Bioprocess Biosyst Eng (2013) 36:965974
DOI 10.1007/s00449-012-0832-z
ORIGINAL PAPER
Continuous biohydrogen production from fruit wastewater
at low pH conditions
Vasileios Diamantis Abid Khan Spyridon Ntougias Katerina Stamatelatou
Anastasios G. Kapagiannidis Alexander Aivasidis
Received: 5 September 2012 / Accepted: 17 September 2012 / Published online: 2 October 2012
 Springer-Verlag Berlin Heidelberg 2012
Abstract Biohydrogen production from a simulated fruit
wastewater (soluble COD = 3.17 0.10 g L-1) was car-
ried out in a continuous stirred tank reactor (CSTR) of 2 L
operational volume without biomass inoculation, heat pre-
treatment or pH adjustment, resulting in a low operational
pH (3.75 0.09). The hydraulic retention time (HRT)
varied from 15 to 5 h. A strong negative correlation
(p \ 0.01) between the biogas production rate and the HRT
was observed. Biogas production rates were higher at
30 C than at 25 C (p \ 0.01), when the CSTR was
operated under the same HRT. The biogas hydrogen con-
tent was estimated as high as 55.8 2.3 % and
55.4 2.5 % at 25 and 30 C, respectively. The main
fermentation end products were acetic and butyric acids,
followed by ethanol. Significant differences (p \ 0.01)
during the operation of the CSTR at 25 or 30 C were
identified for butyric acid at almost all HRTs examined.
Simulation of the acidogenesis process in the CSTR (based
on COD and carbon balances) indicated the possible met-
abolic compounds produced at 25 and 30 C reactions and
provided an adequate fit of the experimental data.
Keywords Biohydrogen  Fermentative bacteria  Butyric
acid  Fruit wastewater  Acidic fermentation
V. Diamantis (&)  S. Ntougias  K. Stamatelatou 
A. G. Kapagiannidis  A. Aivasidis
Laboratory for Wastewater Management and Treatment
Technologies, Department of Environmental Engineering,
Democritus University of Thrace, Vas. Sofias 12,
67100 Xanthi, Greece
e-mail: bdiamant@env.duth.gr
A. Khan
India Institute of Technology, Roorkey, India
Abbreviations
PEM Proton exchange membrane
VFAs Volatile fatty acids
CSTR Continuous stirred tank reactor
HRT Hydraulic retention time
SRT Solids retention time
ASBR Anaerobic sequencing batch reactor
OLR Organic loading rate
UASB Upflow anaerobic sludge blanket
TSS Total suspended solids
ADM1 Anaerobic digestion model 1
Introduction
Several technologies have been applied for biological
hydrogen (biohydrogen) production, including photo- and
dark-fermentation. Levin et al. [1, 2] compared the biogas
production rates in various biohydrogen systems through
standardization of hydrogen production units and calculation
of systems size that is needed for operation of proton
exchange membrane (PEM) fuel cells accounting for high
volumetric hydrogen production rate, concluding that dark
fermentation systems are the most practical for biohydrogen
production. Biogas removal and separation, reactor design
improvement and/or genetic engineering can enhance sys-
tem operation [3].
Optimization of biohydrogen production rate is essen-
tial, since high hydrogen flow rate is an important param-
eter to power PEM fuel cells. Wang and Wan [4] have
recently reviewed the main factors that influence fermen-
tative hydrogen production, such as inoculum, substrate,
reactor type, temperature and pH. To maximize the
hydrogen production yield, fermentative bacteria must be
directed towards low-molecular volatile fatty acids (VFAs),
123
966
particularly acetic and butyric acids [3]. In a single-stage
anaerobic digestion process for biohydrogen production,
the organic content, after hydrolysis, is fermented by aci-
dogenic bacteria, forming a mixture of VFAs, CO2 and H2.
The type of VFAs produced highly affects biohydrogen
process, e.g. propionic acid accumulation can lead to sys-
tem impairments [5] since it scavenges hydrogen during its
formation.
Large-scale implementations for fermentative hydrogen
production postulate continuous operation processes. From
this point of view, continuous stirred tank reactors (CSTRs)
