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Hydrogen and methane production via a two-stage

processes (H2-SBR D CH4-UASB) using tequila
vinasses
German Buitron*, Gopalakrishnan Kumar, Andres Martinez-Arce,
Gloria Moreno
Laboratory for Research on Advanced Processes for Water Treatment, Instituto de Ingeniera, Unidad Academica
Juriquilla, Universidad Nacional Autonoma de Mexico, Blvd. Juriquilla 3001, Queretaro 76230, Mexico

abstract
Keywords:

The feasibility of producing hydrogen and methane via a two-stage fermentation of tequila

Anaerobic digestion

vinasses was evaluated in sequencing batch (SBR) and up-flow anaerobic sludge blanket

Biogas

(UASB) reactors. Different vinasses concentrations ranging from 500 mg COD/L to 16 g COD/

Hydrogen methane organic matter

L were studied in SBR by using thermally pre-treated anaerobic sludge as inoculum for

removal

hydrogen production. Peak volumetric hydrogen production rate and specific hydrogen

Tequila vinasse

production were attained as 57.4
4.0 mL H2/L-h and 918
63 mL H2/gVSS-d, at the
substrate concentration of 16 g COD/L and 6 h of hydraulic retention time (HRT). Increasing
substrate concentration has no effect on the specific hydrogen production rate. The
fermentation effluent was used for methane production in an UASB reactor. The higher
methane composition in the biogas was achieved as 68% at an influent concentration of
1636 mg COD/L. Peak methane volumetric, specific production rates and yield were
attained as 11.7
0.7 mL CH4/L-h, 7.2
0.4 mL CH4/g COD-h and 257.9
13.8 mL CH4/g COD
at 24 h-HRT and a substrate concentration of 1636 mg COD/L. An overall organic matter
removal (SBR UASB) in this two-stage process of 73e75% was achieved.
Copyright 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
reserved.

Introduction
Recent technological developments have driven the human
society towards unavoidable dependence on the fossil fuel
energy resources, which are drastically being depleted due to
over consumption. This has led the researchers in energy
sector to find alternative energy sources or renewable energy
sources as the main task. Currently, hydrogen gas has gained
the credit of being a solution to the future energy demands
and also bearing the possibilities of socio economic,

technological and environmental benefits, because of its

unique features, such as high energy content, no green house
gases emission and also demonstrated applications in the fuel
cells to produce electricity [1e5].
Tequila industry in Mexico represents one of the largest
economic sectors of the nation. The Tequila Regulatory Commission reported that a production of 200 million liters of tequila in Mexico so far in 2010 [6]. For every liter of tequila
production, generation of 10 L of wastewater has been recorded.
This wastewater causes various environmental problems due to
high organic content, acidity (pH < 3.9) and high salt content [7].

* Corresponding author. Tel.: 52 442 1926165; fax: 52 442 1926185.

E-mail address: GbuitronM@ii.unam.mx (G. Buitron).
http://dx.doi.org/10.1016/j.ijhydene.2014.04.139
0360-3199/Copyright 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Buitron G, et al., Hydrogen and methane production via a two-stage processes (H2-SBR CH4UASB) using tequila vinasses, International Journal of Hydrogen Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014.04.139

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Anaerobic digestion is an effective method of treating

different organic pollutants. This process offers several advantages over aerobic systems like economic and energy
saving, less biological sludge production, fewer nutrients requirements and smaller reactor volumes [8]. Biohydrogen
production from various wastewater and solid organic wastes
have been reported previously, such as from condensed
molasses soluble [9], food industry wastewater [10], rice straw
[10], mushroom waste [11], de-oiled jatropha waste [12] and
water hyacinth [13]. In addition, it has been shown as an
alternative method of treating the wastes meanwhile generating the energy. In this spotlight tequila vinasses could act as
a feasible feed stock for the fermentative energy production.
Buitron and Carvajal [6] studied hydrogen production from
tequila vinasses using initial concentrations up to 5 g chemical oxygen demand (COD)/L. In that study, it was observed
that the amount of biogas and hydrogen production was
affected by the initial concentration fed to the reactor, and the
intensity of this effect was dependent on the HRT and the
temperature utilized.
Generating methane from the effluent of H2 fermentation
which is rich in organic acids could significantly enhance the
total energy value of the process. Besides, it could also reduce
the COD of the effluent to a certain extent. Two-stage fermentation system (H2 CH4) has been recently gained more
attention due to the reduction of toxicity of the waste materials
to very lower extent and also generation of more amount of
energy compared to single stage fermentation [14e19]. Thus, in
this work the co-generation of bio-hydrogen and methane was
evaluated using the wastewater of tequila industry as carbon
source. Additionally, the effect of high substrate concentrations
on organic matter removal was also studied.

