Renewable Energy 75 (2015) 496e504

Contents lists available at ScienceDirect

Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Energy recovery from agro-industrial wastewaters through

biohydrogen production: Kinetic evaluation and technological
feasibility
S.D.M. Lucas a, G. Peixoto b, c, *, G. Mockaitis c, d, M. Zaiat c, S.D. Gomes a

State University, 2069, Universita

ria St., Jardim Universita

rio, 5819-110, Cascavel, PR, Brazil
Post-Graduation in Agricultural Engineering, Western Parana
~o Paulo State University (FCFAR/UNESP), Araraquara e Ja Road, km
Department of Bioprocesses and Biotechnology, Faculty of Pharmaceutical Sciences, Sa
~o Paulo, Brazil
1, CEP 14.801-902, Araraquara, Sa
c
~o Carlos School of Engineering,
Laboratory of Biological Processes, Center for Research, Development and Innovation in Environmental Engineering, Sa
~o Paulo, 1100, Joa
~o Dagnone Ave., Santa Angelina, 13563-120, Sa
~o Carlos, Sa
~o Paulo, Brazil
University of Sa
d
National Research Council of Canada (NRC), 6100 Royalmount Avenue, H4P 2R2, Montr

eal, QC, Canada
a

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 14 July 2014
Accepted 9 October 2014
Available online

Biohydrogen production from cassava, dairy and citrus processing wastewaters (WWs) without nutritional supplementation was evaluated in anaerobic single-batch reactors at 37
C for 70 h. Hydrogen
production from cassava, dairy and citrus WW was 31.41, 37.25 and 28.95 mL g1 of chemical oxygen
demand (COD). The kinetic parameters indicated that H2 production rates for cassava processing WW
(0.32 mL h1) and dairy WW (0.31 mL h1) were similar, whereas citrus processing WW exhibited the
App
highest value (0.59 mL h1). The carbohydrate degradation rate k1 was highest for dairy WW
1
(0.045 h ), but the most efcient overall conversion of organic matter to H2 kApp
2 was observed with
cassava WW (0.014 h1). The rate of conversion of the organic matter of the cassava WW together with
its ready availability resulted in a recovery of 0.59 109 MJ year1, the highest in this study. Cassava WW
showed the highest hydrogen production potential (97.9 mL), when compared with dairy (76.1 mL) and
citrus WW (66.6 mL). The economic estimation based on the gasoline energetic equivalent indicated that
a single process of H2 production allowed the maximum economic yield of US$ 0.009 cents L1 WW.
Alternatively, a sequential hydrogen and methane process could increase energy recoveries and economic yields to values near 10.48 kJ g1 COD and US$ 0.61 cents L1 WW (US$ 6.10 m3 WW).
2014 Elsevier Ltd. All rights reserved.

Keywords:
Bioenergy
Hydrogen
Agro-industrial wastes
Kinetics
Energy recovery
Economic yield

1. Introduction
Currently, worldwide energy demands are primarily supplied by
fossil fuels, which are directly linked to greenhouse gas emissions.
As main consequence, the search for renewable energy sources has
become critical. Hydrogen gas is an interesting energetic alternative because its utilization does not involve the emission of
greenhouse gases, and organic wastes can be used as feedstock in
its production. Among the methods employed for H2 production,
dark fermentation using organic wastes as raw materials could be a

* Corresponding author. Department of Bioprocesses and Biotechnology, Faculty

of Pharmaceutical Sciences, S~
ao Paulo State University (FCFAR/UNESP), Araraquara
~o Paulo, Brazil. Tel.:55 16 3301
e Ja Road, km 1, CEP 14.801-902, Araraquara, Sa
4645; fax: 55 16 3301 4669.
E-mail addresses: shaianelucas@gmail.com (S.D.M. Lucas), peixotog@fcfar.unesp.
br (G. Peixoto), gusmock@usp.br (G. Mockaitis), zaiat@sc.usp.br (M. Zaiat), simone.
gomes@unioeste.br (S.D. Gomes).
http://dx.doi.org/10.1016/j.renene.2014.10.025
0960-1481/ 2014 Elsevier Ltd. All rights reserved.

strategy to overcome some economic issues involved in the production process [1].
Some wastes could show some nutritional deciencies to be
used directly as raw materials in a hydrogen production process [2]
and [3]. Thus, various important ratios of nitrogen and phosphorous have been reported in the literature to compensate the
possible lack of such nutrients. The most common ratios for H2
production are C:N 47:1 [4], C:N:P 100:0.5:0.1 [5], C/
N 100:2.2 [6] and C:N:P 100:0.8:0.3 [7]. Hawkes et al. [8] stated
that the COD:N ratios in H2 production studies varied between 11:1
and 200:1 and found that the COD:P ratios presented an even
greater variation (73:1 to 970:1). Micronutrients and trace metals
are essential for cell maintenance and are required for metabolism
and growth, transport processes, enzyme cofactors and dehydrogenases. These requirements were inferred from the
elemental composition of anaerobic bacteria, which includes Cu,
Zn, Mo, Ni and V [9]. Iron has been reported as essential for H2
production [10] because is the element that forms ferredoxin and

