You are on page 1of 10

Bioprocess Biosyst Eng (2013) 36:965974

DOI 10.1007/s00449-012-0832-z


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 Abbreviations

wastewater (soluble COD = 3.17 0.10 g L-1) was car- PEM Proton exchange membrane
ried out in a continuous stirred tank reactor (CSTR) of 2 L VFAs Volatile fatty acids
operational volume without biomass inoculation, heat pre- CSTR Continuous stirred tank reactor
treatment or pH adjustment, resulting in a low operational HRT Hydraulic retention time
pH (3.75 0.09). The hydraulic retention time (HRT) SRT Solids retention time
varied from 15 to 5 h. A strong negative correlation ASBR Anaerobic sequencing batch reactor
(p \ 0.01) between the biogas production rate and the HRT OLR Organic loading rate
was observed. Biogas production rates were higher at UASB Upflow anaerobic sludge blanket
30 C than at 25 C (p \ 0.01), when the CSTR was TSS Total suspended solids
operated under the same HRT. The biogas hydrogen con- ADM1 Anaerobic digestion model 1
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, Introduction
followed by ethanol. Significant differences (p \ 0.01)
during the operation of the CSTR at 25 or 30 C were Several technologies have been applied for biological
identified for butyric acid at almost all HRTs examined. hydrogen (biohydrogen) production, including photo- and
Simulation of the acidogenesis process in the CSTR (based dark-fermentation. Levin et al. [1, 2] compared the biogas
on COD and carbon balances) indicated the possible met- production rates in various biohydrogen systems through
abolic compounds produced at 25 and 30 C reactions and standardization of hydrogen production units and calculation
provided an adequate fit of the experimental data. of systems size that is needed for operation of proton
exchange membrane (PEM) fuel cells accounting for high
Keywords Biohydrogen  Fermentative bacteria  Butyric volumetric hydrogen production rate, concluding that dark
acid  Fruit wastewater  Acidic fermentation 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].
V. Diamantis (&)  S. Ntougias  K. Stamatelatou  Optimization of biohydrogen production rate is essen-
A. G. Kapagiannidis  A. Aivasidis
tial, since high hydrogen flow rate is an important param-
Laboratory for Wastewater Management and Treatment
Technologies, Department of Environmental Engineering, eter to power PEM fuel cells. Wang and Wan [4] have
Democritus University of Thrace, Vas. Sofias 12, recently reviewed the main factors that influence fermen-
67100 Xanthi, Greece tative hydrogen production, such as inoculum, substrate,
reactor type, temperature and pH. To maximize the
A. Khan hydrogen production yield, fermentative bacteria must be
India Institute of Technology, Roorkey, India directed towards low-molecular volatile fatty acids (VFAs),

