Universidade de São Paulo

Biblioteca Digital da Produção Intelectual - BDPI

Departamento de Hidráulica e Saneamento - EESC/SHS Artigos e Materiais de Revistas Científicas - EESC/SHS

2012

Hydrogen and Methane Production, Energy

Recovery, and Organic Matter Removal from
Effluents in a Two-Stage Fermentative
Process

APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, TOTOWA, v. 168, n. 3, p. 651-671, OCT, 2012

http://www.producao.usp.br/handle/BDPI/35231

Downloaded from: Biblioteca Digital da Produção Intelectual - BDPI, Universidade de São Paulo
Appl Biochem Biotechnol
DOI 10.1007/s12010-012-9807-4

Hydrogen and Methane Production, Energy Recovery,

and Organic Matter Removal from Effluents
in a Two-Stage Fermentative Process

Guilherme Peixoto & Jorge Luis Rodrigues Pantoja-Filho &

José Augusto Bolzan Agnelli & Marlei Barboza & Marcelo Zaiat

Received: 27 March 2012 / Accepted: 12 July 2012

# Springer Science+Business Media, LLC 2012

Abstract This study evaluates the potential for using different effluents for simultaneous H2
and CH4 production in a two-stage batch fermentation process with mixed microflora. An
appreciable amount of H2 was produced from parboiled rice wastewater (23.9 mL g−1 chemical
oxygen demand [COD]) and vinasse (20.8 mL g−1 COD), while other effluents supported CH4
generation. The amount of CH4 produced was minimum for sewage (46.3 mL g−1 COD),
followed by parboiled rice wastewater (115.5 mL g−1 COD) and glycerol (180.1 mL g−1 COD).
The maximum amount of CH4 was observed for vinasse (255.4 mL g−1 COD). The total energy
recovery from vinasse (10.4 kJ g−1 COD) corresponded to the maximum COD reduction
(74.7 %), followed by glycerol (70.38 %, 7.20 kJ g−1 COD), parboiled rice wastewater
(63.91 %, 4.92 kJ g−1 COD), and sewage (51.11 %, 1.85 kJ g−1 COD). The relatively high
performance of vinasse in such comparisons could be attributed to the elevated concentrations
of macronutrients contained in raw vinasse. The observations are based on kinetic parameters of
H2 and CH4 production and global energy recovery of the process. These observations
collectively suggest that organic-rich effluents can be deployed for energy recovery with
sequential generation of H2 and CH4.

G. Peixoto (*) : J. L. R. Pantoja-Filho : J. A. B. Agnelli : M. Zaiat

Laboratory of Biological Processes, Center for Research, Development and Innovation in Environmental
Engineering, São Carlos School of Engineering, University of Sao Paulo, João Dagnone,
1100 Santa Angelina, CEP 13563-120 São Carlos, Brazil
e-mail: peixoto@sc.usp.br
J. L. R. Pantoja-Filho
e-mail: pantoja@usp.br
J. A. B. Agnelli
e-mail: gutoagnelli@gmail.com
M. Zaiat
e-mail: zaiat@sc.usp.br

M. Barboza
Department of Chemical Engineering, Federal University of Triângulo Mineiro, Frei Paulino 30, Abadia,
CEP 38025-180 Uberaba, Brazil
e-mail: marlei.uftm@gmail.com
 Appl Biochem Biotechnol

Keywords Hydrogen . Methane . Glycerol . Parboiled rice wastewater . Vinasse . Sewage

Abbreviations
COD chemical oxygen demand, in milligrams per liter
BOD biological oxygen demand, in milligrams per liter
HRT hydraulic retention time, in hours
C carbon
N nitrogen
P phosphorus
TS total solids, in milligrams per liter
VS volatile solids, in milligrams per liter
FS fixed solids, in milligrams per liter
VSS volatile suspended solids, in milligrams per liter
X microorganisms concentration, in milligrams per liter VSS
S substrate, in milligrams per liter
SMP soluble metabolites production, in milligrams per liter

Introduction

The common use of fossil fuels has increased the concentrations of greenhouse gases in the
atmosphere, a matter of prime concern to the scientific community, the environmental
protection agencies, and the general public. These negative effects of uncontrolled fossil
fuel utilization have stimulated research on the development of alternative processes to
derive clean energy, mainly through renewable energy sources. The anaerobic fermentation
process offers the possibility of energy generation using many renewable substrates, such as
glycerol [1], by-products of wheat flour processing [2], molasses [3], rice straw [4], grass
silage [5], cassava stillage [6], soft-drink wastewater [7], and domestic wastewater [8],
among others. The anaerobic process primarily generates methane as its final gaseous
product, and for this purpose, the process involves the joint actions of two major types of
bacterial consortia, namely, the so-called acidogenic bacteria (which break down substrates
into mainly H2, acetic acid, and CO2) and methanogenic bacteria (which convert acetic acid,
H2, and CO2 to methane gas) [9].
Recent studies demonstrated that the physical separation of the acidogenic and methano-
genic consortia in a two-stage anaerobic fermentation process presents many advantages,
such as improvement of the hydrolysis of complex substrates before methanogenesis [10],
avoidance of drastic pH fluctuations in the second stage [11], increase in the process energy
yield [6], significant reduction of the organic load of substrates [12], and generation of
hydrogen gas [13]. This gaseous product is an interesting energy carrier with an energy
density 2.75 times greater than that obtained from fossil fuels [14].
In the two-stage fermentation process, the first stage (hydrogen production) does not
significantly reduce the organic matter content of the substrates [15–18]. The amount of
chemical oxygen demand (COD) removal is usually below 20 % [19] during the hydrogen
production process, while the remaining undegraded organic matter is largely removed in the
subsequent CH4 fermentation stage.
Hydrogen and methane are both energy sources that can be utilized for the generation of
heat, production of electricity, and transportation fuel, either separately or as a mixture of H2
and CH4 known as hythane [20]. Hydrogen has been receiving increasing attention as a
nonpolluting fuel. On the other hand, methane production comprises approximately 95 % of
the total energy yield from the two-stage anaerobic fermentation process [6, 21].
Appl Biochem Biotechnol

