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 3 9 ( 2 0 1 4 ) 4 8 6 3 e4 8 6 9

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Sequencing batch reactors (SBRs) for BioH2

production: Reactor operation criteria

Vincenzo Piemonte a, Luisa Di Paola a,*, Sudip Chakraborty c,d, Angelo Basile b
a
Faculty of Engineering, University Campus Bio-Medico of Rome, via Alvaro del Portillo 21, 00128 Rome, Italy
b
CNR-ITM, c/o University of Calabria, Via Pietro Bucci, Cubo 17/C, Rende, 87030 CS, Italy
c
Department of Chemical and Materials Engineering, University of Calabria, Via P.Bucci, Cubo 44a, Rende,
87036 CS, Italy
d
Department of Chemical Engineering, Jadavpur University, Raja S.C Mullick Road, Kolkata, 700032 West Bengal,
India

article info abstract

Article history: The production of ultra-pure hydrogen through biological processes is a promising alter-
Received 11 April 2013 native to conventional processes, such as steam reforming, limited by thermodynamic
Received in revised form constraints: in this framework, Sequential Batch Reactors (SBRs) allow a strict control of
30 December 2013 the reaction environment, so as to guide the metabolic pathway towards the production of
Accepted 10 January 2014 the desired products. In this work we have formulated a model for the dynamics of an SBR
Available online 11 February 2014 applied to biohydrogen production through a hexose source conversion by anaerobic
(Clostridia) fermentation. We aimed at providing a useful tool to control the reactor per-
Keywords: formance in terms of SBRs operating conditions: an incorrect choice of key operating pa-
SBR rameters would lead to an unstable reaction system, which is likely to shift to low-
Dynamic simulations productivity working points.
Biohydrogen production Copyright ª 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
reserved.

biohydrogen) [3,8,12,23e30], with special focus on the

1. Introduction
In the last few years, hydrogen has proved to be an energy
vector with many benefits [1e10]: it is clean and abundant
(making up more than 75% of the Universe mass) and it can
been extracted from both renewable (biological [6e8,10e16])
and non-renewable feedstocks [17e22]. Hence, novel tech-
nologies are under development so as to increase the sus-
tainability of large-scale hydrogen productivity as a direct
energy vector.
Great interest has been shown in hydrogen extraction from
biomasses, mainly agricultural wastes (second generation
biotechnological production of H2, a sustainable and envi-
ronmentally friendly method to gather energy while disposing
of wastes.
Biohydrogen production relies on different raw materials:
cassawa wastewater [31e33], food waste [27,34e37] starch
waste and wastewater [29,30,38e40], wheat powder solutions
[41e43] and industrial wastes [25,36,44,45]. (Fig. 1).
Biohydrogen processing depends upon fermentation mi-
croorganisms [46]; biohydrogen fermentation can be distin-
guished into photo- or in dark-fermentation [11] (Fig. 1).
Anaerobic dark fermentation overcomes many drawbacks
of photofermentation, such as the light supply in