966 Bioprocess Biosyst Eng (2013) 36:965974

particularly acetic and butyric acids [3]. In a single-stage mixing 0.02 volumes of peach nectar and pulp with 1 volume
anaerobic digestion process for biohydrogen production, of tap water. The medium was also supplemented with
the organic content, after hydrolysis, is fermented by aci- nutrients and trace metals (concentration in mg L-1):
dogenic bacteria, forming a mixture of VFAs, CO2 and H2. 573 NH4Cl, 41.5 (NH4)2SO4, 168.5 K2HPO4, 73.4 CaCl2
The type of VFAs produced highly affects biohydrogen 2H2O, 83.6 MgCl26H2O, 35.1 (NH4)2Fe(SO4)2, 0.27 Cu
process, e.g. propionic acid accumulation can lead to sys- Cl26H2O, 0.415 ZnCl2, 0.36 MnCl26H2O, 0.285 NiCl2
tem impairments [5] since it scavenges hydrogen during its 6H2O, 0.08 CoCl26H2O, 0.005 Na2MoO42H2O, 0.155
formation. Na2SeO3, 0.285 H3BO3 and stored at 4 C. The total and
Large-scale implementations for fermentative hydrogen soluble COD of the raw wastewater were 3.42 0.05
production postulate continuous operation processes. From and 3.17 0.10 g L-1 (mean SD), respectively, corre-
this point of view, continuous stirred tank reactors (CSTRs) sponding to the typical organic strength of industrial fruit
have been extensively used in several studies on biohydro- canning wastewater [11].
gen production [4]. An alternative configuration, which
offers the ability to decouple the hydraulic retention time Experimental set-up
(HRT) from the solids retention time (SRT) is the anaerobic
sequencing batch reactor (ASBR) [6]. Although the semi- The experimental set-up used for biohydrogen production
continuous mode of operation of this reactor type seems to from synthetic fruit wastewater consisted of a lab-scale,
offer a higher degree of process flexibility with regard to commercially available CSTR, of 2 L operational volume. The
practical applications, the ASBR is generally characterized schematic layout of the bioreactor is illustrated in Fig. 1. The
by a lower applicable organic loading rate (OLR), if com- bioreactor was operated without pH control at pH 3.75 0.09.
pared to an upflow anaerobic sludge blanket (UASB) reactor Temperature control was accomplished using a thermal bath
or a CSTR with biomass retention [7]. Although the increase (Lauda Thermo-star C12) with water recirculation through the
of biomass retention within the bioreactor (i.e. in ASBR and reactors double jacket. The experiments were conducted at
UASB reactor) results in high biogas production rate, there two operational temperatures, i.e. 25.6 0.6 C and 30.8
are indications that methanogens can adapt to extremely 0.4 C. The system performance was assessed at different
adverse conditions given that the SRT is adequately high. HRTs for each operational temperature. All hydraulic flows
Castello et al. [8] found that methane was produced along were controlled using peristaltic pumps.
with hydrogen at pH 5 in a UASB reactor treating cheese
whey. This is why CSTR without biomass retention can Analytical methods
ensure methanogens washout at low pH.
Furthermore, biohydrogen production by mixed cultures Samples were obtained for analysis from the storage tank (raw
potentially requires a pre-treatment step in order to suppress wastewater) and the effluent. Alkalinity, total COD and total
hydrogen-consuming bacterial activity, thus increasing the suspended solids (TSS) concentrations were determined
process operational complexity and cost. Among the different according to standard methods [12], after sample homogeni-
pre-treatment methods reported in hydrogen production pro- zation. Soluble COD was measured after 0.45 lm membrane
cesses, heat-shock pre-treatment is the most widely used [4].
However, enhanced hydrogen production can be achieved by Gas flow
the indigenous microbiota present in feedstocks rich in sugars, meter
Gas analyzer
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, Fruit Effluent
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 Heat recirculator

Magnetic stirrer
A synthetic carbohydrate-rich wastewater was used to con-
duct this study. The wastewater was prepared daily by 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
balancing the COD and carbon in the system. 3
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
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

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

temperature level.

Results and discussion 3.8
Process performance
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 Fig. 2 pH evolution during spontaneous anaerobic acidification of
control and biomass inoculation can lead to consequent synthetic fruit wastewater at different operational temperatures in a
savings of reagent costs and process simplification [16]. In CSTR. Symbols and lines show the experimental data and the
addition, methanogenic microbiota was highly suppressed simulation results, respectively
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 0.6

derived from the indigenous microbiota present in the fruit A

supplement. The biomass in the acidification reactor was
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
(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

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

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

of the oxidation reactions of the organic compounds, the
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 (%)

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 =
-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

Bioprocess Biosyst Eng (2013) 36:965974 969

4.0 fermentation accompanied with high butyric and acetic

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

970 Bioprocess Biosyst Eng (2013) 36:965974

5 5
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)

Biogas production rate

(m3 m-3 d-1)

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)