According to Wang et al. [6], the utilization of domestic and industrial effluents for
hydrogen and methane production in the two-stage fermentation process has considerable
potential to enhance the economic feasibility of waste treatment, either by using the organic
waste for energy production or by increasing the treatment efficiency for the residues.
Wastewater, from both domestic and industrial effluents, is a potential energy source.
Moreover, hydrogen and methane generation from wastewater could be cost-effective and
carried out using local feedstock.
In Brazil, for example, current glycerol generation is 17,600 m3 year−1 according
to Da Silva et al. [22], based on estimates that biodiesel production generates 10 %
(w/w) glycerol. The volume of vinasse generated is usually 14 times that of the
ethanol produced when sugarcane is used as feedstock. Data from the Sugarcane
Agroindustry Union of São Paulo State (UNICA) [23] show that 16,000,000 m3 of
ethanol were produced during the 2008–2009 harvest, resulting in 224,000,000 m3 of
vinasse. Also, in 2008–2009, 2,800,000 m3 of parboiled rice wastewater were
generated [24] according to the reports of the Rural Extension and Agropecuary
Research Business (EPAGRI) [25]. According to the national census on domestic
wastewater conducted in 2008 by the Brazilian Institute for Geography and Statistics
(IBGE) [26], metropolitan regions such as Brasília, DF; São Paulo, SP; and Rio de
Janeiro, RJ generate 301,413; 3,064,976; and 1,782,703 m 3 day −1 of sewage,
respectively.
Considering the availability of the aforementioned wastewaters and the growing need for
sustainable energy sources, this paper discusses data on bench-scale batch reactors to
evaluate the potential of each effluent (sewage, glycerol, parboiled rice wastewater, and
vinasse) for the sequential generation of hydrogen and methane and energy recovery. The
apparent kinetic constants were estimated to derive data for use in process upscaling and
reactor design.

Materials and Methods

Batch Reactors

The reactors were composed of 2-L glass flasks (Duran® flasks), containing 1 L of liquid
volume and 1 L of headspace, to provide a sufficient amount of liquid and gas to be sampled
throughout the experiment.

Effluents

Four effluents were tested (vinasse, glycerol, parboiled rice wastewater, and domestic
wastewater), while a sucrose-based medium was the control substrate. Vinasse and
glycerol are the by-products of ethanol and biodiesel plants, respectively. Vinasse was
derived from an ethanol production plant (Usina Nova Era, Ibaté, SP, Brazil), and
glycerol was the by-product of the esterification reaction of vegetable oil in a
biodiesel plant (Granol, Anápolis, GO, Brazil). Parboiled rice wastewater was obtained
as the residue of the rice parboiling process in a food production plant (Nelson Wendt
Alimentos, Pelotas, RS, Brazil). The domestic effluent was collected from an exper-
imental wastewater treatment plant at the University of São Paulo (São Carlos, SP,
Brazil). The basic characteristics of the above-mentioned effluents and the sucrose
medium are presented in Table 1.
 Appl Biochem Biotechnol

Table 1 Effluents and control substrate characterization

Parameters Sewage Vinasse Glycerol Rice wastewater Sucrose

COD (mg L−1) 262 20,731 1,108,230a 5,354 357

pH 6.9 3.8 ND 4.6 5.5
Alkalnity (mg L−1 CaCO3) 124 0 ND 0 0
TS (mg L−1) 1,971 18,170 ND 657 0
VSS (mg L−1) 1,612 14,440 ND 537.5 0
FS (mg L−1) 359 3,270 ND 119.6 0
Total N (mg L−1 NTK) 42 187.5 0 104.6 0
Total P (mg L−1 PO43−) 4.7 133 0 124.5 0

ND not determined
a
For an aqueous solution of 1 g L−1 glycerol

Nutritional Medium

A nutritional medium containing (in milligrams per liter) CH4N2O (6), NiSO4·6H2O (0.15),
FeSO4·7H2O (0.75), FeCl3·6H2O (0.075), CoCl2·2H2O (0.012), CaCl2·6H2O (0.618), SeO2
(0.0108), KH2PO4 (1.608), KHPO4 (0.39), and Na2HPO4·2H2O (0.828) was added at the
beginning of the acidogenic and methanogenic stages. The pH was adjusted by adding
sodium bicarbonate or chloridric acid.

Inoculum

The acidogenic inoculum containing 30 g L−1 of volatile suspended solids (VSS) was
obtained from fixed-bed anaerobic reactors used for hydrogen production from sucrose-
based synthetic wastewater [16].
The methanogenic inoculum (37 g VSS L−1) was obtained from an upflow anaerobic
sludge blanket reactor (UASB) treating poultry slaughterhouse wastewater [27]. The sludge
was taken directly from the middle of the blanket zone of the reactor.

Experimental Procedure

The batch reactors were filled with filtered (0.45-μm membrane) wastewater and diluted
with tap water to the COD level of 300 mg L−1 in each flask. The COD was equalized to
allow a posterior comparative analysis to be conducted [16].
The tests were performed in five batch reactors (in duplicate). Argon was initially flowed
into each reactor (20 min) to generate an anaerobic environment. The reactors were placed in
a thermostatic chamber at 25.0±0.9 °C. Gas and liquid samples were periodically taken from
the reactors for analysis. The internal pressure of the flasks was measured using a pressure
gauge (Desin Instruments® TPR-18) and BS 2200 interface (detection range of 0 to
500 mbar). Less than 10 % of the overall bulk liquid volume was sampled for analyses
throughout the experiment.
In the acidogenic step (0 to 42 h), the liquid phase (1 L) consisted of the diluted effluent
(pH 5.5), the nutritional medium as presented in the “Nutritional Medium” section, and
400 mg VSS L−1 of the acidogenic biomass.
Before beginning the methanogenic step (48 to 224 h), the reactors from the first step
were opened, and their liquid phase was filtered (0.45-μm membrane) to remove the
Appl Biochem Biotechnol

acidogenic biomass. The pH was adjusted to 7.0, and the flasks were complemented with the
nutritional medium (the “Nutritional Medium” section) and incubated with methanogenic
biomass (500 mg VSS L−1).

Analytical Methods

Effluents characterization based on COD, solids, total Kjeldahl nitrogen, and total phospho-
rus (PO43−) was performed in accordance with the APHA [28], following the COD-5220,
Solids-2540, 4500-Norg B. and 4500-P C. protocols. Total alkalinity titration analysis was
also performed ([29], modified by [30]).
Total carbohydrate content was determined by the colorimetric method developed by
Dubois et al. [31], and the composition of the biogas (H2, CO2, and CH4) was determined by
gas chromatography (GC 2010 Shimadzu®) following the protocol described by Peixoto et
al. [7]. Organic acids were analyzed with a Shimadzu® high-pressure liquid chromatography
system using the same configuration and protocols used by Peixoto et al. [7].
The solvent concentrations were measured with a gas chromatograph (GC 2010
Shimadzu®) equipped with a flame ionization detector and a sample introduction
system with a Combi PAL® headspace apparatus (model AOC 5000 and 30-m×0.25-mm
HP-INNOWAX® with a 0.25-μm film thickness) [16, 17].