using a sequencing batch reactor (SBR-1). Three different

substrate concentrations (soluble COD) were employed: 0.5,
1.0 and 5.0 mg COD/L. As no inhibition was observed, a second
set of experiments (SBR-2) was carried out using a lower HRT
(6 h) and six concentrations of vinasses (soluble COD),
covering a higher range than the previous set: 2, 6, 8, 12, 14 and
16 g COD/L. The schematic diagram of the reactor set-up is
shown in Fig. 1A. The reactor operation and methodologies
were previously reported [6]. In first set of experiments, the
reactor had a reaction volume of 4 L, whereas for the second
one, the volume was 600 mL. The SBR-1 was inoculated with
heat pre-treated inoculum (3.7 gVSS/L). SBR-2 was inoculated
with a glucose-adapted biomass (1.5 gVSS/L) which was harvested at the end of the experiments SBR-1. An automatic
control system maintained the temperature at 35  C and the
pH of 5.5. The nutrient medium supplied was prepared as
described by Mizuno et al. [20]. SBRs were run under each
condition for several days and the reported data represent the
average of at least three representative cycles when the
hydrogen production had been stabilized.

Materials and methods

Selection of hydrogen-producing bacteria and tequila
vinasses
Anaerobic sludge from a brewery wastewater treatment plant
was used as seed inoculum in this study. Heat treatment at
100  C for 24 h was employed to eliminate the hydrogen
consuming methanogens and to enrich the hydrogenproducing microorganisms. After thermal treatment, the
sludge was activated with glucose until a stable hydrogen
production was observed as mentioned by Ref. [6]. The
vinasses used in this study contains the following characteristics: organic matter concentration between 19.8 and 20.9 as
gBOD5/L and between 29.9 and 30.5 as g COD/L; glucose 4.6 g/L;
phenols from 44 to 81 mg/L; sulphates 915 mg/L; NeNH3
110 mg/L; and pH from 3.2 to 4.0. The BOD5/COD ratio was 0.67
indicating that organic matter could easily be biodegraded.

Hydrogen fermentation of tequila vinasses

It has been reported that the hydraulic retention time (HRT)
influenced hydrogen production from tequila vinasses [6].
Thus, two sets of experiments, at two different HRTs, were
performed each set at different the tequila vinasses concentrations. The first experiment was conducted with 18 h-HRT

Fig. 1 e (A) Reactor set-up for hydrogen production (1- SBR

reactor, 2- Bio controller, 3- biogas measuring device, 4Acid and base for pH control, 5-pH electrode, 6-agitator, 7thermometer, 8- thermal jacket for temperature control, 9feedstock tank). (B) Reactor set-up for methane production
(1- UASB reactor, 2- recirculation, 3- biogas measuring
device, 4-Phase separator, 5- influent, 6-water jacket, 7water tank at 35  C, 8-thermostat for temperature control,
9-thermal jacket).