* Corresponding author.
E-mail addresses: v.piemonte@unicampus.it (V. Piemonte), l.dipaola@unicampus.it (L. Di Paola), zsudip.c@gmail.com (S. Chakra-
borty), a.basile@itm.cnr.it (A. Basile).
0360-3199/$ e see front matter Copyright ª 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijhydene.2014.01.075
4864 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 3 9 ( 2 0 1 4 ) 4 8 6 3 e4 8 6 9
Fig. 1 e Biohydrogen production.
photobioreactors [47]. Dark fermentation is carried out by
microorganisms with metabolic pathways that are strictly
(Clostridia [24,34,38,39,48e54]) or not strictly anaerobic (Enter-
obacteria [27] or thermophylic microorganisms).
The commonest microflora in dark fermentation belongs
to the Clostridia phylum: its versatile enzymatic activity is
based on the high versatility of hydrogenase and it exploits
several carbohydrate-containing feedstocks. On the other
hand, the variety of metabolic pathways can lead to a wide
range of end-products apart from biohydrogen; moreover, at
low pH biohydrogen productivity decreases [55]. Eventually,
also the feedstock composition affects the fermentation
products distribution [37], high C/N ratios cutting down the
biohydrogen yield [37] and the unstable metabolism of
biohydrogen-producing bacteria may lead to the substrate
conversion being shifted from H2 to the production of ethanol
and lactate [56]: thus, a careful control of operating conditions
is mandatory to avoid bioreactor instability.
Substrate concentration must lie within a lower and a
higher threshold: the first is required for germination and to
prevent re-sporulation in cultivated spore-forming bacteria,
whereas the second detects the fermentation inhibition con-
ditions. Substrate concentrations out of these limits lead to a
limited H2 yield. Furthermore, product inhibition may arise as
well: for instance, in batch conditions both substrate and
product inhibition takes place at substrate concentrations
between 3.5 and 7.5 g of sucrose COD/L [37].
In this work, we propose an SBR dynamics model for bio-
hydrogen production from a hexose source conversion by
anaerobic Clostridia fermentation. We report the effect on the
reactor efficiency of some operating parameters, such as the
reactor loading and the reaction times, as well as suggesting a
reaction set-up for the optimal biohydrogen conversion.
2. State of art of anaerobic bioH2 production
Many works deal with biohydrogen production in sequential
bioreactors [40,52,57e59], but sustainable hydrogen produc-
tion still remains a challenge, owing to the substrate inhibi-
tion of enzymatic activity shrinking the product (H2) yield; on
the other hand, the fermentation possesses a green character,
allowing waste disposal at mild operating conditions.
Comparative reports on fermentative systems point to
anaerobic sequencing batch reactors (ASBR) as highly reliable
and efficient [28,38,40,48,49,60], providing a high H2 yield and a
good biomass retention [37e39,61]. ASBRs allow toperating
parameters e temperature, controlled pH, organic loading
rate, cycle duration, hydraulic retention time (HRT) e to be
controlled accurately and substrate inhibition kinetics (such
as the glucose inhibition of hydrogenase) to be dealt with.
Sequential Batch Reactors (SBRs) work typically in the field
of wastewater treatment [62] but play a role in other
biotechnological applications involving slowly growing mi-
croorganisms whose growth kinetics is affected by substrate
inhibition [63]. SBRs work at low substrate concentration with
a strict control of other operating conditions [64], such as pH
(in the acidogenic biohydrogen fermentation, for instance, a
pH inhibition occurs at pH < 4 above 26
C).
The conventional configuration of an SBR system consists
of one or more discontinuous series reactors [47]. Compared
with a conventional continuous-flow activated sludge system
[65] the SBR stages (equalization, reaction, settling) develop
over time rather than space. The complete operation
sequence (work cycle) is the system design variable, compa-
rable to the total volume of a conventional continuous-flow
facility. The time span of each SBR stage corresponds to the
volume of the corresponding unit in the continuous-flow
system volume: the SBR work cycle comprises five stages
[6e8,10e12,14,23,26,66,67]: feed, reaction, settling, draw, and
idle.
Recently, a study explored the hydrogen production from
waste using an anaerobic sequencing batch reactor (ASBR)
under thermophilic operation and at constant pH [68]: the
thermophilic operation of acidogenic fermentation for
hydrogen production requires more nutrient supply than for
mesophilic hydrogen production.
3. Theoretical background and mathematical
modelling
Clostridia convert hexose through different reaction steps,
resulting in different fermentation products; when hexose
(glucose) is converted into acetate, the yield for hydrogen is
4 mol H2/mol glucose:
C6 H12 O6 /2CH3 COOH þ 4H2
This reaction path occurs if the hydrogen partial pressure
is low [23]; at higher hydrogen partial pressure, glucose con-
verts into butyrate with a lower H2 yield (2 mol H2/
mol glucose):
C6 H12 O6 /CH3 CH2 CH2 COOH þ þ2CO2 þ 2H2
The complete fermentation route leads to ethanol, with no
hydrogen:
C6 H12 O6 /2C2 H5 OH þ þ2CO2
Thus, the distribution of the fermentation end-products
(acetate, butyrate and ethanol) determines, in different Clos-
tridia cultures and at different operating reaction conditions,
different H2 yields (1.5 mol H2/mol glucose [24,34,44,49]).
 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 3 9 ( 2 0 1 4 ) 4 8 6 3 e4 8 6 9 4865