S.D.M. Lucas et al. / Renewable Energy 75 (2015) 496e504

Abbreviations
COD
TS
VSS
FS
WW
C
N
P
Ca
Mg
Mo
Na
K
Ni
Cu
Fe
Zn

chemical oxygen demand (oxidizable organic matter),

in milligrams per liter
total solids, in milligrams per liter
volatile suspended solids, in milligrams per liter
xed solids, in milligrams per liter
wastewater
carbon
nitrogen
phosphorus
calcium
magnesium
molybdenum
sodium
potassium
nickel
copper
iron
zinc

hydrogenase enzymes. Other nutrient requirements include concentrations of Ca (100e200 mg L1), Mg (75e150 mg L1), Na
(100e200 mg L1), Fe (20e100 mg L1) and K (200e400 mg L1)
[11] and [12]. Some studies have employed raw wastes as substrates
for H2 production [7,13,14]. In most cases, the carbon sources were
supplemented with complex nutritional additives [5,15e19] to
improve process hydrogen yielding, thereby decreasing its economic feasibility.
Food processing WW could be interesting raw materials
sources for dark fermentation targeting hydrogen production
because of their high carbohydrate and nutrients content [20].
These WW could require less or none nutrient supplementation
to achieve a feasible hydrogen production process. Cassava starch
processing WW could be an ideal raw material for the dark
fermentation process, due its rich composition of carbohydrates,
nutrients (N, P) and minerals (Fe, Zn, Mg) [5] and [21]. High
content of easily degradable carbohydrates and soluble organic
substances (5e50 g L1 COD) in dairy WW can support bacterial
growth [22] and [23] for hydrogen production. Citrus WW, obtained from orange peels processed for pectin production,
has 17% of soluble sugars [24], being a rich source of
carbohydrates.
In Brazil, the current production of cassava starch WW is
184,100,000 cubic meters per year based on data provided by FAO
(Food and Agriculture Organization of the United Nations) (2008)
[25] and estimates that cassava processing generates 7 cubic meters
per ton of product [26]. Dairy WW generation is typically 2.5 times
the volume of milk processed [27]. Data from FAO (2008) [25]
indicate that 31,200,000 tons of milk were processed in 2008,
resulting in 78,000,000 cubic meters of WW. In addition, in 2013,
82,072.94 cubic meters of citrus processing WW was generated
[24] according to IBGE (Brazilian Institute for Geography and Statistics) reports [28].
Given the availability of the aforementioned WWs and the
growing need for clean energy sources, this paper discusses the use
of bench-scale batch reactors to evaluate the potential of nonsupplemented substrates (cassava, dairy and citrus WW) for the
production of hydrogen and the recovery of energy. In addition,
apparent kinetic parameters were assessed for scaling up the process, and simple, economic estimations were performed to evaluate
the process economic sustainability. Since there are no works in

Co
Se
S
V
CC
CCOD
S
X

m
rs
Cx
YX=S
umax
Ks
App
k1
App

k2
SMP

497

cobalt
selenium
sulfur
vanadium
carbohydrate concentration, in milligrams per liter
concentration of oxidable organic matter, in milligrams
per liter
limiting substrate concentration, in milligrams per liter
biomass
specic growth rate of the microorganisms
rate of substrate utilization
biomass concentration
yield of substrate to biomass
maximum specic growth rate of the microorganisms
half-velocity constant
apparent kinetic constant for carbohydrate
degradation
apparent kinetic constant for COD reduction
soluble metabolites production

hydrogen production through dark fermentation of cassava, dairy

and citrus WW without nutrient supplementation, the present
paper lls a gap in the discussion of technical and economic
feasibility of biohydrogen production.
2. Materials and methods
2.1. Experimental procedure
Experiments were conducted in 2 L batch reactors (Duran
asks). Headspace of the bioreactors (1 L) was ushed with nitrogen gas to maintain anaerobic conditions. All experiments were
performed in shaker fermenters at 37
C with orbital shaking at
150 rpm for 70 h. Each bioreactor was inoculated with an anaerobic
biomass obtained from a UASB reactor treating poultry slaughterhouse WW (26 g VSS L1). Prior to the inoculation step, the inoculum received a heat-shock pretreatment to inhibit the H2consuming bacteria, according to the protocol described by Wang
and Wan [29].
Cassava WW was sampled from a cassava starch processing
plant (Toledo, PR, Brazil). Dairy WW was obtained from the
cleaning procedure of a UHT milk processing plant (Francisco
~o, PR, Brazil), and citrus WW was sampled at a plant
Beltra
extracting pectin from citrus (Limeira, SP, Brazil). Reactors were
lled with 900 mL of ltered (0.20 mm membrane) wastewater
diluted with tap water to its working concentration (approximately 2 g L1 COD) and with 100 mL of inoculum. pH was
adjusted to 5.5 by adding sodium bicarbonate or hydrochloric
acid. Control substrate (sucrose) was complemented with the
nutritional medium shown in Table 1. Gas and liquid samples were
periodically obtained from the reactors for analyses. Internal
pressure of each ask was measured using a pressure gauge
(Desin Instruments TPR-18, Barcelona e Spain) with detection
range of 0e500 mbar.
For the estimation of economic impact of WW nutritional supplementation, a control medium containing sucrose as carbon
source was utilized (Table 1). This control medium was chosen
based on previous successful evaluations [30e32]. Additionally, the
medium consisted of a low cost substance and contained considerably lower nutrient concentrations than those found in the
assayed WWs, as shown in Table 1.