Please cite this article in press as: Buitron G, et al., Hydrogen and methane production via a two-stage processes (H2-SBR CH4UASB) using tequila vinasses, International Journal of Hydrogen Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014.04.139

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Effluent utilization for methane production

The effluent generated in the SBR-1 tests and in the beginning
of the SBR-2 period (2 g COD/L) was utilized for methane
production. The effluent was collected every day, during two
weeks, and was stored at 4  C. The characterization of this
mixed effluent is presented in Table 1. This effluent was used
to feed the Upflow Anaerobic Sludge Blanket Reactor (UASB).
The schematic diagram of the reactor set-up is shown in
Fig. 1B. The reactor had a working volume of 500 mL. At the
bottom of the reactor a funnel was placed to concentrate the
sludge bed and allow expansion of the bed by recycling it.
Anaerobic granular sludge from a brewery wastewater treatment plant (200 mL) was inoculated with a concentration of
12 g/L in terms of VSS. The nutrient solution contained (in mg/
L): NH4Cl (266); KH2PO4 (100); MgCl2$6H2O (60); CaCl2$2H2O (30);
FeCl2$4H2O (20); CoCl2$6H2O (5); MnCl2$4H2O (1); NiCl2$6H2O
(1); ZnCl2 (0.5); H3BO3 (0.5); Na2SeO3 (0.5); CuCl2$2H2O (0.4) and
Na2MoO4$2H2O (0.1) and 3 g/L of sodium bicarbonate as a
buffer to maintain the pH in the range 6.8e7.5 was added
continuously throughout the experiment [21]. Mesophilic
condition (35  C) was maintained in the reactor with an
external heating through a copper pipe with hot water and an
insulating jacket. To determine the effect of substrate concentration in methane production three different values of
initial substrate concentrations: 420, 1085 and 1636 mg COD/L
were evaluated diluting the fermenter effluent with tap water.
The reactor was started with the lower substrate concentration (420 mg COD/L) and it was maintained until a constant
biogas production was obtained for at least five days. There
forward, the concentration has been increased. Two HRT were
tested: 24 h (420, 1085 and 1636 mg COD/L) and 18 h, using only
1636 mg COD/L.

Analytical methods
Total and volatile suspended solids (TSS and VSS), pH and
alkalinity were determined according to the Standard
Methods [22]. Total and soluble COD were determined according to Hach procedure with a spectrophotometer DR 2010.
Dissolved organic carbon (DOC) was determined with a Shimadzu TOC-5050 carbon analyzer. Volatile fatty acids (VFAs)
were analyzed with a high performance liquid chromatography (HP 1100) equipped with an ultraviolet (210 nm) detector
and a 5 mm  150 mm Grace Prevail Organic column. K2HPO4
of 25 mM, with a pH of 2.5, was used as a mobile phase at a
flow rate of 0.6 L/min as mentioned in our previous studies
[4,7]. In both experiments, biogas production was measured in
a cylinder using the water displacement method with a

Table 1 e Characteristics of the effluent of hydrogen

reactor.
Parameter
TSS, mg/L
VSS, mg/L
Total COD, mg/L
Soluble COD, mg/L
TOC, mg/L
Alkalinity, mg CaCO3/L

Value
338
297
2300
1636
416
510

16
14
39
17
16
10

Parameter
pH
Acetate, mg/L
Propionate, mg/L
Butyrate, mg/L
Ethanol, mg/L
Acetone, mg/L

Value
8
71
49
40
7
109

0.5

6

5

6

2

12

saturated NaCl solution at pH 3. The amount of biogas produced was measured by the volume of water displaced in an
inverted measuring cylinder. After each cycle the buffering
capacity of the reactor (given by the alkalinity ratio: intermediate alkalinity/total alkalinity) was determined by titration.

Results and discussion

Effect of vinasses concentration on hydrogen fermentation
Substrate concentration plays an important role in the
hydrogen fermentation as mentioned in many reports
[2,7,23,24], increasing substrate concentration should increase
the production performances in a sequential manner based on
the amount of substrate feed in to the reactor and also available to the microbes. The concentration of the substrate is not
universal, and it varies depend on the substrates. The possible
reason for this discrepancy is the difference in the nature and
carbohydrate content of the substrates employed in the
fermentation reactions. However, it is necessary to find out
the optimal concentration of any substrate, before used in the
continuous reactions. Thus, two sets of experiments (SBR-1
and SBR-2), at two different HRTs, were performed; for each
set the reactor was operated at different the tequila vinasses
concentrations.