Here we present a mathematical model for a sequencing where Cin is the inlet substrate concentration, CRPW;n1 the sub-
batch reactor where anaerobic microorganisms convert a strate concentration in the liquid phase at the end of the
hexose substrate. The work cycle includes four phases: feed, (n  1)-th cycle, Fin the inlet flow rate, ke the endogenous
reaction, settling and effluent discharge (see Fig. 2). respiration coefficient, Y the growth yield coefficient, kmax the
During the feed phase, the water stream containing the maximum removal rate in the Haldane equation at CW ¼ CW ,
substrate is loaded into the reactor, while microorganisms VW the liquid volume inside the SBR, Vmax the reactor volume
start to consume the hexose source in the water stream. at the end of the feed phase, VW0 the residual volume, X the
During the reaction phase, microorganisms convert the hex- biomass concentration, X0 the initial value of the biomass
ose source into the fermentation products. At the end of the concentration, Xopt is the residual biomass concentration at
reaction stage, suspension inside the reactor is allowed to the beginning of the generic n-th feed stage. Similarly, the
settle and then the liquid part is discharged; before starting a mass balance equations for the reaction phase (Fin ¼ 0) are:
new cycle, the biomass (microorganisms) concentration is
dCW
adjusted to the initial value. In this work, we focus on the feed ¼ rS (7)
dt
and reaction phases, affected by the biodegradation process.
We have modelled the substrate degradation rate by the
dX
Haldane equation for substrate inhibited kinetics, rearranged ¼ YrS  ke X (8)
dt
as [64]:
cW t ¼ 0 CW ¼ CFP
W X ¼ XFP VW ¼ Vmax (9)
cW
rS ¼ kmax $Xð2 þ bÞ$
2 (1) FP
where CFPW and X are respectively the substrate and biomass
1 þ b$ccW
 þ
cW
cW
W concentration in the liquid phase at the end of the feed phase.
X represents the biomass concentration, the substrate The set of differential Eqs. (2)e(9) was integrated numerically
pffiffiffiffiffiffiffiffiffiffiffiffi
concentration cw ¼ KS $KI corresponds to the maximum by the gPROMS package (Process System Enterprises, London,
removal rate kmax and b ¼ KI/KS accounts for inhibition, being UK); the numerical values of model parameters are reported in
KS the Haldane saturation constant and KI the inhibition Table 1.
constant.
For a perfectly mixed tank reactor (uniform concentra-
tions), the mass balance equations for the feed stage are:
4. Results and discussion
dðCW VW Þ
¼ Fin $Cin  VW rS (2)
dt To obtain a direct estimation for the order of magnitude of
characteristic times of the reactions, Fig. 3 reports the tran-
dðXVW Þ sient tract of the typical “sawtooth” concentration profiles.
¼ YrS VW  ke XVW (3)
dt The two different operating stages of our interest emerge
clearly in the Figure: in the first one (loading phase) the sub-
dVW
¼ Fin (4) strate concentration in the liquid phase increases steeply e
dt
the substrate accumulation rate exceeds the removal rate e
with the initial conditions: whereas during the reaction stage the biomass progressively
degrades the substrate.
t ¼ 0 CW ¼ 0 X ¼ X0 VW ¼ VW0 ðfirst cycleÞ (5)
We applied the mathematical model to determine the ef-
fect of key operating parameters: the reaction time treaction, the
t ¼ 0 CW ¼ CRP X ¼ Xopt VW ¼ VW0 ðnext cyclesÞ (6)
W;n1;
feed flow rate Fin and the inhibition constant b.
Fig. 4 reports the effect of the reaction time (ranging within
1e5.5 h): when it is shorter than 3 h the reactor shifts to a “low
H2 production efficiency” regime, characterized by a high

Table 1 e Numeric values of the model parameters.