498

S.D.M. Lucas et al. / Renewable Energy 75 (2015) 496e504

Table 1
Nutrient market price, control medium composition and raw wastewater
characterization.
Parameters
pH
COD (mg L1)
TS (mg L1)
VSS (mg L1)
FS (mg L1)
C (Sucrose)
N
P
Ca
Mg
Na
K
Ni
Cu
Fe
Zn
Co
Se

Market pricea
(US$ kg1)

Control
medium

Cassava
starch

Dairy

Citrus

0.97
0.83
20.83
0.16
2.04
0.90
2.14
3.80
11.33
1.13
4.05
83.18
241.29

5.50
2474
0
0
0
1979
11.51
9.42
2.06
e
e
e
0.500
e
2.75
e
0.04
0.036

4.02
8017
7600
6780
820
HCS
81.37
66.0
60.7
24.0
485
670
0.020
0.160
13.3
2.00
e
e

10.61
2370
2280
1870
410
HCS
10.39
34.0
33.2
2.04
184
39
0.036
0.008
1.25
1.01
e
e

3.80
2340
16500
15010
1490
HCS
173.1
44.0
240
27.8
640
935
e
0.020
0.07
0.08
e
e

COD e chemical oxygen demand, TS e total solids, VSS e volatile suspended solids,
FS e xed solids, HCS e heterogeneous carbon sources.
e Below the detection limit.
a
Average commodity price dened for the rst 6 months of 2013 [31,32].

Market price presented in Table 1 provides a comparison price

parameter and an estimation of the average costs of raw materials
utilized to supplement the nutrient composition of the control
medium. Because the real value of each nutrient source is subject to
a variation which includes logistics and processing factors, the
comparison of the prices of the mineral resources is a potential
method to provide a standardized price for each nutrient source.
2.2. Analytical methods
WW characterization based on COD, solids, total Kjeldahl nitrogen, total phosphorous and minerals was performed in accordance with the Standard Methods for Examination of Water and
Wastewater [33].
The total carbohydrate content was determined by the colorimetric method developed by Dubois et al. [34], and the composition of the biogas (H2 and CO2) was determined by gas
chromatography (GC 2010 Shimadzu) following the protocol
described by Peixoto et al. [7]. Organic acids and solvents were
analyzed with a Shimadzu high-pressure liquid chromatography
(HPLC) system using the same conguration and protocols used by
Peixoto et al. [7].
2.3. Data analysis
A modied Gompertz equation [35], showed by Equation (1),
was used to t the cumulative H2 production curves for each batch
reactor to obtain the H2 production potential (P), the H2 production
rate (Rm) and the lag phase (l).




Rm $e
$l  t 1
Ht P exp  exp
P

(1)

In Equation (1), H is the cumulative H2 production (in milliliters), l is the lag-phase time (in hours), P is the H2 production
potential (in milliliters), Rm is the H2 production rate (in milliliters
per hour), t is the incubation time (in hours) and e is Euler's number
(2.71828). The parameters (P, Rm and l) were estimated with the
software STATISTICA 8.0.360 (Statsoft, Inc. Tulsa, Oklahoma) using

a Newton-Gauss algorithm. Up to one hundred iterations were

employed to converge the data predicted using the mathematical
model with the experimental values.
The data for carbohydrate consumption and COD removal were
tted using a simplied Monod kinetic equation. This kinetic model
is modied for low substrate concentrations [36], and is developed
as described by the Equations (2)e(5).

dS
C
S
rS  X $mmax $
dt YX=S
KS S

(2)

KS S KS* KS > > S

(3)

CX
1
$m
$ kApp
YX=S max KS*

(4)

dS
rS  kApp $S
dt

(5)

Considering Monod's model for microorganism kinetics (Equation (2)) and assuming that the substrate concentrations used in
this work were lower than KS, it is possible to assume a simplied
hypothesis (Equation (3)). Thus, by rearranging all of the constants
of Monod's equation (Equation (4)), an apparent rst-order kinetic
equation is obtained (Equation (5)). The substrate variable (S) in
Equation (5) is replaced by CC, the concentration of carbohydrates,
and CCOD, the concentration of oxidizable organic matter (both in
App
mg L1). Apparent kinetic parameter k1 will assume the form
App
k1 for the kinetic constant for carbohydrates and kApp
2 for the
kinetic constant for COD; t represents time.
Experimental design was completely randomized for the three
WWs (cassava starch, dairy and citrus) and the control medium
(sucrose), performed in triplicate. H2 production potential, the H2
production rate, lag phase, H2 yield, specic H2 production rate,
carbohydrate consumption and COD removal were subjected to
one-way analysis of variance (ANOVA) to determine the differences
among the groups. The Dunnet test (p < 0.05) was used to determine signicant differences between the tests and control.
Energy recovery from the WWs (in kJ g1 of COD) was calculated
using 0.09 mg mL1 and 142 kJ g1 as the H2 density and heating
value, respectively. For economic estimations, a kilogram of
hydrogen (119.9 MJ) was compared with a gallon of gasoline
(121.7 MJ) [37] because their energy content is approximately equal
on a lower heating value basis [38]. Consequently, the retail price of
energy from hydrogen was considered to be US$ 3.45 per 121.7 MJ,
the current price of a gallon of gasoline [39]. The maximum economic yield obtained with hydrogen produced from WW without
supplementation was calculated according to Equation (6), which
maintains the xed ratio of US$ 0.028 MJ1 (US$ 3.45 per 121.7 MJ).