Data Analysis

A modified Gompertz equation [32] was used to fit the cumulative hydrogen production
curves for each reactor to obtain the hydrogen production potential (P), hydrogen production
rate (Rm), and lag phase (λ):


Rm  e
HðtÞ ¼ P exp  exp  ðl  tÞ þ 1 ð1Þ
P

In Eq. 1, H is the cumulative hydrogen production (in milliliters), λ is the lag phase time
(in hours), P is the hydrogen production potential (in milliliters), Rm is the hydrogen
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 λ) were estimated with the software STATISTICA® 8.0.360
(Statsoft, Inc., Tulsa, OK, USA) using a Newton–Gauss algorithm. Up to 100 iterations were
used to converge the data predicted using the mathematical model to the experimental value.
The hydrogen yield (YH2 ) was calculated as the relationship between the hydrogen volume (in
milliliters H2) and the initial organic matter concentration of the wastewaters in terms of grams
COD. The specific hydrogen production rate, Rs (in milliliters H2 per hour per gram VSS), was
obtained by dividing Rm by the amount of VSS added to the reactor.
Regarding methane production, the first-order kinetics model described by Borja et al.
[33] was used to fit the experimental data. According to this model, the volume of CH4
accumulated (G) (in milliliters, at 1 atm, 25 °C) at a given time t (in hours) can be expressed
by the following equation:
h  0 i
G ¼ Gm 1  exp KG  t ð2Þ

In Eq. 2, Gm is the maximum CH4 volume accumulated at an infinite digestion time, G is

0
0 at t00, and the rate of gas production is 0 when t is equal to infinity. KG is the kinetic
 Appl Biochem Biotechnol

constant for methane production (per hour) that includes the biomass concentration, as
shown in Eq. 3:
0
KG ¼ KG  X ð3Þ

In Eq. 3, KG is the specific methane production kinetic constant (in milliliters CH4 per
gram VSS per hour) and X is the biomass concentration (in grams per liter VSS). The values
0
of KG and Gm for each load were calculated numerically from the experimental data
obtained by nonlinear regression using the software Origin® 8.0891 (OriginLab Corp.,
Northampton, MA, USA). The methane yield ( YCH4 ) was calculated as the relationship
between the methane volume (in milliliters CH4) and the initial organic matter concentration
in the wastewater (measured in terms of grams COD). The specific methane production rate,
KG (in milliliters CH4 per gram VSS per hour), was obtained using Eq. 3.
The energy recovery, or energy yield (in kilojoules per gram COD), was calculated using
0.09 and 0.72 mg mL−1 as the H2 and CH4 densities and 142 and 55.6 kJ g−1 as the H2 and
CH4 heating values, respectively.
The COD removal and carbohydrate consumption data were fitted by a first-order
reaction equation as described by Eqs. 4 and 5:
dCCOD
 ¼ k1 CCOD ð4Þ
dt

dCC
 ¼ k2 CC ð5Þ
dt
In Eqs. 4 and 5, CCOD is the concentration measured in terms of COD, CC is the
concentration of carbohydrates, k1 is the first-order reaction constant for COD removal, k2
is the first-order reaction constant for carbohydrate consumption, and t is the time.

Results and Discussion

Acidogenic Step

H2 Generation

As the acidogenic stage proceeded, parboiled rice wastewater, vinasse, and sucrose promot-
ed H2 production with acclimation phases lasting 8, 10, and 14 h, respectively. Logan et al.
[34] also observed different times for the acclimation phase using different substrates in
batch reactors. In their study, glucose, sucrose, and molasses presented times ranging from
approximately 19 to 27 h, while potato starch required 40 h before hydrogen production
commenced.
Hydrogen production was not detected during the 42-h biogas monitoring period in the case
of domestic wastewater. As shown in Fig. 1d, a slight amount of butyric acid (7.13 mg L−1)
generation was detected between 24 and 36 h, while the amounts of other acids and solvents
remained the same as initially detected. Available organic matter was, therefore, not satisfac-
torily converted into soluble metabolites and biogas. Nevertheless, a small undetected amount
of H2 must have been produced, as there was a 14.8 % reduction in the concentration of the
initial organic matter.
Appl Biochem Biotechnol

Fig. 1 a COD profile, b carbohydrates profile, c volumetric H2 profile, d sewage soluble metabolites
production (SMP), e glycerol (SMP), f parboiled rice wastewater (SMP), g vinasse (SMP), and h sucrose
(SMP). Dashed lines first-order decay fit, dotted lines modified Gompertz equation fit, 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, EtOh ethanol

Van Ginkel et al. [15], using batch reactors (23 °C and pH 6.1) fed with domestic
wastewater, produced 0.04 L H2 g COD−1 because the COD was concentrated at 25 times
the original concentration, yielding a COD of 2.6 g L−1 and a soluble glucose concentration
of 294 mg L−1. These authors suspected that the glucose concentrations in raw wastewater
were insufficient to facilitate the germination of the spore formers that generate H2. The
 Appl Biochem Biotechnol

