case 2, however, exhibited a different trend.
Initially this case exhibited a small hydrogen production rate
(mL H2/L culture), even smaller than that of the default case.
This could be attributed to the fact that the ratio of hydrogen
production time-to-algae growth time for the first cycle of
Case 2 is the smallest of all other cases; which itself is a result
of Case 2 optimization.
Moreover, the Aspen Plus® optimized cases exhibited
higher hydrogen production rates (mL H2/L culture) for
ncycle>1 than the default case [28], with a smaller drop in later
cycles than the default case. The default case had a 47% drop
in ratio of hydrogen production-to-culture volume between
the first and fourth cycles, whereas the optimized cases had
only a drop of 22%. This was attributed to the greater algae
growth times, which helped maintain a high algal cell density,
beneficial for algae growth. The obtained results thus confirm
that the proposed methodology was able to overcome one of
the main hurdles for hydrogen production, i.e. the lower
hydrogen rates observed in later cycles.
Finally, because of their comparable initial algae concen-
trations, both the Base Case and Case 1 were selected for
Hydrogen balance Base Case Case 1 Difference
H in Input water 2761 2761 0.0%
ðkmol=hÞ
H in Output water 2759 2758 0%
ðkmol=hÞ
Converted to H2 1.77 2.07 16.9%
ðkmol=hÞ
Converted to algae and methionine 0.03 0.06 100%
ðkmol=hÞ
error 0.00011% 0.00011%
14106 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 6 ( 2 0 2 1 ) 1 4 0 9 6 e1 4 1 0 8
further material balance study. Table 8 provides the material
balance for the carbon and hydrogen elements of these two
cases. The conversion values for algae, modeled as glucose in
the system, and methionine were obtained by subtracting the
final concentrations from the initial concentrations. The
negative value obtained for the algae suggests a correspond-
ing algae depletion in the system. Carbon dioxide constitutes
the sole carbon input in the system, whereas algae and
methionine are the sole carbon outputs. Water constitutes the
sole hydrogen input whereas hydrogen and algae are the main
outputs.
As shown, the optimized case (Case 1) had a higher carbon
dioxide input and a lower output, indicating a greater carbon
and CO2 conversion. Moreover, the percentage of converted
carbon for algae/methionine production switched from 0%/
100% to 37%/63%, indicative of a positive algae growth in the
system. These results are attributed to the greater algae
growth time of Case 1. Similarly, the optimized case showed a
greater conversion of input hydrogen atoms, with 17% greater
hydrogen gas production, along with a doubled hydrogen
conversion to algae, stressing the importance of adequate
algae levels for optimal hydrogen production. In addition, the
optimal case yielded a 157% increased carbon consumption
for a 17% increase in hydrogen production, corresponding to a
120% increase in the ratio of converted carbon to produced
hydrogen.
Finally, the error was calculated according to Equation (10).
The low values indicate that the material balance of both
carbon and hydrogen is maintained within the bounds of the
specified tolerances, highlighting the precision of the
simulator.
Equation 10: Calculation of error for a given variable
input  output  converted
error ¼
input
In conclusion, the optimization work performed using
the developed process model and simulation led to higher
hydrogen production rates and yields than those previously
obtained experimentally. Also importantly, it improved
hydrogen production from the later process cycles which
always remained a hurdle in previous experimental works.
Application and perspectives
The results of the current work propose new operating con-
ditions and process parameters for improved hydrogen pro-
duction from green microalgae using the sulfur deprivation
method. They may be validated and further used in experi-
mental and pilot designs. The proposed methodology may
also allow for the inclusion of multi process variables, such as
temperature, light intensity, new bioreactor designs, or novel
biomass strains, as well as the introduction of organic carbon
sources, namely organic pollutants, for enhanced hydrogen
production. Moreover, this work provides the basis for thor-
ough process sizing and cost estimation, along with thermo-
economic optimization. Finally, this work can be included in
a more global process comparison scheme concerning the use
of algae for hydrogen production.
Conclusion
This article presents the simulation and optimization of an
algae-based phototrophic, cyclic two-step hydrogen produc-
tion process. Aspen Plus® and its built-in design specifications
and calculator blocks were used in this endeavor, with the
adoption of an empirical logistic model for process kinetics.
Input process variables were controlled to optimize an
objective function that averages hydrogen production rates (l
H2/h) and yields (l H2/g algae). The combined simulation-
optimization work yielded breakthrough results with 54%
higher hydrogen production yields, 17% higher hydrogen
production per mass of inputted algae, and a 120% increase in
the ratio of converted carbon to produced hydrogen. Further,
the optimized processes overcame the drop in hydrogen pro-
duction observed with greater cycle numbers, as encountered
in many experimental works, thus providing the basis for
improved hydrogen production. In addition, control of algae
growth and hydrogen production times, as well as sulfur in-
jection, were found to be important for optimized operation.
Finally, this work proves the importance of properly designing
the process in order to maximize the biolytic hydrogen pro-
duction from algae.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
The authors gratefully acknowledge the financial support of
the Baha and Walid Bassatne Endowment Research Fund and
the University Research Board (URB) at the American Univer-
sity of Beirut. The authors would like to indicate that they
have no competing interests to declare.
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