HI

S:V:E:G
C:V

(6)

Equation (6) shows the H(I) as maximum economic yield obtained by selling the amount of hydrogen produced with one liter of
WW (in US$ L1), where S is the waste strength (g COD L1), V is the
waste generated in one year (in liters), E is the energetic yield of H2
produced with the residue (in MJ g COD1 L1), G is the current
price of a gallon of gasoline (in US$) and C is the energetic content
of a gallon of gasoline (121.7 MJ).
Concerning the raw materials used to supplement the control
medium (Table 1 e Section 2.1), their costs were calculated according to the amount of nutrients added to the WW. The prot was
inferred as the difference between the production costs and the
competitive energy retail price. In all of the estimations, it was

S.D.M. Lucas et al. / Renewable Energy 75 (2015) 496e504

assumed that no cost was involved in the acquisition of the agroindustrial wastes; thus, nutritional costs were exclusively
considered.

3. Results and discussion

3.1. Feasibility of H2 production
The H2 production, substrate degradation and chemical oxygen
demand proles were monitored throughout the course of the
batch fermentations (Fig. 1AeC).
According to Fig. 1A, sucrose promoted the highest volume of
hydrogen accumulated during the experiment. The same observation was made in the studies by Fernandes et al. [16] and Peixoto
et al. [30], who also used sucrose as the control substrate. Moreover,
95.21% of the sucrose was degraded, contributing to the second
highest apparent kinetic constant kApp
1 determined in this study
(Table 2).
In these sets of experiments, the H2 production rate and potential from sucrose were 0.58 mL h1 and 187.33 mL, respectively
(Table 2). Chen et al. [40], employing batch reactors at 36
C, pH 5.5
and 2.2 g L1 COD (sucrose) for H2 production, obtained a
maximum potential of 68 mL. This value is signicantly lower than
the value obtained in the present study. On the other hand, Chen
et al. [40] observed a shorter lag phase (10 h) and a higher H2 yield
(202 mL H2 g1 COD). In our study, the longer lag phase (21.57 h)
and lower H2 yield (75 mL H2 g1 COD) were most likely related to
the adaptation of the biomass and the lower substrate/biomass (S/
X) ratio of 0.79 g COD g1 VSS compared with the S/X ratio of 7.3 g
COD g1 VSS reported by Chen et al. [40]. According to Chen et al.
[41], higher S/X ratios are favorable for dark fermentation because
they signicantly affect the metabolic characteristics of microorganisms and provide higher efciency in the H2 yield.

499

Despite the higher H2 production achieved with sucrose

(Fig. 1A), its conversion resulted in a yield of 1.78 mol H2 mol1
sucrose, which is very low if compared with the maximum theoretical yield of 8 mol H2 mol1 of substrate with acetic acid as an
end product or 4 mol H2 mol1 of substrate with butyric acid as the
soluble metabolite produced. The possible explanation for such a
low yield is presented in Fig. 2A, which depicts multiple metabolic
pathways that generate citric, malic, succinic, lactic, formic, caproic
acid and ethanol instead of hydrogen. Additionally, low organic
matter removal was reported (Fig. 1C) as a consequence of low
conversion to hydrogen.
As presented in Fig. 1A, cassava processing WW yielded the
largest volume of hydrogen among the WWs (97.85 mL H2). This
enhanced H2 production was most likely related to elevated
intrinsic carbohydrates noted at the beginning of the assay (Fig. 1B).
In addition, Table 2 indicates that cassava WW displayed the
highest kinetic constant for COD removal (0.014 h1), implying a
high substrate conversion rate. Another possible factor contributing
to the high H2 production is the S/X ratio of 1.041. This ratio was
higher than that of either dairy (0.71) or pectin (0.70). In most
cases, an elevated S/X ratio favors H2 production as previously
demonstrated by Chen at al [40].
It is likely that the composition of the cassava processing WW
accounted for the highest volumetric H2 production reported. As
reported in early studies, including that of Sreethawong et al. [6],
the effect of a nutrient in the medium is not only related to its
presence but also to its relative proportion in the substrate. In this
context, among the assayed WWs, cassava processing WW contained a COD:N ratio of 100:1.015, which was close to the best ratio
(100:2.2) determined by the work of Sreethawong et al. [6]. In that
study, the H2 production from cassava WW in anaerobic
sequencing batch reactors was optimized at a COD:N ratio of
100:2.2, resulting in the maximum H2 yield of 237.5 mL H2 g1 COD.
This H2 yield is higher than the 31.41 mL H2 g1 COD obtained in

Fig. 1. e (A) Volumetric H2 temporal prole; (B) Carbohydrate temporal prole; (C) COD temporal prole; Dotted lines () represent the modied Gompertz equation t; Dashed
lines () indicate the rst-order decay t; (WW) wastewater.