Parameter Value Reference
b 1, 2 and 2.57 [44,69]
X0 ¼ Xopt 1000 mgVSS/L
Cin 30e350 mg/L
Cw 19.24 and 24.7 mg/L
Fin 10 L/h
kmax 0.093 mg/mgVSS$h [44]
tload 0.2 h
treaction 0.27e3.45 h
Vmax 4.01 h
Vmin 2.01 h
Y 0.3 [69]
Ke 0.0034 h1 [44]
Fig. 2 e SBR work cycle.
4866 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 3 9 ( 2 0 1 4 ) 4 8 6 3 e4 8 6 9
Fig. 3 e The concentration profile in the transitory stage of
the liquid phase.
substrate outlet concentration (curves C and D); on the other
hand, if the reaction time is higher (treaction ¼ 3.45 h), the
substrate conversion is higher (curve A); at treaction ¼ 3.43 h
(the switching time) there is a clear shift, after some work
cycles, from a high to a low efficiency regime (curve B).
At the low efficiency regime (curve D) it is worth noting the
substrate concentration in the reaction volume CW is only
slightly lower than that of the inlet, pointing to a very low
residual biomass activity.
The low efficiency stationary state, thus, is characterized
by a low degradation rate due to a strong effect of the sub-
strate inhibition on the enzymatic kinetics: cycle by cycle, the
substrate accumulates into the reaction volume, being the
substrate supply larger than the removal rate. This effect is
strongly determined by the inhibition constant b: over a given
threshold value for b, the endogenous metabolism over-
whelms the biomass growth rate, leading to the biomass
death after some cycles; in this case, the outlet and the inlet
substrate concentrations match.
Long reaction times keep the substrate concentration as
low as required to maintain the reactor in a “high efficiency”
Fig. 4 e Dynamic SBR simulation (tload [ 0.2 h) at different
treaction: A) 3.45 h; B) 3.43 h; C) 3 h; D) 1 h.
Fig. 5 e Effect of the endogen metabolism on biomass
growth in strong inhibition conditions (b [ 1).
regime (curve A); otherwise, too long reaction times reduce
the biohydrogen productivity. For this reason, we try and find
a trade off between a high efficiency and a satisfactory pro-
ductivity per day.
The effect of endogen metabolism is clearly depicted in
Fig. 5 at high inhibition condition: the substrate concentration
reaches the inlet value, whereas the biomass concentration
drops to zero, owing to the endogenous metabolism not being
compensated by the substrate conversion.
We specifically tested the effect of the inhibition constant b
on the reactor dynamics (results in Fig. 6). As expected, the
reactor performance declines at increasing the kinetic inhi-
bition (b ranges from 2 to 2.57): a large treaction allows a stable
dynamic condition to be achieved (point A in the Figure)
characterized by a high reactor performance.
For instance, at treaction ¼ 4.02 h at b ¼ 2.0, the system shows
an unstable behaviour characterized by a switch towards a
low efficiency regime (curve B, Fig. 6); remember that b ¼ 2 and
treaction > 3.43 h ensure a stable high efficiency regime (Fig. 4).
Fig. 6 e Dynamic SBR simulation at b [ 1 for different
values of the operating reaction time at treaction
(tload [ 0.2 h). A: treaction [ 4.45 h; B: treaction [ 4.02 h; C:
treaction [ 3.45 h.
 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 3 9 ( 2 0 1 4 ) 4 8 6 3 e4 8 6 9 4867

per working cycle processes a lower amount of substrate

Table 2 e Productivity Optimization Parameters at two
which, at the same time, requires a lower reaction time to
different inhibition constant values.
have a large substrate conversion. These two opposite effects
Fu,in[g/h] Switch Operating cycle H2
lead to an optimum condition that maximizes the bio-
time [h] per day productivity [g/d]
hydrogen productivity: on the one hand, at low substrate
b ¼ 2.57
concentration, the rate of reaction increases at increasing
3.5 3.43 6 60.90
substrate concentration, whereas, over a threshold substrate
1.75 1.45 14 71.05
1.0 0.67 27 78.3 concentration, the reaction rate starts to fall down due the
0.5 0.35 43 62.35 growing inhibition effects provoked by the substrate mole-
0.3 0.27 51 44.37 cules. Thus, a correct analysis of the problem results in an
b ¼ 2.0 efficient reaction system design for the system maximum
3.5 5.42 54 40.6 efficiency. So, we demonstrated that SBRs are capable of
1.75 1.74 12 60.9
providing a proper reaction environment for biohydrogen
1.0 0.81 253 66.7
0.5 0.52 33 47.85
production, given that its design accounts for all the features
0.3 0.39 470 34.8 of the reaction kinetics.

