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33 (2009) 14 20

Available at www.sciencedirect.com

http://www.elsevier.com/locate/biombioe

Anaerobic digestion of the vinasses from the

fermentation of Agave tequilana Weber to tequila:
The effect of pH, temperature and hydraulic
retention time on the production of hydrogen
and methane
Froylan M. Espinoza-Escalantea,, Carlos Pelayo-Ortza, Jose Navarro-Coronaa,
Yolanda Gonzalez-Garcaa, Andre Boriesb, Humberto Gutierrez-Pulidoc
a

Department of Chemical Engineering, University of Guadalajara, Blvd. M. Garca Barragan No. 1421,
Guadalajara CP 44430, Mexico
b
INRA-Unite Experimentale de Pech-Rouge, 11430 Gruissan, France
c
Department of Mathematics, University of Guadalajara, Blvd. M. Garca Barragan No. 1421,
Guadalajara CP 44430, Mexico

ar t ic l e i n f o

abs tra ct

Article history:

The objective of this work was to study the effect of three operational parameters

Received 19 February 2008

(pH, hydraulic retention time (HRT) and growing temperature) on a semi-continuous

Received in revised form

bioreactor treating Tequilas vinasses by anaerobic digestion (AD). The response was measured

21 March 2008

through four response variables: total reducing sugars (TRS) consumption, VFAs, hydrogen

Accepted 6 April 2008

and methane production. Trials were done according to a factorial design. The experi-

Available online 22 May 2008

mental results were studied through a multiple response optimization (MRO) analysis to find

Keywords:
Anaerobic digestion
Environmental factors optimization
Energy
Agave tequilana weber (var. azul)

1.

single and multiple optimums for the above-mentioned variables. Mathematical models that
can describe the effect of the operational parameters on each response variable were found. In
this study it is shown that hydrogen production is favored at thermophilic growth (55 1C),
operating the reactor at a slight acidic pH range and at the higher HRT in the boundaries of the
experimental region.

Introduction

In Mexico, Tequilas distilleries are one of the most important

agroindustries, and they actually affront real problems
because of their high-strength vinasses; in 2007 284.2 hm3 of
Tequila (Bureau for Regulation of Tequila) [1] were produced,
which represents a 10-fold production of vinasses. Given the
proximal organic load of 60 g dm3 as the chemical oxygen
demand due to remaining organic suspended solids and the
Corresponding author. Fax: +52 33 39425924.

E-mail address: froymario@gmail.com (F.M. Espinoza-Escalante).

0961-9534/$ - see front matter & 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biombioe.2008.04.006

& 2008 Elsevier Ltd. All rights reserved.

dissolved organic compounds and the high generation

volumes reported above, this kind of vinasses could represent
a real pollution problem. However, given the advances on
biological processes to treat wastewaters, this problem can be
seen as an opportunity of energy generation, through
hydrogen and methane production. Tequilas vinasses have
an important content of fructose and glucose, on their
metabolic transformation relies most of the main byproducts
under study.

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It must be considered that every biological process as

anaerobic digestion (AD) is subject to multiple fluctuations
caused by pH, alkalinity, temperature, organic load, concentration of toxic compounds or inhibitors, etc. and
hydraulic retention [2]. The stability of the processes depends on the activity of mixed microflore [3], whose
metabolic behavior can be driven by controlling the environmental factors.
Since pH affects the bacterial growth rate, pH change may
cause drastic shifts in the relative numbers of different
species in a heterogeneous population present in an AD
reactor [4]. The pH is strongly dependent on the buffering
capacity of the processes. The most important buffering
component within the optimal pH for methanogenic archea is
bicarbonate. The archeal community structure is closely
correlated with the VFA concentration, and the pH had an
intense impact on the bacterial community structure [5].
Bioreactors operation at acidic pH range (e.g. 5.5) has been
reported as optimal for hydrogen production [4,6]. It has been
demonstrated that those substrates at pH 5.0 can be
introduced straight to the bioreactor without neutralization
for the AD process. This is considerable, since wastewaters
from the food, drinks and fermentation industries are often
acidic (pH 45), as the case of Tequilas vinasses, owing to the
production of short-chain organic acids in the holding
balancing tanks [7].
Temperature has an effect not only on hydrogen and
methane production but also on the decomposition of organic
materials from which the methanogenic substrates are
produced [8]. Lowering the operational temperature generally
leads to a decrease in the maximum specific growth and
substrate utilization rate [4,9]. In addition, methanogenic
sludge yield has been shown to decrease with increasing
temperature [4]. However, thermophilic treatment also has
some disadvantages. For example, it is not so stable and
produces somewhat low-quality effluent compared with the
mesophilic process. Moreover, thermophilic AD is characterized by more toxicity and susceptible to variations in
operational and environmental conditions, such as temperature fluctuations [10].
With respect to hydraulic retention time (HRT), the effect on
the AD process is controversial, Dinopoulou et al. [11]
reported that high HRTs favors acetate accumulation, meanwhile Hwang et al. [3] suggested an increase in acetate in HRT
near to the washout of the microorganisms. Besides, it has
been described that at the near-washout point, the microbial
activity is maximal [12,13].
The objective of this work was to study the effect of three
bioreactors operational parameters: pH, HRT and growing
temperature. In order to describe the bioreactors behavior
in response to these environmental factors, a factorial design
was applied; the statistical analysis consisted of multiple
response optimization (MRO) techniques to find out the
maximal total reducing sugars (TRS) consumption, VFAs,
hydrogen and methane production rates at once. Single
and multiple optimums for these variables were found with
MRO. The use of this information should depict the biases on
hydrogen or methane production depending on the common
bioreactors control parameters affecting the microbial metabolism.

33 (2009) 14 20

2.

Methods

2.1.

Tequilas stillage

15

Vinasses coming from a Tequila distillery of Jalisco, Mexico,

processing Tequila 100% Agave, were frozen at 20 1C until
their utilization. The Tequilas stillage used in this study has
64 g total COD dm3, 19 g TRS dm3 and 3 g VFAs dm3.

2.2.

Analytical methods

The pH was measured using a potentiometer (Orion 520A+).

For VFAs and TRS analysis, samples were centrifuged at
10,000 rpm for 10 min, a 1 mL supernatant aliquot was taken
into a clean 2 cm3 vial; for VFAs analysis 1 mL of external
standard (2-ethyl butyric acid) was added. Samples were
thoroughly mixed using a vortex and 20 mL of sample was
injected using an autosampler system. VFAs and TRS were
detected and quantified with a Varian HPLC equipment with a
30 cm Amminex 87 HPX column. For VFAs the following
conditions were used: UV dectector at 201 nm wavelength and
sulfuric acid 0.01 N as mobile phase at a flow rate of
0.6 dm3 min1 with column at 45 1C. TRS were detected and
quantified using a refractive index detector, with cell temperature of 40 1C; this last system was coupled to the UV
detector system. Digestion gases were measured using a
PerkinElmer Autosystem XL with a thermal conductivity
detector chromatographer using molesieve 13X column with
nitrogen as carrier gas.

2.3.

Fermentation tests

A 1 dm3 glass vessel was used as the model reactor with a

working volume of 0.6 dm3. A 90% vinasse: 10% inoculum
mixture was added to the reactor and then purged with
gaseous nitrogen for 15 min and then sealed. A tedlar bag was
used for gas capture and a magnetic bar was used for
continuous stirring of the reactor content. Temperature was
controlled with a water bath to 35 or 55 1C; pH was adjusted
twice a day with a saturated solution of NaOH or HCl and HRT
was controlled semi-continuously accordingly to the experimental design matrix (Table 1). For HRT control, the equivalent volume of a continuous reactor was taken out from the
reactor, twice a day. For example, for those reactors operating
at an HRT of 5 d, considering the volume (0.540 dm3) of
vinasses in the reactor, the fifth part (0.108 dm3) of the
inoculums free vinasses was taken out from the reactor
daily, i.e. 94 cm3 every 12 h.

2.4.

Inoculum

A digested sludge (14.16 g VSS L-1 and 1.74 g COD/g VSS) from
a mesophilic reactor from a brewing plant was used as
inoculum.

2.5.

Experimental design

Tequilas vinasses were treated according to a factorial design

[14]. Three independent variables (factors) were selected:

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33 (2009) 14 20

Table 1 Experimental design and TRS consumption (in %), cumulative production of acetic and butyric acids, H2 and CH4
Treatments in coded values

Treatments in real values

Response variables

X1

X2

X3

X1

X2

X3

TRS consumption (%)

VFAs (g)

CH4 (mL)

H2 (mL)

1
0
1
1
0
1
1
0
1
1
0
1
1
0
1
1
0
1

1
1
1
0
0
0
1
1
1
1
1
1
0
0
0
1
1
1

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

4.5
5.5
6.5
4.5
5.5
6.5
4.5
5.5
6.5
4.5
5.5
6.5
4.5
5.5
6.5
4.5
5.5
6.5

1
1
1
3
3
3
5
5
5
1
1
1
3
3
3
5
5
5

35
35
35
35
35
35
35
35
35
55
55
55
55
55
55
55
55
55

65.4
64.5
59.3
76.9
87.4
89.4
95.5
95.8
94.1
21.4
43.6
65.3
92.0
86.0
95.3
89.6
88.1
99.8

11.0
21.3
15.1
12.8
17.8
12.7
17.5
15.2
32.9
21.7
70.1
87.8
130.8
138.4
147.6
142.7
147.0
156.5

ND
ND
ND
ND
ND
ND
ND
ND
2775
ND
ND
ND
299
529
335
375
2009
638

ND
ND
ND
ND
ND
ND
ND
ND
6654
ND
ND
ND
395
1477
847
1007
8307
776

X1 pH, X2 hydraulic retention time (days) and X3 temperature (1C).ND not detected.

three levels of pH (X1), adjusted from 3.5 up to the required

level with concentrated NaOH or HCl, (Table 1); three levels of
HRT (X2) and two levels of Temperature (X3). Specific mathematical models were fitted to estimate the effect of the
treatments on each response variable through the MRO using
the codified levels of each factor. All tests were carried out in
duplication.

unacceptable) then D 0 (that is, the overall product is

unacceptable). It is for these reasons that the geometric
mean, rather than some other function of the dis such as the
arithmetic mean, is used.

2.6.

3.1.
Optimization of total reducing sugars (TRS)
consumption

Computing the multiple response optimization

Multifactor variance analysis (ANOVA), linear regression

models and response surface plots for each variable response
plots were computed using a statistical software program
(Statgraphics Centurion XV). The best model for each
response variable was obtained by the forward stepwise
regression procedure [15]. Then the optimal treatment condition for each variable was obtained using that model.
After doing the analysis for each response, an MRO analysis
[16] was done using Statgraphics Centurion XV for computing
and plotting. For MRO analysis, a desirability function was
considered; it involves transformation of each estimated
response variable (Y^ i ) to a desirability value di, where 0pdip1.
The value of di increases as the desirability of the
corresponding response increases. The individual desirabilities are then combined using the geometric mean:
D d1 Y 1  d2 Y 2      dk Y k 1=k

(1)

with k denoting the number of responses. This single value of

D gives the overall assessment of the desirability of the
combined response levels. Clearly the range of D will fall in
the interval [0, 1] and D will increase as the balance of the
properties becomes more favorable. D also has the property
that if any di 0 (that is, if one of the response variables is

3.

Results and discussion

The experimental matrix shown in Table 1 and the respective

TRS consumption rates were subject to multiple regression
and forward stepwise procedures to obtain the equation
describing this parameter as a function of pH, HRT and
temperature:
TRS 87:82 5:20 pH 20:28 HRT  14:31 HRT2 4:39 pH T
4:26 HRT T  5:57 HRT2 T  4:8 pH HRT T
2

(2)

The determination coefficient (R ) of 0.855 and p-value of

0.0000 suggest a good fitting of Eq. (2) to describe TRS
consumption as a function of pH, HRT and temperature at
95% of confidence. The constant in the model implies the
consumption when the values of every variable are set to 0.0,
which is pH 5.5, HRT 3 d and temperature at 45 1C. It can be
observed that the most important factor for this response
variable is HRT, this being more favorable than those values
approaching +1 (5 d), with an optimal value at 0.95 (4.9 d). pH
has linear behavior with a positive impact on consumption as
its level is increased from 1 (4.5) to +1 (6.5), having an
optimal value at 0.91 (pH 6.4). Temperature by its side is a cofactor, it can potentiate the influence of any combination of
pH and/or HRT; the optimal value for temperature was found

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33 (2009) 14 20

T=-1.0

TRS

109

TRS

99
89
79
69
59

-1

-0.6

-0.2
pH

0.2

0.6

0.6
0.2
-0.2
T
HR
-1 -0.6

48.0
54.0
60.0
66.0
72.0
78.0
84.0
90.0
96.0
102.0

Fig. 1 Contour plot for TRS consumption given the possible combinations (in codified levels) of two operational parameters:
pH and HRT when growing temperature level is set to 1.0.

Fig. 2 Contour plot for VFAs production given the possible combinations (in codified levels) of two operational parameters:
HRT and the growing temperature when pH level is set to 1.0.

to be 0.95 (35.5 1C). The maximal consumption rate achieved

was 100%, Fig. 1.

3.2.

Optimization of volatile fatty acids production

The results of the mixed experimental design for cumulative

VFA production are shown in Table 1. In order to evaluate the
effect of the three parameters on VFAs, acetic+butyric acids,
propionic acid was not detected (in g dm3), a mathematical
model was fitted as described above; the following equation
was obtained:
VFA 66:59 23:74 HRT 62:26 T 20:70 HRT T  19:53 HRT2 T
(3)
2

The determination coefficient R was 0.834. Eq. (3) is an

appropriate representation of VFA yield at 95% of confidence
given its p-value of 0.0000. From this model it can be assumed
that the three factors have an important effect on VFA
production, and given all the positive coefficients, high values
of each factor imply a higher accumulation of VFAs. It can be
observed that temperature, because of its high-value coefficient compared to the other terms in Eq. (3), and given its
p-value (less than 0.0001), is the most important factor. HRT by
its own has a positive impact on VFAs accumulation when it
is set to positive values (in codified levels, Table 1).

According to a single optimization analysis, the maximal

accumulation of VFAs is achieved when working at the
highest value of HRT (5 d) and temperature (55 1C), Fig. 2, but
a low level of pH (4.5). In Table 1 it can be observed that at
mesophilic temperature (35 1C) there were lower VFA accumulation rates than in those treatments at thermophilic
temperature (55 1C).

3.3.

Optimization of methane production

In the same way as other response variables, methane

production was subject to a multiple regression analysis
and forward stepwise procedure, the resultant equation was
CH4 256:22 pH 483:06 HRT 483:06 HRT2
379:76 pH HRT  206:20 pH T  313:86 pH HRT T

(4)

The corresponding determination coefficient was 0.692, and

the p-value at 95% of confidence was less than 0.0000, with all
terms being statistically significant because of lower p-values
than a, with a of 0.06. According to the optimization analysis
for methane production, the highest cumulative production
that can be achieved in the experimental region studied is
obtained when pH and HRT values are set to 1.0 (6.5 and 5 d,
respectively) and temperature is set at 1 (35 1C), this result is
only true for the experimental region; as shown in Fig. 4,

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BIOMASS AND BIOENERGY

higher methane production levels can be expected when

working at longer HRT.
When analyzing the experimental results shown in Table 1,
in those treatments at 35 1C, it was possible to detect the
presence of methane only in one case; meanwhile, it was
present in six cases when reactors were operated at 55 1C. The
explanation of these results will be discussed on the basis of
three arguments:
(a) it could be suggested that even when the bioreactor is
operated at long HRT in order to favors methanes
production, values of pH below 6.5 would not favor the
production of this gas. This last result has already been
presented by many other authors, one of the most
conclusive could be that of Hulshoff Pol [17], who reported
that methanogenic activity is adversely affected at pH
values below 6.0 and above 8.0; and
(b) temperature has an important role on production of
methane. It has been shown that at thermophilic temperature the HRT shortens from 5 to 3 days for methane
generation, which could be a consequence of the higher
rates of free energy available to complete the methanogenic reactions, it could be attributed to the slow-growing
methanogens (e.g. genera Methanosaetaceae) able to survive at these temperatures, which in consequence explains the small detected quantities of this gas.

3.4.

Optimization of biohydrogen production

An important objective of this study was to determine the

effect of operational parameters which improve hydrogen
production. The experimental results of the H2 production
(cm3) are shown in Table 1. After applying the multiple
regression analysis and the forward stepwise procedure, the
final model, Eq. (5), was obtained:
H2 572:83 pH 1395:24 HRT 1630:63 T 802:77 pH HRT
1395:24 HRT2  860:61 pH HRT T  1933:0 pH2 T

(5)

The corresponding determination coefficient was 0.668,

statistically significant at 90%, with all terms of p-values below

33 (2009) 14 20

a with a of 0.10. According to ANOVA, the three single factors

have an important effect on hydrogen production. The
optimization analysis showed that the highest cumulative
production that can be achieved in the experimental region
studied is obtained when pH is set to 0.0 (pH 5.5) and HRT
and temperature are set at +1 (5 d and 55 1C respectively),
Fig. 3.
A difference in growing temperature is observed between
our results and those previously reported [4,6], where the
optimal temperature was approximately 35 1C, whilst we
found that the optimal condition is 55 1C. It is necessary
to emphasize, however, that the last two works were made
into a batch bioreactor studying a temperature range from 35
to 45 1C, and in this work a semi-continuous reactor and a
wider range (3555 1C) was studied; this could explain
the differences in our results with respect to growing
temperature.
With respect to HRT, the results presented in Table 1
suggest a more pronounced effect of pH than HRT for
hydrogen production. Noteworthy, at 35 1C the generation of
this gas is favored at pH 6.5, because the only detectable
quantity of hydrogen was observed at this value when
working at HRT of 5 d, whilst at 55 1C the most favorable pH
was 5.5, also at HRT of 5 d. At this point, the most important
factor seems to be pH, stating that HRT, as with methane
production, must be 5 d or higher. And since pH is a definitive
environmental factor to determine the microbial populations
at any given temperature, differences on hydrogen generation
quantities can be attributed to the predominant populations
in the reactor at each operational condition. This could
explain why at thermophilic temperature HRT as low as 3 d
is suitable for hydrogen production, because of fast-growing
bacteria.

3.5.

Multiple response optimization analysis

The MRO was applied to determine the global optimum

treatment when optimizing hydrogen and methane production; TRS and VFAs were also considered to obtain a general
metabolic profile. With these restrictions was transformed

Fig. 3 Contour plot for hydrogen production given the possible combinations (in codified levels) of two operational
parameters: HRT and growing temperature when the pH level is set to 0.0.

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33 (2009) 14 20

BIOMASS AND BIOENERGY

Fig. 4 Contour plot for methane production given the possible combinations (in codified levels) of two operational
parameters: pH and HRT when the growing temperature factor is set to 1.0.

HRT=1.0

Desirability

Desirability

1
0.8
0.6
0.4
0.2
0

0.2

-1

-0.6

-0.2

0.2

0.6

pH

-1

-0.2
-0.6
T

1
0.6

0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0

Fig. 5 Contour plot for global desirability given the possible combinations (in codified levels) of two operational parameters:
pH and growing temperature (T) when the HRT factor is set to +1.

each estimated response variable from Eqs. (2) to (4) using

Eq. (1). Two additional analyses were done, considering
hydrogen or methane as the solely gas to be optimized;
however, the final results of both analysis were too similar to
the global optimization analysis referred above, thus only this
last is considered.
According to the optimization results, it was observed that
the higher desirability value, 0.850, is achieved with the
following combination of treatments in codified levels:
X1 0.30, X2 1.0 and X3 1.0; this is, the operational
parameters must be set to pH 5.8, HRT 5 d and growing
temperature 55 1C, Fig. 5. These treatments contribute to a
94% TRS consumption, accumulation of 154 g VFAs dm3, and
a cumulative hydrogen and methane production of 5600 and
1230 cm3, respectively.
Not surprisingly, it has been reported that higher product
concentration and microbial growth rates are consequences
of high operational temperatures because of a less pronounced product inhibition [11,18]. With respect to the HRT,
as described above, longer HRT will result in high metabolic
rates due to a prolonged contact between substrates and
microorganisms; moreover, final product consideration must
be taken into account; a depleted hydrogen production rate
could be observed by the action of the homoacetogens, for
example.

4.

Conclusion

It was observed that pH, HRT and growing temperature have a

pronounced impact on the microbial metabolic behavior. According to this study, the 55 1C temperature is optimal for hydrogen
and mesophilic temperature (35 1C) for methane production; an
acidic range must be considered in order to favor the AD process.
It must be considered for further studies, the introduction of more
operational factors, such as alkalinity and organic load, in order to
obtain a clearer insight into the microbial behavior. However, it
was demonstrated that those effluents from food, drinks and
fermentation industries, such as Tequilas vinasses, can be
effectively treated by AD processes generating biogas as the
byproduct of the treatment; given their physico-chemical characteristics, only minor adjustments must be applied to the
effluent as well as in the operative process. In the case of
Tequilas vinasses, the pH must be adjusted from 3.5 (the original
pH) to 5.8 and a small cooling treatment should be considered,
which implies an economical advantage for the process.

Acknowledgments
The vinasses supply by Tequila Herraduras is greatly
appreciated. We acknowledge the technical support of the

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BIOMASS AND BIOENERGY

Universidad de Occidente Unit Los Mochis through the head

of the DES-UOCAINTE, Ms.Sc. Bertha Alicia Perez Salazar. The
gratitude of one of the main author is extended to CONACyT
for the Ph.D. scholarship 184794.
R E F E R E N C E S

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