S.D.M. Lucas et al. / Renewable Energy 75 (2015) 496e504

0.85
0.90
0.89
0.87
0.001
0.004
0.000
0.001

0.002
0.014
0.002
0.001
57
57
32
78

Numbers followed by a are signicantly different from numbers followed by b. Numbers indicate average value. () e standard deviation. ns e Not signicant.

2474
3115
2044
2300
Sucrose
Cassava
Dairy
Citrus

21.57
19.08
19.95
18.38

2.68
0.64
7.77
2.21

ns
ns
ns
ns

0.58
0.32
0.31
0.59

0.22
0.10
0.24
0.19

ns
ns
ns
ns

187.3
97.85
76.12
66.58

20.8
17.74
28.74
18.02

a
b
b
b

0.19
0.11
0.11
0.18

0.07
0.03
0.08
0.15

ns
ns
ns
ns

0.98
0.97
0.98
0.98

0.02
0.01
0.01
0.01

1979
2492
1635
1840

125
46
17
12

ns
ns
ns
ns

0.042
0.031
0.045
0.036

0.007
0.003
0.002
0.007

0.95
0.99
0.91
0.94

0.03
0.04
0.04
0.01

Initial concentration
(CCOD, mg L1)

COD

Apparent kinetic R2
constant
1
(kApp
1 , h )
Initial concentration
(CC, mg L1)

Carbohydrates

R2
Specic production
rate (RS, mL g
VSS1 h1)
Production
potential
(P, mL)
Production
rate
(Rm, mL h1)
Lag phase
length
(l, h)

Substrate Hydrogen

Table 2
Modied Gompertz equation parameter values and rst-order kinetics for carbohydrate and COD removal for the evaluated substrates during acidogenic fermentation.

a
a
b
b

Apparent kinetic R2
constant
1
(kApp
2 , h )

0.04
0.02
0.05
0.04

500

our study. It is likely that the reactor conguration with improved

mass transfer, an inoculum previously adapted to cassava WW and
the higher substrate availability (20 g COD L1) contributed to the
enhanced performance reported by Sreethawong et al. [6].
Despite the study developed by Sreethawong et al. [6], in which
COD corresponded to all the organic matter present in the WW, C
exclusively represented the carbon source utilized for H2 production in the experiments of Argun et al. [5]. Their results demonstrated that the N and P requirements were proportional to each
other, and the H2 production increased with higher C/N and C/P
ratios, yielding a maximum of 281 mL H2 g1 starch at C/N 200
and C/P 1000. In the cassava processing WW used in our study,
the C/N and C/P ratios were 98 and 121, respectively. According to
their ndings, these ratios found in the evaluated WW imply an
excess of nitrogen and phosphorous. Because these macronutrients
(N and P) are directly related to microbial growth, an excess of these
elements in dark fermentation would promote biomass growth
instead of H2 production; thus, it is possible that the hydrogen yield
noted in our study could be increased.
According to Fig. 2B, butyric acid was the predominant soluble
metabolite produced in the dark fermentation of cassava processing wastewater, achieving a peak of 717.13 mg L1 (t 46 h).
Caproic acid production was lower, achieving its maximum concentration (57.10 mg L1) by 70 h of the batch experiment. Similar
results for butyric acid production were obtained by Sreethawong
et al. [6], who also used cassava WW to produce hydrogen in
environmental conditions similar to those employed in this study.
These authors achieved 1100e2850 mg L1 of butyric acid in their
fermentation. Our HAc/HBu ratio in the fermentation of cassava
processing WW was 0.16, which is similar to that reported by Lee
et al. [42]. These authors obtained 0.19 as the HAc/HBu ratio in the
fermentative H2 production from starch in batch reactors. Su et al.
[43] obtained HAc/HBu ratios varying from 0.13 to 0.22 in the
evaluation of various hydrolyzed cassava starch concentrations
(10e25 g L1). It is likely that starch-rich substrates play an
important role in driving the metabolic pathway towards butyrate
production, resulting in low HAc/HBu ratios. However, for
improved yields in H2 production, higher concentration of acetate
rather than butyrate is required as stoichiometrically demonstrated
by Equations (7) and (8).
C6H12O6 2H2O / 2CH3COOH 2CO2 4H2

(7)

C6H12O6 / CH3CH2CH2COOH 2CO2 2H2

(8)

Among the agro-industrial efuents assayed, dairy WW

demonstrated the largest yields of hydrogen (37.25 mL H2 g1 COD)
and energy recovery (0.48 kJ g1 COD). In comparison, cassava and
citrus WWs achieved maximum hydrogen yields of 31.41 and
28.95 mL H2 g1 COD and energy recoveries of 0.40 and 0.36 kJ g1
COD, respectively. The H2 yield of 37.25 mL H2 g1 COD
(0.11 mmol g1 COD) obtained from dairy WW was similar to the
0.12 mmol g1 COD reported by Mohan et al. [22]. These authors
also evaluated dairy WW as the primary carbon source for H2
production given its elevated concentration of carbohydrates
(lactose), proteins (casein) and lipids, which are suitable, low-cost
sources for H2 production.
Regarding the relatively enhanced performance achieved in the
fermentation of dairy WW for H2 production, it is likely that the C/N
ratio of 196.70 and C/P of 60.11 stimulated the metabolism of the
microorganisms to convert carbohydrates to hydrogen instead of
cellular growth. This assertion is supported by the suitable C/N
range for H2 production determined by Argun et al. [5] and by the
highest value of the carbohydrate degradation rate (0.045 h1)
shown in Table 2.