same phenomenon could be applied to this study because of the insignificant concentration
of carbohydrates in the domestic wastewater (Fig. 1b). The remaining carbon sources for H2
production were mainly proteins and lipids, the most common compounds present in
domestic wastewater.
Okamoto et al. [35] evaluated lipids (chicken skin and fat), proteins (eggs and lean meat),
and carbohydrates for hydrogen production. They obtained values of 1.43, 1.56, 1.31, and
1.75 mL H2 g COD−1 for chicken skin, fat, eggs, and lean meat, respectively, which are
extremely low compared with the value of 8.8 mL H2 g COD−1 obtained for carbohydrates,
e.g., carrots. H2 production from carbohydrates (carrots), proteins (lean meat), and lipids
(fat) showed lag phases lasting 12, 30, and 100 h, respectively. The longer times required for
H2 generation from proteins and lipids may also explain why no H2 was detected during the
42-h acidogenic time course.
In the current study and in that conducted by Okamoto et al. [35], the H2 production from
proteins was extremely low or null, the Stickland reaction must have probably occurred
during the process. The sole degradation of amino acids permits hydrogen production from
proteins, but the Stickland reaction in clostridia uses molecular hydrogen as the electron
donor for the reductive deamination of amino acids, such as glycine during its degradation
[36]. Even if H2 is produced solely by the degradation of amino acids, the hydrogen can be
consumed during the Stickland reaction. In addition, the degradation of these compounds
leads to the formation of long-chain fatty acids, which inhibit the growth of anaerobic
bacteria [37]. The absence of hydrogen production in the sets containing glycerol was also
noticed during the same time course in which domestic wastewater was evaluated.
Regarding Fig. 1c, the experimental run time was probably not long enough to establish
H2 production because glycerol is difficult to break down via hydrolysis to yield short-chain
fatty acids and produce H2. Although H2 was not detected, the organic matter concentration
in the acidogenic stage presented in Fig. 1a revealed the degradation of 12.6 % in the batch
reactors based on glycerol. The same trend was observed in the batch reactors containing
domestic wastewater, and a low amount of H2 must have been produced that could not be
detected with the analytical methods employed.
The data in Fig. 1e indicate that acidogenic activity existed between 24 and 36 h, as indicated
by the production of acetic (4.08 mg L−1), butyric (7.49 mg L−1), lactic (7.43 mg L−1) and
propionic (38.81 mg L−1) acids. These results indicate that the propionic acid pathway was
predominant during the acidogenic stage and did not favor H2 production because propionic
acid synthesis requires molecular hydrogen from the medium.
According to Barbirato et al. [38], propionate can be produced from glycerol by three
bacterial strains: Propionibacterium acidipropionici, Propionibacterium acnes, and Clos-
tridium propionicum. Lay [39] reported that Clostridium sp. was the most common micro-
organism involved in H2 production. The current findings support the hypothesis that the
inoculum obtained from the upflow anaerobic-packed bed reactor probably contained at
least one of the Clostridium species that produced propionate from glycerol.
Seifert et al. [40] achieved hydrogen production using glycerol through a dark fermen-
tation process at 37 °C and pH 6. Their experiments were conducted in batch mode using
glycerol (5 g L−1), an inoculum containing 2.3 g L−1 VSS and 0.73 g L−1 N, which resulted
in the production of 0.241 L H2 L−1 at a rate of 0.014 L H2 L−1 h−1, with a lag phase of 5.5 h
and a yield of 0.198 mol H2 mol glycerol−1. In the study by Ito et al. [1] conducted in batch
reactors, the lag phase was negligible and the extent of hydrogen conversion was even better,
with a substrate yield of 1.05 mol H2 mol glycerol−1 at 5.0 g L−1 glycerol.
Compared with the work of Seifert et al. [40] and Ito et al. [1], the present experiment had
limitations. Among these limitations were the glycerol concentration of approximately
Appl Biochem Biotechnol

0.3 mg L−1 and the inoculum concentration of 422.5 mg L−1 VSS. The differences in the
concentration of the carbon source, biomass concentration, inoculum type, and length of the
acidogenic stage influenced the production of hydrogen from glycerol, as evident from the
current study.
The use of parboiled rice wastewater as the substrate for H2 production resulted in the
production potential of 6.82 mL H2 with the immediate generation of mostly acetate and
succinate as the soluble metabolites (with peaks of production at t06 h and t012 h,
respectively) (Fig. 1f). Hydrogen is released, while, at the same time, acetate or butyrate
are also produced (based on Eqs. 9 and 10), but the high simultaneous generation of
succinate (Fig. 1f) suggests the existence of an alternative metabolic pathway.
Considering glucose as the main carbohydrate obtained by the hydrolysis in the parboi-
lization process of rice, it was then possible to propose the reaction presented in Eq. 6 to
describe the metabolic conversions described above:

C6 H12 O6 þ 2H2 O ¼ C4 H6 O4 þ 2CO2 þ 5H2

ð6Þ
ΔG0 ¼ 179:99 kJ mol1

Despite the observations in Fig. 1f, the amount of lactate increase throughout the assay
was not related to the H2 production, as suggested by Argun et al. [41], who stated that the
generation of lactate as the end product does not support H2 production.
In the parboiled rice wastewater essays, H2 generation corresponded to 14.66 mL H2 g−1
of volatile solids (VS) with a hydrogen yield of 23.90 mL H2 g COD−1, while the
corresponding values obtained by Okamoto et al. [35], who used batch reactors inoculated
with pretreated anaerobic-digested sludge plus a specific medium, were 19.3 mL H2 g VS−1
and 16.8 mL H2 g COD−1 for 15 % total solids (as the rice content in the medium). Although
the results are in good agreement, the higher H2 yield in the current study is probably related
to the thermal hydrolysis involved in the rice parboiling process, which improved the
conversion of starch into glucose, thereby facilitating H2 production. This hypothesis is
supported by the highest obtained value of the carbohydrate consumption rate based on
parboiled rice wastewater (Table 2). In contrast, in the reports of Okamoto et al. [35], the
substrate was from the particulate rice instead of the solubilized carbon source. Thus, starch,
the main carbohydrate in particulate rice, had to be hydrolyzed before being transformed into
a substrate readily available for H2 generation.
Using rice slurry as the feedstock for hydrogen production, Fang et al. [42] obtained 248 mL
H2 at a production rate of 11 mL h−1. The substrate contained 5.5 g carbohydrate L−1, degraded
at 37 °C with a pH of 5.5 by an inoculum from an anaerobic digester containing 566.66 mg VSS
L−1. The H2 yield achieved under these conditions was 300 mL H2 g carbohydrate−1, a value
much higher than the 131.15 mL H2 g carbohydrate−1 obtained in the current study. The S/X
ratio probably contributed greatly to such diverse observations because this ratio was 9.71 in the
reports by Fang et al. [42], while the ratio was 0.11 in the current experiment. Chen et al. [43]
reported the optimum S/X ratio for sucrose to be 7.3 g COD g VSS−1. These values show that
the S/X ratio used by Fang et al. [42] was closer to the optimum value found by Chen et al. [43].
In the case of vinasse, the H2 production rate was 0.84 mL h−1, with an H2 production
potential (P) of 6.25 mL. The remarkable H2 production rate (Rm), coupled with the highest
obtained rate of organic matter degradation (0.0142 h−1), is probably related to the elevated
VSS increase, corresponding to 55 mg L−1 (16.17 %), as registered at the end of the
acidogenic stage (data not shown).
The components of the wastewater, mainly nitrogen, probably favored the development of
hydrogen-producing microorganisms, an observation further supported by the H2 production
Table 2 First-order kinetics for COD and carbohydrates removals and modified Gompertz equation parameter values for the evaluated substrates during the acidogenic step