have been extensively used in several studies on biohydro-
gen production [4]. An alternative configuration, which
offers the ability to decouple the hydraulic retention time
(HRT) from the solids retention time (SRT) is the anaerobic
sequencing batch reactor (ASBR) [6]. Although the semi-
continuous mode of operation of this reactor type seems to
offer a higher degree of process flexibility with regard to
practical applications, the ASBR is generally characterized
by a lower applicable organic loading rate (OLR), if com-
pared to an upflow anaerobic sludge blanket (UASB) reactor
or a CSTR with biomass retention [7]. Although the increase
of biomass retention within the bioreactor (i.e. in ASBR and
UASB reactor) results in high biogas production rate, there
are indications that methanogens can adapt to extremely
adverse conditions given that the SRT is adequately high.
Castello et al. [8] found that methane was produced along
with hydrogen at pH 5 in a UASB reactor treating cheese
whey. This is why CSTR without biomass retention can
ensure methanogens washout at low pH.
Furthermore, biohydrogen production by mixed cultures
potentially requires a pre-treatment step in order to suppress
hydrogen-consuming bacterial activity, thus increasing the
process operational complexity and cost. Among the different
pre-treatment methods reported in hydrogen production pro-
cesses, heat-shock pre-treatment is the most widely used [4].
However, enhanced hydrogen production can be achieved by
the indigenous microbiota present in feedstocks rich in sugars,
such as sweet sorghum extract [9] or cheese whey [10].
She present study aims to optimize biohydrogen pro-
duction from synthetic fruit wastewater in a CSTR, without
biomass inoculation, heat pre-treatment and pH control,
and to examine the effect of hydraulic retention time and
operational temperature on process performance (biogas
production and hydrogen composition).
Materials and methods
Wastewater characteristics
A synthetic carbohydrate-rich wastewater was used to con-
duct this study. The wastewater was prepared daily by
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Bioprocess Biosyst Eng (2013) 36:965974
mixing 0.02 volumes of peach nectar and pulp with 1 volume
of tap water. The medium was also supplemented with
nutrients and trace metals (concentration in mg L-1):
573 NH4Cl, 41.5 (NH4)2SO4, 168.5 K2HPO4, 73.4 CaCl2
2H2O, 83.6 MgCl26H2O, 35.1 (NH4)2Fe(SO4)2, 0.27 Cu
Cl26H2O, 0.415 ZnCl2, 0.36 MnCl26H2O, 0.285 NiCl2
6H2O, 0.08 CoCl26H2O, 0.005 Na2MoO42H2O, 0.155
Na2SeO3, 0.285 H3BO3 and stored at 4 C. The total and
soluble COD of the raw wastewater were 3.42 0.05
and 3.17 0.10 g L-1 (mean SD), respectively, corre-
sponding to the typical organic strength of industrial fruit
canning wastewater [11].
Experimental set-up
The experimental set-up used for biohydrogen production
from synthetic fruit wastewater consisted of a lab-scale,
commercially available CSTR, of 2 L operational volume. The
schematic layout of the bioreactor is illustrated in Fig. 1. The
bioreactor was operated without pH control at pH 3.75 0.09.
Temperature control was accomplished using a thermal bath
(Lauda Thermo-star C12) with water recirculation through the
reactors double jacket. The experiments were conducted at
two operational temperatures, i.e. 25.6 0.6 C and 30.8
0.4 C. The system performance was assessed at different
HRTs for each operational temperature. All hydraulic flows
were controlled using peristaltic pumps.
Analytical methods
Samples were obtained for analysis from the storage tank (raw
wastewater) and the effluent. Alkalinity, total COD and total
suspended solids (TSS) concentrations were determined
according to standard methods [12], after sample homogeni-
zation. Soluble COD was measured after 0.45 lm membrane
Gas flow
meter
Gas analyzer
Fruit Effluent
wastewater
2L
Heat recirculator
Magnetic stirrer
Fig. 1 Schematic layout of the experimental set-up
Bioprocess Biosyst Eng (2013) 36:965974 967