S.D.M. Lucas et al. / Renewable Energy 75 (2015) 496e504

501

Fig. 2. (A) sucrose soluble metabolite production (SMP); (B) cassava WW (SMP); (C) dairy WW (SMP); (D) citrus WW (SMP); HCi citric acid; HMa malic acid; HSu succinic acid; HLa
lactic acid; HFo formic acid; HAc acetic acid; HPr propionic acid; HBu butyric acid; HVa valeric acid; HCa caproic acid; EtOH ethanol.

Despite the additional H2 production using the other carbon

sources present in dairy WW [22], the results presented in Fig. 1A
suggest that after 96% of the soluble carbohydrates had been
degraded (Fig. 1B), no further hydrolysis occurred. The proteins and
lipids that remained of the industrial process [22] most likely did
not contribute to the H2 production because the hydrolysis of these
large molecules became the limiting step in the process.
In addition to biogas determination, soluble metabolite monitoring revealed that butyric and caproic acids were signicantly
produced (Fig. 2C). The dynamic of metabolite production indicated
that when the concentration of propionic acid decreased, the H2
production increased as observed at the beginning of the dairy
wastewater fermentation (Fig. 2C; t 10 h). This behavior was
expected because propionic acid formation consumes hydrogen
from the system, as demonstrated in Equation (9).
C6H12O6 2H2 / 2CH3CH2COOH 2H2O

(9)

In contrast, the increase of 277.93 mg L1 of butyric acid at

t 14 h possibly yielded hydrogen to the system, as described by
the metabolic route presented in Equation (8). In addition, a
simultaneous increase in caproic acid production (70.50 mg L1) at
t 14 h was noted (Fig. 2C). It is likely that this caproic acid production was related to H2 consumption and production pathways
(Equations (10)e(11)); in anaerobic bacteria metabolism, it is
feasible to generate 1 mol of caproate by consuming 1 mol of acetate, 1 mol of butyrate and 2 mols of hydrogen (Equation (10)) [44].
Moreover, it is also possible to produce hydrogen and caproate at a
hydrogen yield of 1.33 mol H2 mol1 glucose (Equation (11)) [45] if
it is assumed that Clostridium sp. is part of the fermentation
consortium.
CH3(CH2)2COO CH3COO2H2 H/CH3(CH2)4COO
2H2O
(10)
3Glucose / 2Caproate 6CO24H22H2O

(11)

According to Ding et al. [45], simultaneous hydrogen and caproic

acid production is feasible, but it results in low amounts of H2.
These authors reported that caproic acid could also be formed
through the secondary fermentation of ethanol and acetate or
ethanol and butyrate. Because butyrate was one of the major
products found in this set of experiments, it is likely that the
acidogenic bacteria used butyrate as an alternative terminal
acceptor for the reducing equivalents during caproic acid synthesis.
Moreover, during the fermentation of sucrose and dairy WW, an
elevated production of ethanol was noted that potentially resulted
in the observed production of caproic acid (Fig. 2A and C). This
observation is consistent with the assumption that some caproateproducing bacteria form a syntrophic relationship with ethanolproducing bacteria [40]. Another fact supporting the existence of
caproate-producing bacteria in the mixed culture used for H2
production in this study is that the spore-forming Clostridium
kluyveri is known to be resistant to the heat and chemical treatments used for selecting H2-producing bacteria [45].
Citrus WW displayed the lowest H2 production potential
(66.58 mL H2) and H2 yield (28.95 mL H2 g1 COD) among the
assayed WWs. This WW composition likely restricted hydrogen
production because citrus WW is primarily composed of insoluble
particles with low natural biodegradability [24]. In this study, the
orange peel used for the extraction of pectin contains 16.9% soluble
sugar, 9.21% cellulose, 10.5% hemicellulose and 42.5% pectin, its
most important component. Despite the low biodegradability of
pectin, this abundant carbon source (42.5% in pectin WW) could
produce larger amounts of hydrogen if an acid or enzymatic hydrolysis step were performed prior to the fermentation.
A large amount of malic acid was detected at the beginning of
the experiment (Fig. 2D). It is likely that the presence of this substance in the fermentation medium was intrinsically linked to the
natural composition of citrus fruits. According to Fig. 2D, malate has
presented a marked decay prole as soon as fermentation was
initiated, which most likely was because it could be readily
metabolized by the bacterial consortium. Malate was most likely

2345.37
2178.12
1756.89
2716.92
1687.60
2053.83
1368.06
2076.96
350.64
0.00
0.00
71.04
54.68
121.29
58.87
121.71
461.73
129.12
337.78
63.48

177.98
34.12
95.16
363.75
Sucrose
Cassava
Dairy
Citrus

CODcarb: COD of the residual carbohydrates measured.