Substrate COD Carbohydrates Hydrogen

−1 −1 2 −1 −1 2
CCOD (mg L ) k1 (h ) R CC (mg L ) k2 (h ) R λ (h) Rm (mL h−1) P (mL) Rs (mL g−1 VSS h−1) R2

Sewage 262±5 0.0043±0.001 0.9709 NS – – – – – – –

Glycerol 368±15 0.0037±0.001 0.9942 NS – – – – – – –
Rice 285±17 0.0091±0.002 0.9292 52±2 0.2192±0.012 0.9975 8.27±0.57 0.13±0.03 6.82±0.32 0.28 0.9380
Vinasse 375±14 0.0142±0.001 0.9257 37±4 0.1036±0.013 0.9778 10.7±0.82 0.84±0.06 6.25±0.26 2.95 0.9918
Sucrose 375±8 0.0123±0.009 0.9944 327±7 0.1212±0.018 0.9298 14.12±1.4 0.18±0.02 9.34±0,40 0.42 0.9684

NS not significant
Appl Biochem Biotechnol
Appl Biochem Biotechnol

efficiency of the biomass (2.95 mL H2 g−1 VSS h-1), which was the highest among the
wastewaters tested (Table 2).
The metabolism assumed by the microorganisms involved in H2 production from
vinasse seems to be associated with the acetic–butyric pathway because this metabo-
lism was detected mainly as 22.44 and 17.24 mg L−1 of acetic and butyric acid,
respectively (Fig. 1g). The HAc/HBu ratio of 1.3 was consistent with the relatively
ideal hydrogen generation achieved with vinasse because the best H2 yield from
carbohydrates can be achieved with high HAc/HBu ratios [44]. The HAc/HBu ratio
must not be considered in isolation because the significant generation of other end
products, such as ethanol or propionic acid, may lower H2 production even with the
higher HAc/HBu ratio. In this study, the generation of 15.70 mg L−1 of ethanol was
also detected, which certainly affected the H2 production as proposed by Sreethawong
et al. [45] according to Eq. 7:

C6 H12 O6 ! 2CH3 CH2 OH þ 2CO2 ð7Þ

For the removal of organic matter, the flasks containing vinasse presented an efficiency of
approximately 42.72 %, well within the range (29.1 to 56.5 %) achieved by Mohan et al.
[46]. The elevated organic matter removal reported is probably related to the high biode-
gradability of vinasse. As reported by Buitirón and Carvajal [47], vinasse is generally
composed of organic matter between 19.8 and 20.9 g of biological oxygen demand
(BOD) L−1 and 29.9 to 30.5 g COD L−1; the phenol concentration ranges from 44 to
81 mg L−1; the sulfate concentration is 915 mg L−1; the N–NH3 is 110 mg L−1; and the
pH ranges from 3.2 to 4.0. Additionally, the BOD5/COD ratio is approximately 0.67,
indicating that organic matter could easily be biodegraded [48].
As expected, the sucrose solution supported high amounts of H2 production, as it had the
highest carbohydrate content among the effluents assayed (Fig. 1b). Hawkes et al. [49] had
already demonstrated that an elevated carbohydrate concentration was critical for H2 pro-
duction; therefore, the results presented in this paper are in good agreement with their report
[49].
The apparent carbohydrate degradation constant for the sucrose solution was relatively
low compared with the constants for the other substrates evaluated. This could be attributed
to the lower carbohydrate concentrations of the other substrates, causing the carbohydrates
to be consumed in a shorter amount of time (Table 2).
Chen et al. [43] used 250-mL serum bottles at 36 °C, pH 5.5 and 0.3 g L−1 COD (as
sucrose) to obtain a hydrogen production potential of 2 mL. This value is significantly lower
than the value obtained in the present study (Table 2), but the abovementioned investigators
[43] observed a shorter lag phase (6 h) and a higher hydrogen yield (45 mL H2 g COD−1).
The longer lag phase and the lower specific hydrogen production yield in the present study
are probably related to the high S/X ratio of the medium (0.86) compared with the S/X ratio
of 0.5 reported by Chen et al. [43]. According to these investigators, pure carbohydrate
substrates, such as sucrose, support hydrogen production at lower S/X ratios. Although the
current experiment reached a higher production potential, the hydrogen yield was approx-
imately 44 % lower than that achieved by Chen et al. [43]. The solvent-forming micro-
organisms, such as Klebsiella sp., in the inoculum could probably have converted the carbon
source to ethanol and other solvents instead of releasing the H2 in the biogas. This idea is
supported by the study of Fernandes et al. [16] regarding the existence of Klebsiella sp. in
anaerobic reactors operated under conditions almost similar to those used in the present
study.
Table 3 Electron mass balances (in terms of COD) according to the results from the acidogenic step of the evaluated substrates

Substrate CODcarb CODH2 CODSMP (mg COD) CODsum CODtotal Missinga electron
(mg COD) (mg COD) (mg COD) (mg COD) equivalents (%)
Hci HMa HSu Hla HFo Hac HPr Isob Hbu Isov Val EtOH

Sewage 0.0 0.0 0.0 3.7 7.0 20.0 1.2 30.8 13.9 20.1 35.0 28.4 23.9 7.7 192.7 223.0 12.2
Glycerol 0.0 0.0 0.0 3.2 7.9 27.3 1.2 13.7 73.2 0.0 33.4 11.5 26.1 40.8 243.8 290.3 16.0
Rice wastewater 0.0 3.3 8.4 0.0 7.2 50.1 1.3 16.6 15.6 18.6 31.1 0.0 22.8 5.1 173.7 210.7 17.5
Vinasse 0.0 2.9 8.2 4.7 8.4 19.1 1.1 19.6 57.2 0.0 53.5 0.0 0.0 22.5 191.7 233.3 18.0
Sucrose 5.3 5.1 0.0 0.0 0.0 3.1 0.0 19.5. 25.9 0.0 47.7 0.0 38.2 90.8 188.1 235.8 20.2

