UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO FACULTAD … · UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO...

UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO

FACULTAD DE QUÍMICA

VI Seminario Latinoamericano y del Caribe en Biocombustibles, Cuernavaca Morelos

Pre-treatment and enzymatic hydrolysis of blue agave bagasse

(BABETHANOL) [Pre-tratamiento e Hidrólisis Enzimática del

residuo de agave azul ("BABETHANOL")]

Oscar Hernández-Meléndez1, VirginieVandenbossche2, Julien Brault2, Carmina Montiel-

Pacheco1, Eduardo Vivaldo-Lima1, Martín Hernández-Luna1, Eduardo Barzana1,Luc Rigal2,

Gérard Vilarem2

1Facultad de Química, Universidad Nacional Autónoma de

México, 04510México D. F., México.

2Université de Toulouse, INPT, ENSIACET, Laboratoire de

Chimie Agro-Industrielle, 4 Allée Emile Monso B.P.44362,

31030 Toulouse, France.




Agave tequilana Weber is a plant cultivated in Mexico whose stem plus basal part of the

leaves (termed “piña”, because it resembles a pineapple) is used for the production of

the distilled alcoholic beverage known as “Tequila”.




When the plants are about 6-8 years old, the “piña” is subjected to the following processes:

1) cooking in brick ovens conducted by steam injection for around 36-48 hrs, obtaining

temperatures of 100ºC. After that period, the steam is shut off and the “piña” is left in the

oven for further two days to complete the cooking process.




2) autoclave cooking operation (after “piña” washing with steam for 1 hr for waxes removal

from the agave cuticle) wherein agave is contacted with steam for additional 6 hrs to obtain a

pressure of 1.2 kg/cm2 and a temperature of 121ºC. The agave remains in the autoclave for

other 6 hrs without additional steam; cooking is carried out slowly in the remaining heat.




After stages 1) or 2), modern systems in which cooked agave is passed through a cutter

to be shredded (except in factories that did this operation before cooking of the “piña”,

namely, inuline extraction) with a combination of milling and water extraction, the sugars

are extracted to start the fermentation stage.




Factories that do this operation before cooking of the “piña”, namely, inuline extraction.




The disposal of the fibers from agave constitutes a serious waste problem. The Tequila

industry has adopted policies, practices and strategies to achieve sustainability that called

for a balance between the economic and environmental domains. The problem in the

Tequila industry has been the continuous growth, due mainly to the increasing worldwide

demand of Tequila.




The mexican consumption of Agave tequilana Weber for tequila production in 2009 was

estimated around 924700 ton. Approximately 11% (dry weight) corresponds to blue agave

bagasse residue (BAB).The chemical value of these 101717 ton of BAB has not been

adequately exploited [Consejo Regulador del Tequila, CRT].

BAB Chemical composition:

Inuline: 24.0%

Fibres: 11.0%

Fibres:

Cellulose: 43.0%

Hemicelluloses: 17.0%

Lignin: 17.0%

Mineral matter: 4.1%

Others: 18.9%




Bagasses from Agave atrovirens as well as Agave tequilana are suitable candidates for

conversion to ethanol. In recent publications, samples were pretreated or partially hydrolyzed

(the hemicellulose carbohydrate) with either mineral acids (HCl), steam explosion, or by a

combined pretreatment composed of alkaline delignification followed by enzymatic hydrolysis.

“Comparative hydrolysis and fermentation of sugarcane and agave bagasse”. Hernández-

Salas, JM., Villa-Ramírez, MS., Veloz-Rendón, JS., Rivera-Hernández, KN., González-César,

RA., Plascencia-Espinosa, MA., Trejo-Estrada, SR. 2009. Bioresource Technology 100.

1238-1245.

“Efficient chemical and enzymatic saccharification of the lignocellulosic residue from Agave

tequilana bagasse to produce ethanol by Pichia caribbica”. Saucedo-Luna, J., Castro-

Montoya, AJ., Martinez-Pacheco, MM., Sosa-Aguirre, CR., Campos-Garcia, J. 2010. Journal

of Industrial Microbiology and Biotechnology 38. 25-732.

“Bioethanol and xylitol production from different lignocellulosic hydrolysates by sequential

fermentation”. Arrizon, J., Mateos, JC., Sandoval, G., Aguilar, B., Solis, J., Aguilar, MG. 2012.

Journal of Food Process Engineering 35. 437-454.




N'Diaye et al., carried out a significant extraction of the hemicellulose/lignin moieties for Populus

tremuloides (PT) substrate in a BC-45 extruder (CLEXTRAL),consisting of 7 modules, with an

L/D = 31.1. Chemical characterization reported by N'Diaye et al., as well as for BAB, in our case,

following the same characterization methodology is presented.

N'Diaye et al., hydrolyzed PT with aqueous NaOH (3-7%), at 50 - 90ºC. Around 82% of the

potential hemicelluloses were continuously extracted. Lignin extraction yield was not

reported. The major advantage of the twin-crew extruder is that extraction and liquid/solid

separation can be conducted simultaneously in a very efficient manner.

Fibers

(Mesh)

Cellulose Hemicelluloses Lignin Mineral

fraction

PT 6mm 44.92% 36.8% 17.67

%

0.19%

BAB 6mm 43% 17% 17% 4.5%

N'Diaye, S., Rigal, L., Larocque, P., Vidal, PF. 1996.Extraction of hemicelluloses from poplar, populus tremuloides, using

an extruder-type twin-screw reactor: a feasibility study. Bioresource Technology 57, 61-67.