1
CODH2 : mg COD of H2 evolved, calculated with mol H2 x 16 g COD mol H1
.
2 x 1000 mg g
CODsmp: COD of soluble microbial products (SMP).
CODsum: CODH2 : the sum of the COD of residual carbohydrates and COD of SMP e COD evolved from hydrogen production.
CODtotal: the measured COD of the reactor wastewater.
Calculated using the formula [(1 e (CODsum)/(CODtotal))  100%].

0.00
59.71
0.00
0.00
105.47
737.94
153.04
340.85
618.10
933.35
486.89
605.98
63.21
38.40
296.02
313.06
0.00
0.00
0.00
38.58
33.77
30.01
21.56
45.41
0.00
0.68
0.74
0.00
0.00
0.00
13.15
93.41
0.00
3.33
0.00
43.86
0.00
0.00
0.00
327.18

HBu
Isob
HPr
HAc
HFo
Hla
HSu
Hma

CODsmpc (mg COD)

CODH2 b
(mg COD)
CODcarba
(mg COD)
Substrate

As demonstrated in Table 4, the energy recoveries and the

maximum economic yields obtained from non-supplemented
WWs were signicantly different. The main causes for the
distinct economic yields were the substrate strength (S) and energy yield (E) of the assayed WWs, which are the main parameters
of Equation (4) (section 2.3).
According to Table 4, cassava WW was the residue that provided the largest availability (V) and the second highest energy
yield (0.40 kJ g1 COD) among the WWs evaluated. Hence, more
energy was produced, resulting in higher prots, with the biohydrogen production process. Dairy WW exhibited the highest H2
yield (37.25 mL g1 COD). This nding was potentially related to
sufcient concentrations of micronutrients, especially Fe and Ni
utilized for the synthesis of ferredoxin and hydrogenase enzymes
[10]. Additionally, the most suitable C/N ratio [7] (196.70) and C/P
ratio (60.11) was observed with dairy wastewater, explaining the
greater H2 production. However, this WW did not provide the
largest energy production due to its lower availability (V).
Based on Table 1, the hypothetic nutritional supplementation
would cost US$ 22.3 cents L1 of WW. These costs clearly exceeded the competitive retail price, implying that energy from
hydrogen could not be sold protably for US$ 2.84 cents MJ1 (US$
3.45 per 121.7 MJ). Instead, the cost would be at least US$ 69.53,
195.38 and 295.36 MJ1 for cassava, dairy and citrus WW,

Table 3
Electron mass balances (in terms of chemical oxygen demand) of the evaluated substrates.

3.3. Energy recovery and economic implication of H2 production

from agro-industrial wastewaters

Isov

Electron mass balances were performed to ascertain the

plausibility of the data obtained (Table 3). The balance results
indicate low missing electron equivalents. Reactors containing
sucrose were the exception with missing equivalents of 20.46%. In
this case, it is possible that ethanol generation was accompanied
by the production of other undetectable soluble metabolites. This
is supported by the observations made by Peixoto et al. [7], who
found that the fermentation of sucrose with mixed cultures can
lead to the growth of Klebsiella sp., a bacterium that produces
mainly 2,3-Bu(OH)2 [47]. The analytical methods employed in this
study are unable to detect and quantify this metabolite. Thus, the
high level of missing electron equivalents detected was possibly
due to the activity of Klebsiella sp.

0.00
0.00
0.00
12.38

HVa

3.2. Electron mass balances

0.07
0.03
0.02
0.02

HCa

MeOH

EtOH

CODsumd
(mg COD)

CODtotale
(mg COD)

Missingf electron
equivalents (%)

converted to pyruvic acid, which was integrated into the ethanolic

fermentation reactions involving decarboxylation to acetaldehyde
with a subsequent reduction to alcohol [46].
In spite of the high initial concentration of malic acid, butyrate
was the predominant byproduct generated during H2 production,
reaching a peak of 670.60 mg L1. The production of this acid has
been clearly described because it is directly linked to one of the
most common H2 generation processes (Equation (8)). According
to Fig. 2D, caproate (90.76 mg L1), acetate (56.67 mg L1) and
ethanol (65.35 mg L1) were also produced but in smaller
amounts. The HAc/HBu ratio of 0.13 was lower than that reported
with sucrose (0.37) or cassava starch (0.16), contributing to the
lowest H2 production reported in this study. Moreover, the
nutritional ratios of S/X (0.70), C/N (13.51) and C/P (53.18) were
not within the ranges suitable for improved H2 production
[4,18,24,45], consistent with the low performance observed with
this WW.
Although the H2 production potential from citrus WW was the
lowest (Fig. 1A), this substrate still could be used for energy
obtaining through methane production, since the COD of substrate did not exhibit a signicant reduction (Fig. 1C).