CODcarb COD of the residual carbohydrates measured, CODH2 milligrams COD of H2 evolved, calculated with mol H2 ×16 g COD mol H2−1 ×1,000 mg g−1 , CODSMP COD of
soluble microbial products (SMP), CODsum–CODH2 the sum of the COD of residual carbohydrates and COD of SMP–COD evolved from hydrogen production, CODtotal the
measured COD of the reactor effluent
a
Calculated by [(1−(CODsum)/(CODtotal))×100 %]
Appl Biochem Biotechnol
Appl Biochem Biotechnol

Besides ethanol, propionic acid, which also did not favor H2 production, was generated,
consuming hydrogen from the system based on Eq. 8 [50]:
C6 H12 O6 þ 2H2 ! 2CH3 CH2 COOH þ 2H2 O ð8Þ
According to Fig. 1h, the major soluble metabolites produced were ethanol (54.57 mg L−1),
acetic acid (51.37 mg L−1), butyric acid (28.75 mg L−1), and propionic acid (25.12 mg L−1), in
line with the soluble metabolite composition reported by Fernandes et al. [16].
Despite the generation of ethanol, the predominance of acetate and butyrate as the soluble
acid metabolites is in close agreement with the findings of Minton and Clarke [51], which
showed that approximately 50 % of the clostridia had the same acetate and butyrate pathways.
In addition, the ΔG0 of the reactions involving the conversion of carbohydrates to hydrogen,
acetate, and butyrate is very favorable, as demonstrated by Eqs. 9 [52] and 10 [53]:

C6 H12 O6 þ 2H2 O ! 2CH3 COOH þ 2CO2 þ 4H2 "

ð9Þ
ΔG0 ¼ 215:69 kJ mol1

C6 H12 O6 þ 2H2 O ! CH2 CH2 CH2 COOH þ 2CO2 þ 2H2 "

ð10Þ
ΔG0 ¼ 254:80 kJ mol1

Electron Mass Balances for the Acidogenic Step

Table 3 presents an electron mass balance for the batch reactors to ascertain the plausibility
of the data obtained. The balance results show low missing electron equivalents. The
exception was for sets containing vinasse and sucrose, with missing equivalents of 18.00
and 20.23 %, respectively. The major reason for the deficit in the electron mass balances
may be the formation of undetected metabolites or the presence of other alternative electron
sinks. However, as the formation of CH4 or H2S was not detected, it is unlikely that the
missing electron equivalents arose due to the electron flow toward those two common
terminal electron acceptors (i.e., CH4 or H2S) in the anaerobic process. It is more likely
that some soluble metabolites that were not monitored (e.g., higher carbon fatty acids or
solvents) contributed to the missing equivalents calculated.

Methanogenic Step

CH4 Generation

Because the easily degradable carbon sources were completely utilized in the acidogenic
stage (Fig. 2b), the organic acids and ethanol were mainly used for methane production, as
shown in Fig. 2d–h.
The sets containing domestic wastewater produced 10.20 mL of CH4 after the longest lag
phase, as observed for all of the substrates tested (Fig. 2c). The lower CH4 production rate
obtained for the domestic wastewater seems to be related to the organic matter removal and
the degradation rate, which were also the lowest among all of the substrates. Low organic
matter degradation (41.81 %) probably occurred because the compounds were not easily
degraded. The soluble metabolite profile (Fig. 1d) indicates that domestic wastewater had
already undergone the degradation process, as the wastewater traversed a few kilometers
before being sampled from the treatment plant.
 Appl Biochem Biotechnol

Fig. 2 a COD profile, b carbohydrates profile, c volumetric CH4 profile, d sewage soluble metabolites
production (SMP), e glycerol (SMP), f parboiled rice wastewater (SMP), g vinasse (SMP), and h sucrose
(SMP). Dashed lines first-order decay fit, dotted lines Borja first-order kinetic model fit, 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, EtOh ethanol

Regarding the metabolites generated using domestic wastewater (Fig. 2d), the
beginning of the methanogenic step yielded 13.95 mg L−1 of acetic acid, 9.71 mg
L−1 of propionic acid, and lower amounts of succinic, lactic, and valeric acids. The
decrease in the concentration of such acids at t048 h indicates that they were possibly
degraded to support methanogenesis [54].
Based on the dynamics presented in Fig. 2d, CH4 production was probably directly
facilitated by the acetoclastic methanogens that consumed the available acetic acid, includ-
ing that produced from the degradation of propionic acid and ethanol, among others.
Using domestic wastewater with characteristics similar to those of the wastewater
used in the present study (such as a COD of 268.5 mg L−1 and pH of 7.1), Gao et al.
[8] also reported acetic acid to be the major soluble metabolite. The acetic acid
Appl Biochem Biotechnol

concentration was 19.7 mg L−1 for the hydraulic retention time of 8 h in their 6-L
upflow anaerobic fixed-bed reactor. The CH4 production obtained by Gao et al. [8] was
187.8 mL g−1 CODremoved, while the CH4 production was 111.06 mL CH4 g−1 CODremoved in
this study, a relatively comparable result.
According to Fig. 2a, the initial COD of glycerol was the highest among the substrates
tested. Therefore, the glycerol initial COD probably contributed to the volumetric methane
production of 49.9 mL, the highest value found in the current study (Table 4). The methane
yield (180.14 mL CH4 g−1 COD) indicates that the organic matter was well suited for
methanogenesis. Glycerol yielded organic matter conversion into acids at approximately
72 h in the methanogenic stage, with a longer lag phase (86 h) in the CH4 production stage,
mainly because methanogenesis consumes acids rather than producing them.
The study by López et al. [55] showed that obtaining a higher methane yield with
glycerol as the substrate was possible. In their experiments, glycerol derived from biodiesel
manufacturing was anaerobically digested in batch laboratory-scale reactors (35 °C) using
methanogenically active nongranular biomass from a full-scale anaerobic reactor treating
urban wastewater (28,400 mg VSS L−1). They obtained 288 mL CH4 g−1 CODremoved, which
is higher than the value of 252.53 mL CH4 g−1 CODremoved obtained in the present study.
The inoculum and/or reactor configuration used by López et al. [55] may have played a
crucial role in improving CH4 production.
In the glycerol assay (Fig. 2e), a considerable peak in the production of lactic acid
(19.61 mg L−1; t070 h) was observed. However, there was a subsequent decrease that
stabilized later at the end of the assay. The acetic acid concentration (25.00 mg L−1; t060 h)
behaved similarly, and the acetic acid was completely consumed by the end of the experi-
ments. The elevated organic matter content at t048 h (307.38 mg L−1), which was associated
with the production of acetic acid (by-product used by acetoclastic methanogens), may
explain the improved methane production for the glycerol experiments. During the meth-
anogenic stage, there was no generation of propionic acid or ethanol. The propionic acid and
ethanol were completely consumed, which also helps to explain the better performance with
respect to CH4 volumetric production observed in the sets containing glycerol.
The evaluation of the acidogenic stage end products of parboiled rice wastewater as
substrates for methanogenesis revealed a volumetric methane production (26.00 mL) rate of
0.018 L CH4 g VSS−1 h−1, corresponding to the second-highest production rate among the
substrates tested (Table 4). The methanogenic biomass appeared to successfully develop the
ability to generate CH4 from the end products of the acidogenic stage with a short lag time of