filtration, following APHA procedure [12]. The pH was C6 H12 O6 ! 2 C3 H6 O3 homolactic fermentation 4
determined using a WTW 192 pH-meter (WTW, Germany).
C6 H12 O6 ! C3 H6 O3 CH3 CH2 OH
The VFAs (acetic, propionic, n-butyric, i-butyric or n-valeric
CO2 heterolactic fermentation 5
acid) and ethanol concentrations were determined by capillary
gas chromatography, as previously described by Diamantis C6 H12 O6 ! C3 H6 O3
et al. [13]. Biogas flow rate was determined by a wet gas-meter 3=2 CH3 COOH Bifidobacterium 6
(Ritter Kunstoffwerk KWU B). The carbon dioxide and
C6 H12 O6 ! 3 CH3 COOH 7
methane contents in biogas were assessed using an IR Gas
Analyzer (BINOS, Leybold-Heraues GmbH). C6 H12 O6 ! 2 CH3 CH2 OH
2 CO2 alcoholic fermentation 8
Statistical analysis
C6 H12 O6 ! CH3 CH2 CH2 CH2 OH 2 CO2
H2 O butanol fermentation 9
Standard deviations were calculated for mean values.
Pearson correlation coefficients (r) at p \ 0.01 were .
determined to evaluate relationships between variables. The acidogenic microorganisms were considered as one
Students t test (p \ 0.01) was also performed for com- group and the kinetics for the sugar uptake was based on
parison purposes between the two operational temperatures Monod type as in ADM1:
using Microsoft spreadsheets.
km  S
Specific uptake rate = XI 10
Simulation Ks S
where km is the maximum specific uptake rate constant of
Simulation was carried out by modifying the Anaerobic sugars, KS is the half saturation constant, S is the substrate
Digestion Model 1 (ADM1) [14]. The objective of the (sugar) concentration, X is the acidogenic biomass con-
simulation was to indicate the stoichiometric yields of the centration and I the inhibition factor caused by pH.
metabolic products from the juice wastewater at the spe- The inhibition factor in ADM1 [14] was incorporated
cific temperature levels examined, under varying loading through an empirical function which accounts only for
conditions. Moreover, by simulating the experimental data lower inhibition:
through ADM1, the stoichiometry derived conforms to
 2
balancing the COD and carbon in the system. 3
pHpHUL
pHUL pHLL
Juice-based synthetic water consists of sugars. There- Ie ; pH \pHUL 11
fore, glucose molecule was used as the model compound in I 1; pH [ pHUL
the reactions that yield several metabolic products, such as
volatile fatty acids, lactic acid, alcohols, hydrogen and where pHUL and pHLL are the upper and lower limits of pH
carbon dioxide. Since, only acidogenesis step was active in inhibition, where no inhibition and total inhibition is
the experiments presented here at a pH lower than 4, the observed, respectively. This function is applicable in sys-
steps of acetogenesis and methanogenesis were inactivated tems with low pH, such as systems fed on carbohydrate
in the model by setting the concentrations of the respective feedstocks. pH is calculated through an algebraic equation
microorganisms at zero in the bioreactor. The acidogenesis set for the charge balance [14] containing weak acids and
step in ADM1 particularly involves volatile fatty acid bases, cations and anions. In this case, I was influenced
production through three metabolic pathways: strongly by the volatile fatty acids, lactic acid and bicar-
bonate and varied versus time along with the evolvement of
C6 H12 O6 2 H2 O ! 2 CH3 COOH 2 CO2 4 H2 these compounds. The net effect of I was to lower the
1 specific uptake rate (I \ 1) when pH \ pHUL (pHUL =
5.5, [14]) which was the case throughout all experiments.
C6 H12 O6 2 H2 ! 2 CH3 CH2 COOH 2 H2 O Biomass and substrate mass balances in an ideal CSTR
2 were constructed and algebraic equations were used for the
C6 H12 O6 ! CH3 CH2 CH2 COOH 2 CO2 2 H2 dissociation of the weak acids and the pH calculation as in
ADM1 [14]. All kinetic constants were kept the same as in
3
ADM1 [14], except from the lower pH limit which was set
. to zero (meaning that there was no full inhibition but
The modification of the model relies on inserting more partial, according to Eq. 11), the maximum specific uptake
reactions, since more metabolic products were expected, rate constant of sugars km and the proportions of the sugar
i.e., lactic acid, ethanol and butanol: which was metabolized through each of the reactions