Hydrogen production performances of SBR-1

Hydrogen conversion of tequila vinasses by the anaerobic
granular sludge at various substrate concentrations ranging
from 500 to 5000 mg COD/L was evaluated under the HRT of
18 h. The hydrogen production performance is depicted in
Figs. 2 and 3. It could be clearly seen that, increasing substrate
concentration has resulted positively in the volumetric
hydrogen production rate (VHPR). Interestingly, there was no
inhibition occurred at even higher substrate concentration,
which revealed that, it is possible to increase the concentration further, to attain the maximal hydrogen formation. It
could be explained that, the sludge biomass is getting acclimated to the substrate fed, and could efficiently utilize the
feed at its maximal concentration. From Fig. 2, it can be noted
that the peak for VHPR (27.7
0.12 mL H2/L-h) and for the
specific hydrogen production rate (SHPR), in terms of VSS
(177.4 mL H2/gVSS-d), was obtained as at the higher concentration of 5 g COD/L (Fig. 3). The observed hydrogen yield (HY)
for this set of experiments was 190
25 mL H2/g COD. Specific
hydrogen production rate achieved in this study is comparable with the previous studies. Buitron and Carvajal [6], using
tequila vinasses, obtained a VHPR from 14 to 50 mLH2/L-h at
24 h and 12 h of HRT, respectively. Gong et al. [25], working
with a continuous reactor and molasses as substrate, found a
SHPR of 163 and 276 mLH2/gVSS-d, at the organic loading rates
of 3 and 7 g COD/L-d, respectively. In this current work,
maximal specific hydrogen production in terms of COD was
comprised between 4.5 and 5.7 mL H2/g COD-h (Fig. 3). COD
removal for this set of experiments was, in average, 25%. In
order to achieve the maximal production performance, the
substrate concentration has been increased, and it has been
referred as SBR-2, and the results obtained at this phase has
been discussed in the next section.

Please cite this article in press as: Buitron G, et al., Hydrogen and methane production via a two-stage processes (H2-SBR CH4UASB) using tequila vinasses, International Journal of Hydrogen Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014.04.139

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Fig. 2 e Volumetric hydrogen production rate as a function

of the initial concentration.

Hydrogen production performances of SBR-2

Since, there was no inhibition occurred at SBR-1 experiments,
the concentration of the tequila vinasses has been increased
to higher concentrations and the HRT was decreased to 6 h.
The concentration increase was ranged from 2 to 16 g COD/L.
The hydrogen production performance of this high concentration phase is illustrated in Figs. 2 and 3. Here, also
increasing substrate concentration has resulted in the
increased amount of hydrogen formation. The peak VHPR
(57.4
3.9 mLH2/L-h) and SHPR (918 mLH2/gVSS-d) were obtained at the higher concentration of 16 g COD/L employed in
this study. The observed HY was 118
4 mL H2/g COD and the
specific hydrogen production in terms of COD was, in average
3.3 mL H2/g COD-h (Fig. 3). The specific production rate is
comparatively low with a report by Herbert et al. [26], where

Fig. 3 e Specific hydrogen production rates as a function of

the initial concentration.

glucose has been used as feedstock and the rate was achieved
as 4.6 LH2/gVSS-d. The possible reason for the above difference is attributed to the factors such as the nature of the
substrate and the presence of hydrogen-consuming bacteria
in the mixed consortia. Since, glucose is a simple sugar and
monomer, microorganisms could utilize efficiently compared
to tequila vinasses, which is a complex feedstock and bears lot
of toxic substances that could affect the growth of hydrogen
producers.
Maximal HY was obtained in SBR-1 conditions where HRT
was higher than in SBR-2 (18 h versus 6 h). It has been reported
and demonstrated that HRT is key factor in the enrichment of
the hydrogen producing microorganisms [7,27]. In our previous study, HRT of 12 h was proved to be efficient for the
hydrogen production; however, it was achieved at lower
concentration (1 g COD/L). Under SBR-1 conditions, a higher
COD removal was observed (25%) compared with the value
obtained with the SBR-2 condition (18%). No glucose was
found at the effluent of SBR-1 and SBR-2. Considering that all
the glucose was degraded in the fermentation process, and
that glucose represents 16% of the COD of the tequila vinasses,
it is possible to determine that 9% of additional COD, other
than glucose, was degraded in SBR-1, and only 2% in the case
of SBR-2. The main reason for these better performances were
attributed to the longer HRT, which allowed the transformation of recalcitrant material to easily digestable. This
fact also explains the higher HY and SHPR (in terms of COD)
obtained in SBR-1 experiments.