900 C
Ethanol (mg L-1 )

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

Bioprocess Biosyst Eng (2013) 36:965974 971

Organic compound (g L ) 5 The COD was inserted in the model as the sugar concen-

tration (soluble COD) and particulate carbohydrate con-

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

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
NR not reported

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,

Bioprocess Biosyst Eng (2013) 36:965974 973

models with variable stoichiometry have been developed 7. Won SG, Lau AK (2011) Effects of key operational parameters
[48, 49] with the potential to predict bioreactor response in on biohydrogen production via anaerobic fermentation in a
sequencing batch reactor. Bioresour Technol 102:68766883
different pH and organic loading conditions. However, in the 8. Castello E, Garca y Santos C, Iglesias T, Paolino G, Wenzel J,
current study, where the pH was fairly constant, the appli- Borzacconi L, Etchebehere C (2009) Feasibility of biohydrogen
cation of the slightly modified ADM1 with fixed coefficients production from cheese whey using a UASB reactor: links
resulted in adequate simulation of the distribution of the between microbial community and reactor performance. Int J
Hydrogen Energy 34:56745682
metabolic products over a range of organic loading rates at a 9. Antonopoulou G, Gavala HN, Skiadas IV, Angelopoulos K,
low pH level, including the presence of butanol and lactic Lyberatos G (2008) Biofuels generation from sweet sorghum:
acid as fermentative end-products. fermentative hydrogen production and anaerobic digestion of the
remaining biomass. Bioresour Technol 99:110119
10. Antonopoulou G, Stamatelatou K, Venetsaneas N, Kornaros M,
Lyberatos G (2008) Biohydrogen and methane production from
Conclusions cheese whey in a two-stage anaerobic process. Ind Eng Chem Res
In the present study, continuous hydrogen production 11. Diamantis D, Pavlidou E, Aivazidis A (2005) UASB treatment of
fruit canning wastewater: pilot-scale investigations. Environ Eng
through anaerobic acidification of carbohydrate-rich efflu- Manag J 4:339352
ent was evaluated in a CSTR without biomass inoculation, 12. Clesceri LS, Greenberg AE, Eaton AD (1998) Standard methods
heat pre-treatment and pH adjustment. The spontaneous for the examination of water and wastewater. American Public
fermentation of the fruit wastewater carbohydrates resulted Health Association (APHA), Washington, DC
13. Diamantis V, Melidis P, Aivasidis A (2006) Continuous determi-
in a low reaction pH (3.75 0.09), restricting biomass nation of volatile products in anaerobic fermenters by on-line
growth and, on the other hand, enhancing hydrogen pro- capillary gas chromatography. Anal Chim Acta 573574:189194
duction and inhibiting methanogenesis. The biogas pro- 14. Batstone DJ, Keller J, Angelidaki I, Kalyuzhni SV, Pavlostathis
duction rate was between 1 and 5 L L-1 day-1, as SG, Rozzi A, Sanders WTM, Siegrist H, Vavilin VA (2002)
Anaerobic Digestion Model no. 1 (ADM1). IWA Task Group for
influenced by the OLR, and the hydrogen content was Mathematical Modelling of Anaerobic Digestion Process, IWA,
estimated as high as 55.8 2.3 % and 55.4 2.5 % at 25 London
and 30 C, respectively. Reactor operation was stable in 15. Reichert P (1994) AQUASIM: a tool for simulation and data