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968 Bioprocess Biosyst Eng (2013) 36:965974

considered (19), namely m1, m2, , m9. The values of these 5.0

parameters were estimated using Aquasim 2.1d [15] at each 4.8

4.6
temperature level.
4.4
4.2

pH
4.0
Results and discussion 3.8
3.6
3.4
Process performance
3.2
3.0
The influence of HRT and operational temperature on 0 20 40 60 80 100 120

biogas production was studied at spontaneous reaction pH Time (d)

3.75 0.09 (Fig. 2). The CSTR operation without pH
control and biomass inoculation can lead to consequent
savings of reagent costs and process simplification [16]. In
addition, methanogenic microbiota was highly suppressed
under this low pH range (Fig. 3c), since the operational pH
was much lower than the optimum pH for growth of the
methanogens [17]. The acidogenic biomass was mainly
Fig. 2 pH evolution during spontaneous anaerobic acidification of
synthetic fruit wastewater at different operational temperatures in a
CSTR. Symbols and lines show the experimental data and the
simulation results, respectively
0.6

derived from the indigenous microbiota present in the fruit A

0.5
supplement. The biomass in the acidification reactor was
0.4
yellowish-white and the TSS concentration was 0.84
HRT (d)
0.3 g L-1. Under these conditions 1020 and 1528 % 0.3

(depending on the operational HRT) of the soluble COD 0.2

was degraded in the CSTR at 25 and 30 C, respectively
0.1
(Fig. 4). This limited COD removal was expected since the
organic compounds of the raw wastewater (mainly com- 0.0
0 20 40 60 80 100 120
posed by sugars) were fermented to volatile fatty acids,
resulting in strong acidification of the mixed liquor. The 5.0

sum of the VFAs and ethanol concentrations ranged within 4.5 B

Biogas production rate

9811,551 and 562820 mg L-1 (average values, 4.0

3.5
depending on the operational HRT) at 25 and 30 C,
(m3 m-3 d-1)

3.0
respectively, corresponding to 9011,359 and 2.5
1,5322,368 mg L-1 COD. Therefore, 2842 and 4874 % 2.0
of the incoming COD was converted to VFAs and ethanol 1.5

at 25 and 30 C, respectively. Based on the stoichiometry 1.0

0.5
of the oxidation reactions of the organic compounds, the
0.0
conversion factors used were 2.087 g COD g-1 ethanol, 0 20 40 60 80 100 120

1.067 g COD g-1 C2, 1.515 g COD g-1 C3 and 1.820 g 70

COD g-1 C4. C
Biogas composition (%)

60
The biogas production rate showed a linear increase
(R = 0.67, p \ 0.05 and R2 = 0.92, p \ 0.01 for 25 and
2 50

30 C, respectively) with organic loading rate (OLR; 40

Fig. 5c). This was due to the fact that the biogas production 30
rate correlated significantly with the HRT. A strong 20
CO2 CH4 H2
negative correlation (Pearson correlation coefficient r =
10
-0.88, p \ 0.01) between the biogas production rate and
the HRT was observed when the CSTR was operated at 0
0 20 40 60 80 100 120
30 C, while a less pronounced negative correlation Time (d)
(r = -0.74), but still statistically significant (p \ 0.01),
was found at 25 C, indicating a better functionality of the Fig. 3 Variation of a HRT, b biogas production rate and c biogas
composition during spontaneous anaerobic acidification of synthetic
acidogenic biomass at 30 C. By decreasing the operational
fruit wastewater at different operational temperatures in a CSTR.
temperature from 30 to 25 C, the biogas production rate Symbols and lines show the experimental data and the simulation
displayed a respective decrease at any OLR examined results, respectively

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Bioprocess Biosyst Eng (2013) 36:965974 969

4.0 fermentation accompanied with high butyric and acetic

3.5
A acid production was not inhibited at HRTs within
3.0 3.08.34 h, corresponding to organic loading rates of
CODsol (g L-1)