Methanisation of the H2 fermentor effluent and organic

matter removal
Methanisation studies were conducted using the collected
effluent from the hydrogen fermentation reactor. The effluent
contains the VFA generated during hydrogen production and
other residual organic matter. As only 18%e25% of the initial
soluble COD present in the tequila vinasses has been removed
in the H2 fermentation process, the remaining organic matter
could be efficiently digested in the anaerobic digestion process
to generate methane and further decrease the COD concentration of this waste. The collected effluent of the fermentative reactor was fed in to a methanogenic UASB reactor. The
production performance of the methanogenic reactor is

Fig. 4 e Continuous operation of methane production.

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Table 2 e Methane production performances in UASB at various phases.

Phases
I
II
III
IV

HRT

SMPR
(mL CH4/g COD-h)

24
24
24
18

5.7
6.0
7.2
7.4

0.5
0.8
0.4
0.7

shown in Fig. 4 and Table 2. The reactor was run for about 52
days. In this stage three different initial feed concentrations
and two different HRTs (24 h and 18 h) were evaluated. Three
different influent concentrations of soluble COD were used as
400, 1085 and 1636 mg/L. The results showed that there was an
increase in the amount of biogas produced when the organic
loading rate was increased during the HRT of 24 h. The
amount of methane in the biogas varied significantly. The
content of methane in the biogas was observed as 57, 66 and
68% for the concentrations of 400, 1085 and 1636 mg COD/L,
respectively. Maximal methane content was seen at the concentration of 1636 mg COD/L. The production performances
are provided in Table 2. Peak specific methane production rate
(SMPR), volumetric methane production rate (MPR) and
methane yield (MY) were achieved at the high concentration
with 24 h HRT and a substrate concentration of mg COD/L;
however, similar results were attained at HRT 18 h as well. The
values were observed as 11.7
0.7 mL CH4/L-h, 7.2
0.4
mL CH4/g COD-h and 257.9
13.8 mL CH4/g COD for SMPR,
MPR and MY, respectively. As it could be seen from Fig. 4, the
stability was quite better at 24 h HRT comparatively with 18 h
HRT. Decreasing the HRT to 18 h had resulted in the surge of
methane content to 40%. The results achieved in this study
was quite comparable with a study by Koutrouli et al. [28],
where a MP of 8.75 mLCH4/L-h was attained while using olive
pulp waste as substrate. In that study, the concentrations of
acetic, propionic and butyric acids used were 1, 0.7 and 1.3 g/L,
respectively, and these values are much higher than the
concentrations used in this study. A comparative report has
been shown in Table 3, with other types of wastes employed
for two-stage fermentation.

MPR
(mL CH4/L-h)
2.3
6.5
11.7
12.2

0.3
0.9
0.7
1.2

MY
(mL CH4/g COD)

CH4 content
(%)