Table 4 First-order kinetics for COD removal and first-order kinetic model parameter values for methane
production

Substrate COD Methane

0
CCOD k1' (h−1) R2 X (g L−1 VSS) KG (h−1) Gm (mL) KG R2
(mg L−1) (mL g VSS−1 h−1)

Sewage 220±12 0.0033±0.0011 0.9741 0.573±0.089a – 10.2±0.8a – –

Glycerol 307±20 0.0059±0.0009 0.9944 0.585±0.074 0.0074±0.0011 49.9±1.3 0.013 0.9998
Rice 226±15 0.0050±0.0010 0.9795 0.667±0.110 0.0122±0.0024 26.0±1.2 0.018 0.9889
Vinasse 223±16 0.0049±0.0008 0.9331 0.477±0.142 0.0071±0.0013 49.3±2.3 0.015 0.9711
Sucrose 224±19 0.0043±0.0007 0.9927 0.441±0.088 0.0103±0.0018 19.0±2.8 0.023 0.9938

k1' the first-order reaction constant for COD removal in the methanogenic stage
a
Did not present enough degrees of freedom to be fitted into the model
 Appl Biochem Biotechnol

18 h, similar to vinasse. The dynamics of soluble metabolites presented in Fig. 2f indicates

that lactic acid could have been converted into a mixture of citric, propionic, butyric, and
isovaleric acids. Methanogenesis probably occurred mainly via the acetoclastic pathway
because of the continuous decrease in the lactic acid concentration and the increase in the
acetic, propionic, butyric, and isovaleric acid concentrations at t072 h. The acetic acid
concentration probably remained below the concentration of the other acids because acetic
acid was being simultaneously consumed via the acetoclastic pathway, which kept the acetic
acid concentration very low. The quick initiation of biogas generation seems to be related to
ethanol consumption in the beginning of the methanogenic stage (t060 h). In lower
concentrations, the other acids may have been used to produce methane [56].
Using rice straw for CH4 production in 2.5-L batch reactors (25 °C), Lianhua et al. [4]
achieved a CH4 production rate of 370 mL day−1 at 152 h (6 days). In this study, a production
rate of approximately 23.80 mL CH4 day−1 was obtained after 6 days, a value much lower than
the value obtained by Lianhua et al. [4]. This difference was probably based on the availability
of the carbon source. In the study by Lianhua et al. [4], the organic matter available for
methanization consisted of 800 g of pretreated rice straw with a total solids content
corresponding to 7.5 % by weight. In the current experiments, only 53 mL of parboiled rice
wastewater containing a negligible solids concentration was utilized.
According to Table 4, vinasse yielded a methane production of 255.44 mL CH4 g−1 COD,
the highest among the substrates tested. The absence of the generation of long-chain
compounds, such as succinic, valeric, and propionic acids in the methanogenic stage, could
have supported the elevated yield, as the degradation of long-chain acids requires larger
amounts of energy, with a consequent reduction and delay in CH4 production. Such
possibilities were not observed in the current study (Fig. 2g). In general, only acetic
(14.43 mg L−1) and butyric (8.97 mg L−1) acids were completely consumed until 72 h.
The constant decrease in the propionic acid concentration, as observed at the beginning of
the methanogenic stage, was probably due to its conversion to acetate or H2, both of which
serve as electron donors for methane formation.
Lalov et al. [57] achieved a COD removal efficiency of 69.2 % using a 30-mL fixed-film
reactor (37 °C). In this study, the COD removal efficiency was 50.7 %, close to the 60 %
efficiency obtained by Harada et al. [58] using a thermophilic (55 °C) anaerobic treatment of
alcohol distillery wastewater (cane molasses vinasse) in a UASB (140 L) with an influent
COD of 10 g at the organic loading rate of 2.4 kg COD m−3 and at a pH of 7.3.
The CH4 yield obtained in this study was 385 mL g−1 CODremoved, which is unexpectedly
higher than the 290 mL CH4 g−1 CODremoved reported by Harada et al. [58], who used a
continuous reactor that generally has better performance than that obtained in discontinuous
reactors. The vinasse used by Harada et al. [58] probably had a higher concentration of
poorly degradable or toxic organic matter, thereby affecting the development of the
microorganisms responsible for organic matter degradation and CH4 production. The
vinasse was also diluted tenfold with tap water by Harada et al. [58]. The vinasse was
diluted 55 times in the current work, which likely could have significantly lowered the
concentration of toxic compounds.
The end products of sucrose fermentation yielded only 53.67 mL CH4 g−1 COD with
consequently low volumetric methane production (19.0 mL). According to Table 4, these
values were the lowest among all of the substrates tested, with the exception of domestic
wastewater. The specific methane production was 0.023 L g VSS−1 h−1, mainly because of
0
the elevated methane production rate represented by KG (Table 4).
Despite the elevated CH4 production rate, the lag time in the experiments utilizing
degraded sucrose solution was 57 h, which is longer than the lag time observed using other
 Appl Biochem Biotechnol

Table 5 Electron mass balances (in terms of COD) according to the results from the methanogenic step of the evaluated substrates

Substrate CODcarb CODCH4 CODSMP (mg COD) CODsum CODtotal Missinga electron
(mg COD) (mg COD) (mg COD) (mg COD) equivalents (%)
Hci HMa HSu Hla HFo Hac HPr Isob HBu Isov Val EtOH