2.5 514 kg COD m-3 day-1, a range similar to the respective

2.0 applied in the current study.
1.5 In the absence of an external electron donor, acetic acid,
1.0 butyric acid, lactic acid and alcohols have been reported as
0.5 common carbohydrate fermentation end products [20].
0.0 According to the data presented in Fig. 6, the products
0 2 4 6 8 10 12 14 16
distribution at the same operational temperature was not
OLR (kg m-3 d-1)
significantly influenced by the OLR, for the specific range
100 examined. Apart from the maximum OLR tested (lower
90 B HRT examined), statistically significant lower butyric acid
CODsol removal (%)

80 concentrations (p \ 0.01, in Students t test) were observed

70
60
50
40
30
20
10
0 Clostridia spp. which grow optimally at 3037 C [22].
0 2 4 6 8 10 12
OLR (kg m-3 d-1)
Fig. 4 a Soluble COD (CODsol) and b COD removal (%) as a
function of the organic loading rate. Shaded diamonds 25 C, filled
triangle 30 C
(Fig. 5c). Biogas production rates at 30 C were signifi-
cantly higher than those determined at 25 C (p \ 0.01, in
Students t test) when the CSTR was operated under the
same HRT. The biogas hydrogen content was maintained
equal to 55.8 2.3 % and 55.4 2.5 % at 25 and 30 C,
respectively. No methane production was detected at both
operational temperatures. The acidogenic microbiota
developed at the two operational temperatures examined,
resulted in a fairly constant pH, which indicates that the
hydrogen to carbon dioxide ratio was also constant, since
acidogenesis was the only metabolic procedure occurred.
The concentration and distribution of the main acidic
fermentation products as a function of the OLR and time
are shown in Figs. 6 and 7, respectively. Ren et al. [18]
have reported that no pre-treatment of the inoculum can
lead to mixed-acid type fermentation of glucose at pH 6.
The main products were acetic acid and butyric acid, fol-
lowed by ethanol. In a study performed by Antonopoulou
et al. [10], the indigenous bacteria of cheese whey were
capable of mixed-acid fermentation (acetic, butyric and
lactic were the predominant acids) and limited ethanol
fermentation at pH 5. Similarly, higher acetic acid com-
pared to ethanol concentration and vice versa was observed
at pH 4 and 5, respectively, where sucrose-rich synthetic
wastewater was fermented [7]. In agreement with our
results, Wu et al. [19] reported that acidogenic
at operational temperature of 25 C as compared to 30 C,
which was not the case for acetic acid and ethanol fer-
mentation. Similarly, higher butyric acid production was
determined at 35 C compared to 25 C during anaerobic
digestion of food waste [21]. This observation may be due
to the fact that the main butyric acid-producing bacteria are
14 16
Propionic acid fermentation is preferable at neutral pH
[2325], and, as expected, propionic acid was not detected
in the bioreactor. In a similar manner, restricted propionic
acid production was observed during biohydrogen fer-
mentation of organic residues containing fruit waste, where
acetic and butyric acids were the main VFAs detected [26].
The absence of propionic acid production and the high
conversion of COD to VFAs make the effluent an ideal
source for a stable methane production process [27].
Moreover, no i-butyric acid and n-valeric acid production
was observed.
The main H2-producing microorganisms for butyrate
type fermentation are affiliated with the genera Clostrid-
ium, Butyrivibrio and Bacillus [28], while members of the
genera Bacteroides, Peptostreptococcus, Propionibacte-
rium, Ruminococcus and Veillonella as well as some
Clostridia spp. are present in propionate type fermentation
[28, 29]. In ethanol type fermentation, Ethanoligenens and
Acetanaerobacterium are the main H2-producing bacteria
[30]. Indeed, the vast majority of fermentative microbiota
in the acetogenic reactors belongs to Firmicutes, followed
by and Actinobacteria and members of Bacteroidetes/
Chlorobi group.
The results of the present study and literature data on
biohydrogen production in continuous-flow systems being
operated at low pH (\5) are summarized in Table 1.
A wide range of hydrogen production rates has been
reported in various studies, mainly attributed to the dif-
ferent bioreactor design, microbial community structure
and operational conditions applied. The optimum pH range
for biohydrogen production is considered to be within 5.2
and 6.0 [16]. However, Fang and Liu [25] conducted