57
66
68
40

275.0
220.3
257.9
257.6

31.1
29.6
13.8
25.7

The organic matter removal, as COD, was 56%, 65% and

67% for the initial concentrations of 400, 1085 and
1636 mg COD/L, respectively. Maximal COD removal were
observed when the reactor was operated at 24 h-HRT. It has
been shown that methanogens require a minimum concentration of undissociated acetic acid, the true substrate for
acetogenic methanogens, below which they do not function
properly [33]. Thus, as the acetic acid concentration decreases,
the overall performance of the system decreased. In our study,
at the lower COD concentrations, lower amounts of VFA were
obtained, resulting in a decrease of the organic matter
removal efficiency. This trend was also observed for the
methane content in the biogas (Table 2). Using 1636 mgCDO/L
and 18 h-HRT, the removal percentage decreased to 52%. In
this case, the low efficiency can be explained because tequila
vinasses contain some recalcitrant organic matter difficult to
degrade [6]. As the sugars were removed from the first stage,
other recalcitrant components required more time to be processed; thus, a higher HRT is required. It is noteworthy to
mention that no VFAs or very low concentrations were
observed after the methanisation of H2 fermentor effluent via
UASB reactor indicating a good digestion capacity of the
operation. However, still few organic matter was left has to be
considered for further treatment process. Taking into account
the two stage processes (H2CH4), a total COD removal from
73% (SBR-2UASB) to 75% (SBR-1UASB) was achieved. On the
whole, tequila vinasses used for H2 generation via SBR and
CH4 production of the fermented effluent via UASB operations
have resulted in a reasonable organic removal and also
generated high amount of energy, compared to one stage
fermentation process.

Table 3 e Comparison with other two stage fermentation systems used different wastes.
Substrate

Temperature
regime

Food waste

Mesophilic

Paperwaste pulverized
garbage
Household solid waste

Hydrogen production
index

Methane production
index

VS removal (%)

HRT (d)a

Reference

MY: 353.5 mL/gVS,

SMPR: 1.7 mL/gVS-h
MPR: 6.1 m3/m3-d

10e77

Batch

[29]

Thermophilic

HY: 106.4 mL/gVS,

SHPR:14.7 mL/gVS-h
HPR:5.4 m3/m3-d

88

[30]

Mesophilic

HY: 43 mL/gVSadded

MY: 500 mL/gVSadded

86

Organic waste (stillage)

Mesophilic

HY: 69 mL/gVS

MY: 348 mL/gVS

nr

Potato waste

Mesophilic

HPR:119 mL/h

MPR: 187 mL/h

70

Tequila vinasses

Mesophilic

SHPR:38.3 mL/g VSS-h,

VHPR:57 mL/L-h,b

SMPR: 24.3 mL/gVSS-h,

MPR:11.7 mL/L-h,
MY: 257.9 mL/g COD

83

H2-1.2
CH4-6.8
H2-2
CH4-15d
H2-3
CH4-12d
H2-0.25
CH4-1.25
H2-0.75
CH4-1.0

[31]
[32]
[15]
This
study

nr: not reported in the source.

a
Calculated from the source.
b
Calculated from the given values, (conditions: substrate concentration, 16 g COD/L, and HRT 6 h).

Please cite this article in press as: Buitron G, et al., Hydrogen and methane production via a two-stage processes (H2-SBR CH4UASB) using tequila vinasses, International Journal of Hydrogen Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014.04.139

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Conclusions
[11]

In this study, feasibility of utilizing tequila vinasses with high

organic matter concentrations to produce hydrogen via SBR
and the usage of effluent in methane production by UASB
reactor operations have been demonstrated. There was a
direct relationship between the hydrogen production performances and the vinasses concentration. The maximal
methane production was achieved at high COD concentration
and HRT of 24 h. A further reduction in HRT resulted in a lower
methane production. Overall, tequila vinasses used for H2
generation and, CH4 production of the fermented effluent,
have resulted in a reasonable organic removal (73e75%) as
well as in a high amount of gaseous biofuel production,
compared to one stage fermentation process.

[12]

[13]

[14]

[15]

Acknowledgments
The financial support for this project was provided by the
Consejo Nacional de Ciencia y Tecnologia (CONACyT) through
grant 100298. Jaime Perez Trevilla is acknowledged for his
technical assistance.

[16]

[17]

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Please cite this article in press as: Buitron G, et al., Hydrogen and methane production via a two-stage processes (H2-SBR CH4UASB) using tequila vinasses, International Journal of Hydrogen Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014.04.139

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Please cite this article in press as: Buitron G, et al., Hydrogen and methane production via a two-stage processes (H2-SBR CH4UASB) using tequila vinasses, International Journal of Hydrogen Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014.04.139