Sewage 0.0 8.3 7.7 3.5 7.3 19.3 0.0 14.7 14.7 21.1 21.2 25.9 21.7 0.0 152.3 181.5 16.0
Glycerol 0.0 22.1 7.4 2.9 7.4 16.9 1.5 3.7 9.2 29.2 23.4 29.6 32.9 0.0 148.6 189.3 21.5
Rice wastewater 0.0 25.2 4.4 2.0 5.1 10.2 1.2 0.0 9.6 17.0 16.1 18.7 19.7 0.0 79.2 103.5 23.4
Vinasse 0.0 17.0 3.2 5.0 8.3 10.6 1.3 4.1 15.4 22.1 4.3 26.1 15.26 5.0 104.2 129.7 19.6
Sucrose 0.0 16.3 0.0 0.0 2.7 5.5 1.5 4.5 9.6 27.9 23.5 12.8 13.3 8.5 93.8 127.9 26.6

CODcarb COD of the residual carbohydrates measured, CODCH4 milligrams COD of CH4 evolved, calculated with mol H2 ×32 g COD mol H2−1 ×1,000 mg g−1 , CODSMP COD of
soluble microbial products (SMP), CODsum–CODH2 the sum of the COD of residual carbohydrates and COD of SMP–COD evolved on methane production, CODtotal the
measured COD of the reactor effluent
a
Calculated by [(1 – (CODsum)/(CODtotal))×100 %]
 Appl Biochem Biotechnol

substrates. This trend was probably due to the limiting step involved in the production of
acetate from ethanol generated during the acidogenic stage (Fig. 2h). The other volatile fatty
acids, such as butyric, lactic, and propionic acids, followed a similar pattern, as their peak
concentrations appeared early during the methanogenic stage, followed by a constantly
decreasing profile. This decrease was expected, as the acetogenic step consisted of interme-
diary reactions that produce acetate, i.e., via the oxidation of organic acids, alcohols, or
hydrogen or via the reduction of CO2 [59]. As shown in Fig. 2h, more time may have been
required for the metabolites, which were initially elevated in the acidogenic stage, to become
completely converted to substrates for CH4 production. CH4 production was derived mainly
from the oxidation of ethanol into acetate, with posterior acetoclastic methanogenesis.
According to the study by Jones et al. [59] in batch reactors containing sucrose
(1.71 g L−1; pH 7.2; 37 °C) with consortia of Methanosarcina barkeri (350 mg
L−1), Escherichia coli (90 mg L−1), Acetobacterium woodii (70 mg L−1), Desulfovi-
brio vulgaris (65 mg L−1), and Methanobacterium formicicum (90 mg L−1), 70 % of
the CH4 was generated via acetate.

Electron Mass Balances for the Methanogenic Step of the Experiment

Table 5 presents the electron mass balance of the batch reactors to confirm whether the
experimental data were reasonable. The results presented show that there were small missing
electron equivalents for glycerol, sewage, parboiled rice wastewater, and vinasse used in the
batch reactors.
As expected, the sets containing sucrose had large missing electron equivalents
(26.63 %), which likely originated in the acidogenic stage, as shown in Table 3. It is
possible that, in these sets, ethanol generation could have been accompanied by the
production of other undetectable soluble metabolites. This suggestion is supported by
the observations of Fernandes et al. [16] and Peixoto et al. [7], who found that the
natural fermentation of sucrose used to produce inocula for anaerobic packed-bed
reactors led to the growth of Klebsiella sp. That bacterium produces mainly 2,3-Bu
(OH)2 [18], the metabolite that was impossible to detect and quantify by the analytical
methods employed. The elevated missing electron equivalents detected were possibly
due to the activity of Klebsiella sp.

Potential for Energy Recovery and Organic Matter Removal

According to Table 6, all of the effluents showed potential for energy recovery and organic
matter removal in the two-stage fermentation process, but better performance was achieved
in the case of glycerol and vinasse.
Hydrogen was not detected in the first stage of glycerol fermentation; thus, vinasse
seems more suitable for the process, mainly because the biological production of
hydrogen can be an alternative to methane steam reforming and electrolysis, currently
the most common processes used for hydrogen production [60]. Another reason for
using vinasse is that, in the two-stage fermentation process, it would be possible to
reduce the organic load to 5.24 g COD, which is easily treatable in the conventional
wastewater treatment system. The potential of vinasse for methane production was the
highest among the substrates evaluated (Table 4).
Assuming that all of the vinasse generated in São Paulo State (Brazil) during the 2008–
2009 harvest was submitted to the two-stage process and assuming that the hydrogen and
methane produced were burned in the combined cycle turbines used in modern power plants
Appl Biochem Biotechnol

Table 6 Performance parameters of sequential hydrogen and methane production from each of the assayed
wastewaters

Wastewater Hydrogen Methane Combined (global)

Ymax Energetic COD Ymax Energetic COD Energetic COD

(mL g−1 potential removal (mL g−1 potential removal potential removal
COD) (kJ g−1 (%) COD) (kJ g−1 (%) (kJ g−1 (%)
COD) COD) COD)

Sewage – – 14.83 46.36 1.85 41.92 1.85 51.11

Glycerol – – 12.68 180.14 7.20 61.17 7.20 70.38
Rice 23.90 0.30 26.15 115.55 4.62 54.36 4.92 63.91
wastewater
Vinasse 20.79 0.26 42.72 255.44 10.22 50.77 10.48 74.72

[61], it would be possible to produce 681,942.40 GW h of electricity to power 434,258

houses, considering the average Brazilian power consumption of 157 kW h month−1 [62].

Conclusions

Sewage, glycerol, parboiled rice wastewater, and vinasse showed potential for simultaneous
energy recovery and organic matter removal. Vinasse exhibited the highest potential, along
with the highest energy recovery (10.4 kJ g−1 COD) and organic matter removal (74.7 %). In
the acidogenic stage, vinasse presented the highest COD removal kinetic constant
(0.0142 h−1), hydrogen production rate (0.84 mL−1), and specific hydrogen production rate
(2.95 mL g−1 VSS h−1). Glycerol was the second best substrate in terms of energy recovery
and organic matter removal. Like vinasse, glycerol is also an effluent derived from renew-
able fuel production, and its application in the two-stage fermentation process can be further
increased looking at the sustainability of the process. The kinetic models employed to
describe the biogas production, carbohydrate consumption, and organic matter removal fit
the experimental data quite well, yielding ideal parameters for reactor design and process
development involving simultaneous energy recovery and wastewater treatment.

Acknowledgments The authors wish to thank Murilo Daniel de Mello Innocentini and Tatiane Lotufo Leite
for providing some of the industrial effluents used in this work. This work was funded by the Fundação de
Amparo à Pesquisa do Estado de São Paulo (FAPESP) and the Conselho Nacional de Desenvolvimento
Científico e Tecnológico, Brazil (CNPq).

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