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970 Bioprocess Biosyst Eng (2013) 36:965974

5 5
A B
CO2 production rate

H2 production rate*
4 4

(m3 m-3 d-1)

(m3 m-3 d-1)

3 3

2 2

1 1

0 0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
OLR (kg m-3 d-1 ) OLR (kg m-3 d-1)

5
C
Biogas production rate

4
(m3 m-3 d-1)

0
0 2 4 6 8 10 12 14 16
OLR (kg m-3 d-1)

Fig. 5 Volumetric a CO2, b H2 and c total biogas production rates Shaded diamonds 25 C, filled triangle 30 C. Values are presented as
during spontaneous anaerobic acidification of synthetic fruit waste- mean SD. *, estimated
water at different operational temperatures and organic loading rates.

1000 1000
900 A 900 B
Butyric acid (mg L-1 )
Acetic acid (mg L-1)

800 800
700 700
600 600
500 500
400 400
300 300
200 200
100 100
0 0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
OLR (kg m-3 d-1) OLR (kg m-3 d-1)

1000
900 C
800
Ethanol (mg L-1 )

700
600
500
400
300
200
100
0
0 2 4 6 8 10 12 14 16
OLR (kg m-3 d-1)

Fig. 6 Fermentation products during spontaneous anaerobic acidifi- diamonds 25 C. Values are presented as mean SD. a Acetic acid,
cation of synthetic fruit wastewater at different operational temper- b butyric acid and c ethanol
atures and organic loading rates. Filled triangle 30 C, shaded

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Bioprocess Biosyst Eng (2013) 36:965974
Organic compound (g L ) 5 The COD was inserted in the model as the sugar concen-
-1
4
3 The concentration of inorganic nitrogen and inorganic car-
2
1 the model.
0
0 20 40 60 80 100
Time (d)
Fig. 7 COD, ethanol, acetic acid and butyric acid concentrations
during spontaneous anaerobic acidification of synthetic fruit waste-
water at different operational temperatures in a CSTR. Symbols and
lines show the experimental data and the simulation results,
respectively
experiments on hydrogen production from glucose by a
continuous mixed culture at different pH in the range of
4.07.0 and found no significant difference for the specific
hydrogen production rates achieved between pH 4 and 5.5,
while further pH increase drastically lowered H2 produc-
tion. Furthermore, Ren et al. [31] stated that the optimal pH
for hydrogen production can be \5.0, in order to avoid
onset of propionic fermentation. In addition, biohydrogen
production has been reported to be favoured in ethanol-
type fermentation at pH \4.5 [32]. The results in the
present study concerning hydrogen production rate are in
close agreement with those reported in cases of similar
reactor type and operational conditions [25, 32, 33],
reaching up to 2.5 L L-1 day-1 (Table 1). On the other
hand, it should be noted that this is the first study to report
fermentative hydrogen production by a continuous mixed
culture at operational pH \4.
Process simulation
The ADM1 with slight modifications as described in
Simulation was applied to simulate the biohydrogen
production process from synthetic fruit wastewater. The
objective of the simulation was to estimate the breakdown of
the metabolic products during acidogenesis under varying
loading conditions and to indicate quantitatively the pres-
ence of butanol and lactic acid (not measured), so that the
COD and carbon balances (which the ADM1 formulation
was based on) were satisfied. In this way, the breakdown of
the metabolic products through the estimation of the stoi-
chiometric yields indicated the electron and carbon flow
from the initial carbohydrate-rich feedstock to hydrogen as
well as the fermentative products. The substrate fed to the
bioreactor was diluted fruit juice accounting for a COD
concentration as reported in Waste water characteristics.
971
tration (soluble COD) and particulate carbohydrate con-
centration (difference of the soluble from the total COD).
bon (as carbonate alkalinity) and the variations of the COD
loading and hydraulic retention time were also inserted in
The model was used to fit the experimental data of
120 acetic acid, butyric acid, ethanol, pH, biogas production
rate and biogas composition. Preliminary runs of the model
showed that reactions (1), (3), (4), and (79) had the most
significant influence. Therefore, the parameters allowed to
be estimated were the corresponding fractions of the sugars
that were converted through the respective reactions m1, m3,
m4, and m7m9, as well as the maximum specific uptake rate
constant, km, of sugars. That is, seven parameters were
allowed to vary in order to fit six sets of independent
experimental data simultaneously at each temperature
level. Moreover, each set of experimental data reflected the
dynamic response of the bioreactor under varying loading
conditions, rendering the estimation of the parameters
trustworthy over a range of organic loading levels. The
model allowed for temperature effect on the dissociation
constants of water, ammonia and carbonate, which are
more influenced than volatile fatty acids and lactic acid due
to their higher change of enthalpy [38]. The calculation of
the partial pressures of the gases also involves temperature
dependence according to the law of ideal gases.
The values of the fractions of sugars converted to vari-
ous products are depicted in Table 2, as estimated from
fitting the model to the experimental data. The model fitted
the experimental data satisfactorily as seen in Figs. 2, 3 and
7. Acetic acid is produced through reactions (1) and (7) at
30 C, with reaction (7) yielding no hydrogen at all, in
contrast to reaction (1) which yields 2 mol of hydrogen per
mole of acetic acid produced. At 25 C, only reaction (1)
seems to be active, yielding as much hydrogen as possible,
with the acetic acid produced, to compensate for the
hydrogen loss due to lesser butyric acid production. Indeed,
less sugar is converted to butyric acid (through reaction 3)
at 25 C than 30 C. Ethanol production seemed to have
resulted from alcoholic fermentation through reaction (8),
where carbon dioxide but no hydrogen is produced. Alco-
holic fermentation of fruit juices is common due to the
presence of Pichia and Saccharomyces spp. often found in
such feedstocks [3941].
In the present study, the lactic acid was not determined
but would probably be present in appreciable amounts,
since hydrogen and pH balances (as resulted from the
simulation) dictate that another metabolic product (such as
lactic acid) should be present, the production of which
would not be accompanied by hydrogen production
(hydrogen quantities can be predicted through reactions 1
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972 Bioprocess Biosyst Eng (2013) 36:965974