Hence, to reach a high degradation efficiency, a reduction of 5. Conclusions

the reactor operating working cycles per day is necessary,
resulting into a lower biohydrogen productivity in terms of g
of biohydrogen produced per day (with a biohydrogen yield
coefficient, YH2 ¼ 2:9 [44]). Table 2 reports the reactor oper-
ating working cycles per day and the reactor productivity at
different biomass inlet flowrates for two different kinetic in-
hibition constants; a higher kinetic inhibition constant pro-
duces a reduction of the reactor productivity at all inlet
flowrates (see also Fig. 7).
To complete the analysis of the influence of the inlet mass
flow rate (Fu, in) on the system dynamics, we estimated the
switching time by varying the inlet flowrate, the working cy-
cles per day and the biohydrogen productivity (Table 2).
We outlined an optimum set of operating conditions
resulting in the largest biohydrogen productivity per day (see
Fig. 7): at 1 g/h it is observed a maximum of the biohydrogen
productivity (about 78 g/d for b ¼ 2.57) that decreases of 25%
for a flowrate of 3.5 g/h. At b ¼ 2 productivity values are about
15% lower than those at b ¼ 2.57.
Again, a high inlet mass flowrate per working cycle allows
a large amount of substrate to be processed, converted in
biohydrogen, but, a large reaction time results in a high
hydrogen yield. On the other hand, a lower inlet mass flowrate
The enzymatic inhibition effects affect biohydrogen produc-
tion, so the reaction route must be finely controlled in order to
obtain reasonable hydrogen yields. The analysis of the reac-
tion systems, thus, provide a powerful tool to estimate the
course of the biomasses conversion into biohydrogen. In this
work we provided the dynamic modelling of a sequencing
batch reactor (SBR) for biohydrogen production: we analyzed
the influence of key operating parameters, such as the reactor
loading time and the reaction time, on the SBR performance;
for instance, the reaction time strongly influences the pro-
ductivity regime. We derived an optimum set of operating
conditions, which are noticeable for a successful reactor
design. In particular, for an inlet flowrate of 1 g/h and b ¼ 2.57
we found an optimum biohydrogen productivity per day (80 g/
d) that decreases by 25% for a flowrate of 3.5 g/h. At b ¼ 2
productivity values are about 15% lower than those at b ¼ 2.57.
Therefore, an incorrect choice of these operating parameters
would lead to an unstable reaction route, characterized by a
working point corresponding to a very poor reactor efficiency
and biohydrogen productivity. This is an evident case of the
complexity arising from industrial enzyme uses, which
require tight control tools to avoid low efficiency and, even
worse, unstable reaction conditions.

List of symbols

CW Substrate concentration, mg/L

CW substrate concentration at maximum removal rate
kmax, mg/L
Cin inlet substrate concentration, mg/L
CRP
w;n1 substrate concentration at the end of the (n  1)-th
reaction stage, mg/L
CFP
w substrate concentration at the end of the feed stage,
mg/L
Fin inlet flow rate, L/h
ke endogenous respiration coefficient, 1/h
KI inhibition constant (Haldane equation), mg/L
kmax maximum removal rate at CW ¼ CW , 1/h
KS saturation constant in the Haldane equation, mg/L
Fig. 7 e Bio-hydrogen productivity vs. inlet mass flow rate rS substrate conversion rate, mg/hL
at two different inhibition constant values. tload feed stage time, h
4868 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 3 9 ( 2 0 1 4 ) 4 8 6 3 e4 8 6 9
treaction reaction stage time, h
Vw liquid volume, L
Vmax reactor volume at the end of the feed stage, L
X biomass concentration, mg/L
X0 initial biomass concentration, mg/L
XFP biomass concentration at the end of feed stages, mg/
L 2012;37:11556e61.
Xopt biomass concentration at the beginning of feed
stages, mg/L
Y growth yield coefficient, mgbiomass/mgsubstrate
b inhibition parameter (Haldane equation)
adimensional
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