relation to biogas production rate, hydrogen content and the analysis of aquatic systems. Water Sci Technol 30:2130
16. Show KY, Lee DJ, Chang JS (2011) Bioreactor and process
product acids. The mathematical simulation, based on design for biohydrogen production. Bioresour Technol 102:
COD and carbon balances, resulted in the prediction of the 85248533
breakdown of carbohydrates to the fermentative products 17. Nijaguna BT (2002) Biogas technology. New Age International
under both temperature levels and indicated the possible publishers (P) Ltd, New Delhi
18. Ren NQ, Guo WQ, Wang XJ, Xiang WS, Liu BF, Wang XZ,
fermentation reactions performed. This is the first report on Ding J, Chen ZB (2008) Effects of different pretreatment meth-
biohydrogen production by a continuous mixed culture at ods on fermentation types and dominant bacteria for hydrogen
operational pH \4. production. Int J Hydrogen Energy 33:43184324
19. Wu X, Yang H, Guo L (2010) Effect of operation parameters on
anaerobic fermentation using cow dung as a source of microor-
ganisms. Int J Hydrogen Energy 35:4651
20. Redwood MD, Paterson-Beedle M, MacAskie LE (2009) Inte-
grating dark and light bio-hydrogen production strategies:
References towards the hydrogen economy. Rev Environ Sci Biotechnol
1. Levin DB, Pitt L, Love M (2004) Biohydrogen production: 21. Komemoto K, Lim YG, Nagao N, Onoue Y, Niwa C, Toda T
prospects and limitations to practical applications. Int J Hydrogen (2009) Effect of temperature on VFAs and biogas production in
Energy 29:173185 anaerobic solubilization of food waste. Waste Manag 29:
2. Levin DB (2004) Erratum: Biohydrogen production: prospects 29502955
and limitations to practical applications (Int J Hydrogen Energy 22. Rainey FA, Hollen BJ, Small A (2009) Genus 1. Clostridium.
2004;29:173185). Int J Hydrogen Energy 29:14251426 In: De Vos P et al (eds) Bergeys manual of systematic bacteriol-
3. Sinha P, Pandey A (2011) An evaluative report and challenges for ogy: Firmicutes, vol 3, 2nd edn. Springer, New York, pp 738828
fermentative biohydrogen production. Int J Hydrogen Energy 23. Zoetemeyer RJ, Matthijsen AJCM, van den Heuvel JC, Cohen A,
36:74607478 Boelhouwer C (1982) Anaerobic acidification of glucose in an
4. Wang J, Wan W (2009) Factors influencing fermentative hydrogen upflow reactor. Biomass 2:187199
production: a review. Int J Hydrogen Energy 34:799811 24. Aivasidis A, Wandrey C, Hilla E (1989) Studies on reaction
5. Kyazze G, Martinez-Perez N, Dinsdale R, Premier GC, Hawkes techniques concerning reactor design for the anaerobic degrada-
FR, Guwy AJ, Hawkes DL (2006) Influence of substrate con- tion of complex substrates with the example of the methanation
centration on the stability and yield of continuous biohydrogen of effluents in the fermentation industry. Bioprocess Eng 4:6374
production. Biotechnol Bioeng 93:971979 25. Fang HHP, Liu H (2002) Effect of pH on hydrogen production
6. Arooj MF, Han SK, Kim SH, Kim DH, Shin HS (2008) Effect of from glucose by a mixed culture. Bioresour Technol 82:8793
HRT on ASBR converting starch into biological hydrogen. Int J 26. La Licata B, Sagnelli F, Boulanger A, Lanzini A, Leone P, Zitella
Hydrogen Energy 33:65096514 P, Santarelli M (2011) Bio-hydrogen production from organic