Table 1 Design and performance data of continuous-flow systems for biohydrogen production using mixed cultures at low operational pH
Reference Reactor Reactor Substrate HRT pH T Organic loading Biomass Hydrogen production Biogas
type volume (days) (oC) rate (kg concentration rate hydrogen
(L) COD m-3 day-1) (g VSS L-1) content
(%)

[31] CSTR 9.03 Molasses 0.15 4.5 30 46.1 20 NR 41.5

-1 -1
[25] CSTR 1.7 Glucose 0.25 4 36 30 NR 4 L g VSS day 40
4.5 5.1 L g VSS-1 day-1 55
[34] UASB 3.0 Winery 0.08 4.5 35 408 9.411.7 4.72 L g VSS-1 day-1 54
[35] CSTRa 9.0 Glucose 9.0 4.5 35 1.067 1.5 2840 mL g NR
VSS-1 day-1
[33] CSTR 2.0 Glucose 0.42 4.5 30 12 NR 2.76 L L-1 day-1 61
[36] UASB 0.21 Glucose 0.5 4.5 35 21 NR 93.6 mmol L-1 day-1 37.5
0.25 4.4 42 132 mmol L-1 day-1 28.9
0.08 4.4 126 456 mmol L-1 day-1 29.4
-1 -1
[32] CSTR 3.1 Molasses NR 4.14.4 35 15 NR 4 L L day 50
[37] CSTR 6.0 Glucose 0.17 4.04.5 35 33 4.3 3.64.0 L L-1 day-1 40
[7] ASBR 6.0 Sucrose 2.5 4.0 2830 3.07.8 [11.0 0.060.35 L L-1 day-1 5272
1.25 11.0 1.544.38 L L-1 day-1 NR
0.83 16.6 0.340.54 L L-1 day-1 6070
-1 -1
This CSTR 2.0 Glucose 0.2 3.75 30 15 0.84 2.5 L L day 55
study
NR not reported
a
Semi-continuous