N'Diaye, S., Rigal, L., Larocque, P., Vidal, PF. 1996.Extraction of hemicelluloses from poplar, populus tremuloides, using

an extruder-type twin-screw reactor: a feasibility study. Bioresource Technology 57, 61-67.




Production d’éthanol à partir de biomasse lignocellulosique. 1999. Ogier, JC., Ballerini, D., Leygue,

JP., Rigal, L., Pourquié, J. Oil & Gas Science and Technology - Revue de l’IFP. 54, 1, 67-94.




Methodology

The following 3 factors were chosen for our study:

1) NaOH Concentration (%W)

2) Temperature (ºC)

3) Ratio of total liquid to total solid [L/S] (kg/h)/(kg/h)

The influence of the factors for deconstruction/decrystallization was studied by means of

an experimental design, using Doelhert´s matrix.

After neutralization of the solid extrudates, the cellulose, hemicellulose and lignin content

of the extruded BAB fibers was determined by using NDF-ADF methodology.

The raw BAB fibers were characterized for cellulose, hemicellulose and lignin content,

organic matter ratio in filtrate versus organic matter in entrance (MOfiltrat/MOentrance),

as well as purity of cellulose, expressed as the ratio of cellulose on the sum of cellulose,

hemicelluloses and lignin in extrudate [C/(C+H+L)].




Enzymatic hydrolysis of BAB alkaline extrudates

In order to evaluate the alkaline pre-treatment process, extruded BAB samples were

subjected to enzymatic saccharification (experiments carried out by duplicate).

Twenty milliliters of sodium citrate buffer 0.05 M were placed in Erlenmeyer flasks

equipped with coil-cover stoppers and mixed with 0.5 g of extruded BAB.

Sodium azide was added to avoid contamination (0.005 g).

The Erlenmeyer flasks were incubated at 50ºC by 30 minutes in an orbital shaker

incubator.

4 mixtures from commercial cocktails were prepared. All enzymes are cellulose based.

These will be identified here as:

Boosted Enzyme 1

Boosted Enzyme 2

Enzyme 3,and

Boosted Enzyme 4.




After 30 minutes of incubation, 17.5 FPU of cellulose-based cocktail/g of substrate were

added to the Erlenmeyer flasks.

Aliquots were taken after 48 hours of reaction and the samples were refrigerated (-72ºC)

before sugar analysis.

The samples were centrifuged and standards of glucose were prepared for construction

of a calibration curve.

Total reducing sugars were quantified by DNS method by reading of the absorbance at

540 nm by VIS spectrophotometer.

Average results of saccharification experiments are reported as yield from potentially

reducing sugars released if all the carbohydrates present in the BAB were hydrolyzed by

diluted sulphuric acid.




Results and Discussion

Experiment

NaOH/BAB

ratio (dry

basis)*100%

Temperature

(°C)

Total

liquid/Total

solid

Aqueous

NaOH

concentration

Mass

flow of

aqueous

NaOH

SME

(Wh/kg)

Cellulose

content

(%)

Hemicelluloses

content

(%)

Lignin

content

(%)

[%W [(kg/hr)/(kg/hr)] [%W (kg/hr)

1 51.6 50 11.3 15 14 8.6 53 11 16

2 17.2 100 16.9 1.97 12.66 16.5 43 14 14

4 64 57 14 5 11.4 18.4 35 14 17

5 49 57 12.5 10 11.64 16.6 40 14 13

6 22 143 13.5 5 11.49 18.5 50 14 14

7 19 114 7.7 10 10 20.4 46 13 15

8 13 86 7.6 5 10.02 31.3 48 12 17

9 26 86 7 10 11.64 25.0 46 17 9

10 14 129 5.2 8 9.9 17.3 43 10 14

11 9 114 8.3 5 11.49 11.2 50 14 13

12 11 71 6.8 8 9.38 18.7 45 11 13

13 34 100 17.7 8 8.4 16.1 46 12 15




Enzymatic hydrolysis of the carbohydrates present in BAB was significantly

enhanced by the NaOH pre-treatment and sulphuric acid neutralization,

compared to the performance of raw BAB. Significant differences were observed

among the 4 enzymatic cocktails. Two of them provided similar best results.

0

10

20

30

40

50

60

70

80

90

100

BAB

raw

BAB

ext 1

BAB

ext 2

BAB

ext 3

BAB

ext 4

BAB

ext 5

BAB

ext 6

BAB

ext 7

BAB

ext 8

BAB

ext 9

BAB

ext 10

BAB

ext 11

BAB

ext 12

BAB

ext 13

To

tal

red

uc

ing

su

ga

rs o

f p

ote

nti

al

(%)

Samples 4, 5, 9,13

TRS (85-95%) for

NaOH/BAB ratios (26-

64%)

Samples 2, 6, 7

TRS (70-80%) for

NaOH/BAB ratios (14-

22%)

Samples 8, 11,12

TRS (60-70%) for

NaOH/BAB ratios (9-

13%)




Conclusions

In this first exploratory study with BAB, the most important factor for

production of total reducing sugars was the NaOH/BAB ratio. However, it

is not wise to work at such high NaOH/BAB ratios, from an economic point

of view, since this implies recovery of the aqueous NaOH dissolutions.

We proposed to work in the future with a different extruder arrangement in

order to reduce water consumption.

UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO FACULTAD … · UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO...

Documents

Transcript of UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO FACULTAD … · UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO...