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Hydrogen and methane production via a two-stageprocesses (H2-SBR D CH4-UASB) using tequilavinasses

German Buitron*, Gopalakrishnan Kumar, Andres Martinez-Arce,Gloria Moreno

Laboratory for Research on Advanced Processes for Water Treatment, Instituto de Ingenierıa, Unidad Academica

Juriquilla, Universidad Nacional Autonoma de Mexico, Blvd. Juriquilla 3001, Queretaro 76230, Mexico

Keywords:

Anaerobic digestion

Biogas

Hydrogen methane organic matter

removal

Tequila vinasse

* Corresponding author. Tel.: þ52 442 192616E-mail address: GbuitronM@ii.unam.mx

Please cite this article in press as: BuitronUASB) using tequila vinasses, Internation

http://dx.doi.org/10.1016/j.ijhydene.2014.04.10360-3199/Copyright ª 2014, Hydrogen Ener

a b s t r a c t

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

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

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

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

hydrogen production. Peak volumetric hydrogen production rate and specific hydrogen

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 þ UASB) in this two-stage process of 73e75% was achieved.

Copyright ª 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,

5; fax: þ52 442 1926185.(G. Buitron).

G, et al., Hydrogen andmal Journal of Hydrogen E

39gy Publications, LLC. Publ

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 Com-

mission reported that a production of 200 million liters of te-

quila in Mexico so far in 2010 [6]. For every liter of tequila

production, generationof 10 L ofwastewater has been recorded.

Thiswastewater causesvariousenvironmental problemsdue to

highorganic content, acidity (pH< 3.9) and high salt content [7].

ethane production via a two-stage processes (H2-SBRþ CH4-nergy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014.04.139

ished by Elsevier Ltd. All rights reserved.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y x x x ( 2 0 1 4 ) 1e72

Anaerobic digestion is an effective method of treating

different organic pollutants. This process offers several ad-

vantages over aerobic systems like economic and energy

saving, less biological sludge production, fewer nutrients re-

quirements 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 gener-

ating 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 chemi-

cal 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 fermen-

tation system (H2 þ CH4) has been recently gained more

attention due to the reduction of toxicity of thewastematerials

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 andmethane was

evaluated using the wastewater of tequila industry as carbon

source.Additionally, the effectofhighsubstrate concentrations

on organic matter removal was also studied.

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

reactor, 2- Bio controller, 3- biogas measuring device, 4-

Acid and base for pH control, 5-pH electrode, 6-agitator, 7-

thermometer, 8- thermal jacket for temperature control, 9-

feedstock 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, 7-

water tank at 35 �C, 8-thermostat for temperature control,

9-thermal jacket).

Materials and methods

Selection of hydrogen-producing bacteria and tequilavinasses

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 hydrogen-

producing 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 character-

istics: 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

110mg/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 HRT’s, were

performed each set at different the tequila vinasses concen-

trations. The first experiment was conducted with 18 h-HRT

Please cite this article in press as: Buitron G, et al., Hydrogen andmUASB) using tequila vinasses, International Journal of Hydrogen

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

substrate concentrations (soluble COD) were employed: 0.5,

1.0 and 5.0mg 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 har-

vested 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.

ethane production via a two-stage processes (H2-SBRþ CH4-Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014.04.139

i n t e rn a t i o n a l j o u rn a l o f h y d r o g e n en e r g y x x x ( 2 0 1 4 ) 1e7 3

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 treat-

ment plant (200 mL) was inoculated with a concentration of

12 g/L in terms of VSS. The nutrient solution contained (inmg/

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 con-

centration 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 concentra-

tion (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. TwoHRTwere

tested: 24 h (420, 1085 and 1636mg 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 ac-

cording to Hach procedure with a spectrophotometer DR 2010.

Dissolved organic carbon (DOC) was determined with a Shi-

madzu TOC-5050 carbon analyzer. Volatile fatty acids (VFAs)

were analyzed with a high performance liquid chromatog-

raphy (HP 1100) equippedwith 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 wasmeasured in

a cylinder using the water displacement method with a

Table 1 e Characteristics of the effluent of hydrogenreactor.

Parameter Value Parameter Value

TSS, mg/L 338 � 16 pH 8 � 0.5

VSS, mg/L 297 � 14 Acetate, mg/L 71 � 6

Total COD, mg/L 2300 � 39 Propionate, mg/L 49 � 5

Soluble COD, mg/L 1636 � 17 Butyrate, mg/L 40 � 6

TOC, mg/L 416 � 16 Ethanol, mg/L 7 � 2

Alkalinity, mg CaCO3/L 510 � 10 Acetone, mg/L 109 � 12

Please cite this article in press as: Buitron G, et al., Hydrogen andmUASB) using tequila vinasses, International Journal of Hydrogen E

saturated NaCl solution at pH 3. The amount of biogas pro-

duced 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: interme-

diate 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 sequentialmanner based on

the amount of substrate feed in to the reactor and also avail-

able to themicrobes. 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 HRT’s, were performed; for each

set the reactor was operated at different the tequila vinasses

concentrations.