974 Bioprocess Biosyst Eng (2013) 36:965974

wastes in a pilot plant reactor and its use in a SOFC. Int J species from orange fruit and juice by RFLP and sequence
Hydrogen Energy 36:78617865 analysis of the 5.8S rRNA gene and the two internal tran-
27. Diamantis VI, Aivasidis A (2007) Comparison of single- and scribed spacers. FEMS Yeast Res 3:39
two-stage UASB reactors used for anaerobic treatment of syn- 40. Viljoen BC (2006) Yeast ecological interactions. Yeastyeast,
thetic fruit wastewater. Enzyme Microb Technol 42:610 yeastbacteria, yeastfungi interactions and yeasts as biocontrol
28. Moat AG, Foster JW, Spector MP (2002) Fermentation pathways. agents. In: Querol A, Fleet G (eds) Yeasts in food and beverages,
In: Moat AG, Foster JW, Spector MP (eds) Microbial physiology, vol 2. Springer, Heidelberg, pp 83110
4th edn. Wiley, New York, pp 412433 41. Chanprasartsuk OO, Prakitchaiwattana C, Sanguandeekul R,
29. Gerardi MH (2003) Fermentation. In: Gerardi MH (ed) The Fleet GH (2010) Autochthonous yeasts associated with mature
microbiology of anaerobic digesters. Wiley, Hoboken, pp 4350 pineapple fruits, freshly crushed juice and their ferments; and the
30. Ren N, Xing D, Rittmann BE, Zhao L, Xie T, Zhao X (2007) chemical changes during natural fermentation. Bioresour Technol
Microbial community structure of ethanol type fermentation in 101:75007509
bio-hydrogen production. Environ Microbiol 9:11121125 42. Karovicova J, Kohajdova Z (2005) Lactic acid fermentation of
31. Ren NQ, Wang BZ, Huang JC (1997) Ethanol type fermentation various vegetable juices. Acta Aliment 34:237246
from carbohydrate in high rate acidogenic reactor. Biotechnol 43. Stiles ME, Holzapfel WH (1997) Lactic acid bacteria of foods
Bioeng 54:428433 and their current taxonomy. Int J Food Microbiol 36:129
32. Ren NQ, Chua H, Chan SY, Tsang YF, Wang YJ, Sin N (2007) 44. Felis GE, Dellaglio F (2007) Taxonomy of lactobacilli and bifi-
Assessing optimal fermentation type for bio-hydrogen production dobacteria. Curr Iss Intest Microbiol 8:4461
in continuous-flow acidogenic reactors. Bioresour Technol 45. Tashiro T, Takeda K, Kobayashi G, Sonomoto K, Ishizaki A,
98:17741780 Toshino S (2004) High butanol production by Clostridium sac-
33. van Ginkel S, Logan BE (2005) Inhibition of biohydrogen pro- charoperbutylacetonicum N14 in fed-batch culture with pH-stat
duction by undissociated acetic and butyric acids. Environ Sci continuous butyric acid and glucose feeding method. J Biosci
Technol 23:93519356 Bioeng 98:263268
34. Yu H, Zhu Z, Hu W, Zhang H (2002) Hydrogen production from 46. Lowe SE, Jain MK, Zeikus JG (1993) Biology, ecology, and
rice winery wastewater in an upflow anaerobic reactor by using biotechnological applications of anaerobic bacteria adapted to
mixed anaerobic cultures. Int J Hydrogen Energy 27:13591365 environmental stresses in temperature, pH, salinity, or substrates.
35. Kim IS, Hwang MH, Jang NJ, Hyun SH, Lee ST (2004) Effect of Microbiol Rev 57:451509
low pH on the activity of hydrogen utilizing methanogen in bio- 47. Rodriguez J, Juan M, Lema JM, Kleerebezem R (2008) Energy-
hydrogen process. Int J Hydrogen Energy 29:11331140 based models for environmental biotechnology. Trends Biotech-
36. Gavala HN, Skiadas IV, Ahring BK (2006) Biological hydrogen nol 26:366374
production in suspended and attached growth anaerobic reactor 48. Penumathsa BKV, Premier GC, Kyazze G, Dinsdale R, Guwy AJ,
systems. Int J Hydrogen Energy 31:11641175 Esteves S, Rodriguez J (2008) ADM1 can be applied to contin-
37. Song J, An D, Ren N, Zhang Y, Chen Y (2011) Effects of pH and ORP uous bio-hydrogen production using a variable stoichiometry
on microbial ecology and kinetics for hydrogen production in con- approach. Water Res 42:43794385
tinuously dark fermentation. Bioresour Technol 102:1087510880 49. Rodriguez J, Kleerebezem R, Lema JM, van Loosdrecht MCM
38. Everett DH, Wynne-Jones WFK (1941) The thermodynamics of (2006) Modeling product formation in anaerobic mixed culture
acid-base equilibria. Trans Faraday Soc 37:373375 fermentations. Biotechnol Bioeng 93:592606
39. Las Heras-Vazquez FJ, Mingorance-Cazorla L, Clemente-
Jimenez JM, Rodriguez-Vico F (2003) Identification of yeast