20.46
4.14
16.72
10.17

S.D.M. Lucas et al. / Renewable Energy 75 (2015) 496e504

HCi

502

S.D.M. Lucas et al. / Renewable Energy 75 (2015) 496e504

503

Table 4
Estimations of energy recovery, prot and retail price.
Parameters/estimations

Theoretical WW supplementation

Non-supplemented WWs

Alternative process

Cassava WW

Dairy WW

Citrus WW

Cassava WW

Dairy WW

Citrus WW

Vinasse

Bioprocess

Acidogenic
fermentation

Acidogenic
fermentation

Acidogenic
fermentation

Acidogenic
fermentation

Acidogenic
fermentation

Acidogenic
fermentation

Two-stage
fermentation

COD (g L1)
Volume (L y1)
Energy yielda (MJ g COD1 L1)
Energy Recoveryb (MJ y1)
Gross Protc(US$ y1)
Supp. Expensesd (US$ y1)
Retail Pricee (US$ MJ1)
Max. Econ. Yieldf (US$ cents L1 WW)

8.01
184.10 109
0.40 103g
0.59 109
16,770,577.1
41,054,300.0
69.53
e

2.37
78.00 109
0.48 103g
0.09 109
2,528,831.9
17,394,000.0
195.38
e

2.34
0.08 109
0.36 103g
0.00006 109
1760.1
18,302,265.6
295.36
e

8.01
184.10 109
0.40 103
0.59 109
16,770,577.1
0
0.028
0.009

2.37
78.00 109
0.48 103
0.09 109
2,528,831.9
0
0.028
0.003

2.34
0.08 109
0.36 103
0.00006 109
1760.1
0
0.028
0.002

20,731.00
224,109
10.48 103
48.66 109
1,382,414,734.0
0
0.028
0.61

a
b
c
d
e
f
g

Energy yield: (hydrogen yield)  (hydrogen density)  (hydrogen heating value).

Energy recovery: (energy yield)  (COD)  (volume).
Gross prot: (Equation (4))  (volume).
Supplementation expenses: calculated by (control medium market price e Table 1)  (volume).
Retail price: calculated by (supplementation expenses)/(energy recovery).
Maximum economic yield: calculated by (Equation (4) e Section 2.3).
Assuming the same hydrogen yield obtained with intrinsic nutrients.

respectively. In the case of a synthetic (lab made) substrate (sucrose-based medium) mixed with the same volume of cassava WW,
the costs involved in the carbon source and nutrients would be US$
2.63 L1 substrate, resulting in the minimum retail price of US$
1106.25 MJ1. Despite the highest H2 production and energy yield
(75.72 mL g1 COD/0.97 103 MJ g COD1 L1) obtained with this
synthetic substrate, the economic yield (US$ 0.007 cents L1 substrate) would not be sufcient to permit a competitive energy retail
price (US$ 2.84 cents MJ1).
As depicted in Table 4, the two-stage fermentation process,
whereby hydrogen and methane are harvested sequentially, could
result in considerably increased gains. In the two-stage case study,
the energy recovery from vinasse [30] was 10.48 kJ g COD1, which
is approximately 21 times higher than reported in our study.
Therefore, the maximum economic yield could reach 0.61 US$ cents
L1 WW, whereas in the single acidogenic fermentation only 0.009
US$ cents L1 WW was obtained. The main factor that contributes
to the higher performance in the two-stage fermentation process is
the energy recovered in the methanogenic stage. According to the
study by Peixoto et al. [30], the energy recovery from vinasse was
0.26 kJ g COD1 in the acidogenic fermentation, whereas in the
subsequent methanogenic stage, it was 10.22 kJ g COD1. Thus, the
energy from methane corresponded to 97.5% of the total energy
recovered. The energy yield of the substrates evaluated in our study
could potentially be increased by harvesting methane given the
residual organic matter that remained from the H2 production, as
observed in Fig. 1C. Another advantage of a sequential process is
that up to 74% of the organic matter from the entire process can be
removed [30] with the methanogenic stage, thus substantially
reducing the issue of environmental pollution.
4. Conclusions
Biohydrogen production from cassava, dairy and citrus WWs
without nutritional supplementation is technologically feasible.
Dairy WW exhibited the highest substrate degradation rate, H2
yield and energetic potential. This performance is likely related to
the favorable C/N and C/P ratios as well as other intrinsic nutrients.
Nevertheless, the highest energy recovery was estimated for cassava WW (0.59 109 MJ y1) due to its greater availability. We also
found the highest economic yield (US$ 0.009 cents L1 WW) for
cassava WW, which was greatly inuenced by its high organic
matter concentration (8.01 g L1 COD).

Hypothetically, nutritional supplementation of the assayed

WWs would cost US$ 22.3 cents L1 WW. This nding implies an
economic imbalance because this cost surpasses the economic
yields estimated. These observations collectively suggested that
nutritional supplementation is not economically feasible. In addition, low energy recovery and prots were achieved by exclusively
harvesting hydrogen from agro-industrial WWs. However, the
utilization of a sequential process for hydrogen and methane production can be a solution to increase energy yield and economic
gains. Our results demonstrate that recovering energy from
methane and employing readily available, non-supplemented,
carbon-rich WWs are necessary requirements for the economic
sustainability of the process.
Acknowledgments
The authors thank Funda~
ao de Amparo 
a Pesquisa do Estado de
~o Paulo (FAPESP e Process number 2009/15984-0), Conselho
Sa

gico, Brazil
Nacional de Desenvolvimento Cientco e Tecnolo
~o de Aperfeioamento de Pessoal de Nvel
(CNPq) and Coordenaa
Superior (CAPES) for supporting this study.
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