Hydrogen production performances of SBR-1Hydrogen 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 concentra-

tion further, to attain the maximal hydrogen formation. It

could be explained that, the sludge biomass is getting accli-

mated 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 concen-

tration 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 compara-

ble 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 276mLH2/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.

ethane production via a two-stage processes (H2-SBRþ CH4-nergy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014.04.139

Fig. 2 e Volumetric hydrogen production rate as a function

of the initial concentration.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y x x x ( 2 0 1 4 ) 1e74

Hydrogen production performances of SBR-2Since, 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 concen-

tration 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 ob-

tained 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.

Please cite this article in press as: Buitron G, et al., Hydrogen andmUASB) using tequila vinasses, International Journal of Hydrogen

glucose has been used as feedstock and the rate was achieved

as 4.6 LH2/gVSS-d. The possible reason for the above differ-

ence 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 previ-

ous 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 CODof 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. Themain reason for these better performances were

attributed to the longer HRT, which allowed the trans-

formation 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 organicmatter 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 concen-

tration of this waste. The collected effluent of the fermenta-

tive 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.

ethane production via a two-stage processes (H2-SBRþ CH4-Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014.04.139

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

Phases HRT SMPR(mL CH4/g COD-h)

MPR(mL CH4/L-h)

MY(mL CH4/g COD)

CH4 content(%)

I 24 5.7 � 0.5 2.3 � 0.3 275.0 � 31.1 57

II 24 6.0 � 0.8 6.5 � 0.9 220.3 � 29.6 66

III 24 7.2 � 0.4 11.7 � 0.7 257.9 � 13.8 68

IV 18 7.4 � 0.7 12.2 � 1.2 257.6 � 25.7 40

i n t e rn a t i o n a l j o u rn a l o f h y d r o g e n en e r g y x x x ( 2 0 1 4 ) 1e7 5

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 1636mg/L. The results showed that therewas 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 con-

centration of 1636 mg COD/L. The production performances

are provided in Table 2. Peak specificmethane 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 resultswere attained at HRT 18 h aswell. 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 usedwere 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.

Table 3 e Comparison with other two stage fermentation syst

Substrate Temperatureregime

Hydrogen productionindex

M

Food waste Mesophilic HY: 106.4 mL/gVS,

SHPR:14.7 mL/gVS-h

M

S

Paperwaste þ pulverized

garbage

Thermophilic HPR:5.4 m3/m3-d M

Household solid waste Mesophilic HY: 43 mL/gVSadded M

Organic waste (stillage) Mesophilic HY: 69 mL/gVS M

Potato waste Mesophilic HPR:119 mL/h M

Tequila vinasses Mesophilic SHPR:38.3 mL/g VSS-h,

VHPR:57 mL/L-h,bS

M

M

nr: not reported in the source.a Calculated from the source.b Calculated from the given values, (conditions: substrate concentration

Please cite this article in press as: Buitron G, et al., Hydrogen andmUASB) using tequila vinasses, International Journal of Hydrogen E

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 concen-

tration 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 systemdecreased. 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 pro-

cessed; 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 (H2þCH4), a total COD removal from

73% (SBR-2þUASB) to 75% (SBR-1þUASB) 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.

ems used different wastes.

ethane productionindex

VS removal (%) HRT (d)a Reference

Y: 353.5 mL/gVS,

MPR: 1.7 mL/gVS-h

10e77 Batch [29]

PR: 6.1 m3/m3-d 88 H2-1.2

CH4-6.8

[30]

Y: 500 mL/gVSadded 86 H2-2

CH4-15d

[31]

Y: 348 mL/gVS nr H2-3

CH4-12d

[32]

PR: 187 mL/h 70 H2-0.25

CH4-1.25

[15]

MPR: 24.3 mL/gVSS-h,

PR:11.7 mL/L-h,

Y: 257.9 mL/g COD

83 H2-0.75

CH4-1.0

This

study

, 16 g COD/L, and HRT ¼ 6 h).

ethane production via a two-stage processes (H2-SBRþ CH4-nergy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014.04.139

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y x x x ( 2 0 1 4 ) 1e76

Conclusions

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 perfor-

mances and the vinasses concentration. The maximal

methane production was achieved at high COD concentration

andHRT 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.

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.

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