Table 2 Influence of temperature on the utilization of fruit juice

is stimulated by butyric acid [45]. The pH plays a significant
sugars to various fermentative products
role in alcohol fermentation, since at high values, the organic
Ratio of sugar 25 C 30 C acids are mainly produced, while at low pH, butanol pro-
fermentation through:
duction is stimulated [45]. In their work, Tashiro et al. [45]
m1, reaction (1) 0.158 0.057 found that the butanol production was enhanced at pH 5. In
m2, reaction (2) 0 0 this work, the simulation indicates that sugar fermentation to
m3, reaction (3) 0.190 0.494 butyric acid was shifted to butanol, when the temperature was
m4, reaction (4) 0.183 0.149 decreased from 30 to 25 C.
m5, reaction (5) 0 0 The values of km were 58.3 and 60.0 kg COD kg-1
m6, reaction (6) 0 0 COD day-1 at 25 and 30 C, respectively, meaning that the
m7, reaction (7) 0.003 0.069 change in the temperature practically did not affect the
m8, reaction (8) 0.156 0.168 overall microbial growth. The microorganisms were con-
m9, reaction (9) 0.310 0.063 sidered for simulation purposes as one group, consuming
the sugars at a specific rate, according to Eq. (10).
Modeling of acidogenesis phase under non-methanogenic
conditions is not adequately developed in ADM1. Acido-
and 3) and could cause significant pH drop at the experi- genic microorganisms under conditions of low pH tend to
mental levels. Lactic acid bacteria play an important role in swift their metabolism to pathways that yield less acidic
fermented juices [40, 42]. Several lactic acid bacteria, e.g. products (acids with lower pKa) or even cease acid produc-
all Pediococcus, Lactococcus, Enterococcus and Strepto- tion and start producing alcohols, such as ethanol and butanol
coccus spp. as well as many Lactobacillus spp., are strictly [46] as an effort to maintain their intracellular pH. Ther-
homofermentative [43, 44]. Pediococcus spp. have been modynamic limitations can also influence the final distri-
identified in fermented juices [40]. bution of the products, since accumulation of the metabolites
Butanol was not determined either, but its presence would may result in positive Gibbs free energy change stopping or
predict the carbon dioxide observed, as a result of the carbon even reversing the reactions [47]. These factors have been
balance implemented in the simulation. Butanolproduction by taken into account by researchers who have developed
Clostridium species, such as C. saccharoperbutylacetonicum, models aiming to describe the acidogenesis step. As a result,

123
Bioprocess Biosyst Eng (2013) 36:965974
models with variable stoichiometry have been developed
[48, 49] with the potential to predict bioreactor response in
different pH and organic loading conditions. However, in the
current study, where the pH was fairly constant, the appli-
cation of the slightly modified ADM1 with fixed coefficients
resulted in adequate simulation of the distribution of the
metabolic products over a range of organic loading rates at a
low pH level, including the presence of butanol and lactic
acid as fermentative end-products.
Conclusions
In the present study, continuous hydrogen production
through anaerobic acidification of carbohydrate-rich efflu-
ent was evaluated in a CSTR without biomass inoculation,
heat pre-treatment and pH adjustment. The spontaneous
fermentation of the fruit wastewater carbohydrates resulted
in a low reaction pH (3.75 0.09), restricting biomass
growth and, on the other hand, enhancing hydrogen pro-
duction and inhibiting methanogenesis. The biogas pro-
duction rate was between 1 and 5 L L-1 day-1, as
influenced by the OLR, and the hydrogen content was
estimated as high as 55.8 2.3 % and 55.4 2.5 % at 25
and 30 C, respectively. Reactor operation was stable in
relation to biogas production rate, hydrogen content and the
product acids. The mathematical simulation, based on
COD and carbon balances, resulted in the prediction of the
breakdown of carbohydrates to the fermentative products
under both temperature levels and indicated the possible
fermentation reactions performed. This is the first report on
biohydrogen production by a continuous mixed culture at
operational pH \4.
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