ES2552603B2 - Procedure for the transformation of dried Opuntia ficus-indica cactus cladodes to produce second generation bioethanol - Google Patents

Abstract

Process for the transformation of dried Opuntia ficus-indica cactus cladodes to produce second-generation bioethanol. # The invention is based on the application of a series of mechanical pre-treatment, acid thermochemical pre-treatment, saccharification and alcoholic fermentation techniques. Simultaneous (SSF) and distillation, with the aim of extracting both fermentable sugars from the lignocellulosic matrix of cactus cladodes and then converting them to second generation bioethanol. # The invention allows the valorization of Opdountia cactus cactus cladodes. indicates as a non-food, abundant and unexploited lignocellulosic biomass resource for the development of second generation biofuels as renewable and clean energy, to face the notable depletion of fossil energy resources.

Description

Procedure for the transformation of dried opuntia jicus-indica cactus cladodes to produce second generation bioethanol.

SECTOR OF THE TECHNIQUE

The present invention is part of the development of biofuels from non-food Hgnocellulosic biomass to cope with the depletion of fossil energy resources.

STATE OF THE TECHNIQUE

The notable depletion of fossil energy resources and the cost they entail, as well as the regulatory requirements of sustainable development, are the main arguments in favor of the development of this invention.

Bio · refineries are the main producers of renewable energy producing methane, hydrogen, bioethanol and biodiesel (Lens, 2005). On the other hand, lignocellulosic biornase constitutes a suitable substrate. sure. abundant and with great potential for biorefineries (Kumar et al. 2009).

Green plants produce approximately 170 tons of dry biomass / year where 10 tons / year will be exploited for energy purposes by the 2050 horizon (which represents 15% of total global energy demand) (Lens. 2005). Among the lignocellulosic biomass resources we can mention: agricultural waste and co-products (bagasse of sugarcane. Cotton stalk. Wheat straw, rice straw, wood ... etc.). agro-industrial waste. the compost urban solid waste and non-food energy crops that produce 300 million tons of dry biomass per year (Lens. 2005).

Despite the spectacular development of cactus crops in Tunisia, the exploitation of this species is restricted to the production and marketing of prickly pears. The cladodes that make up the majority of the species in biomass are very little exploited despite their nutrient-rich chemical composition. Opuntia is a succulent plant with tissues that allow water storage, which makes Opuntia the most effective plant for converting water into dry matter compared to other fatty plants. Thus, some Opuntia species produce 26 tons of MSlhectare / year.

5 In the same way, the amount of biomass produced by cladodes and cactus fruits in arid, semi-arid climates as well as irrigated soils has been estimated. The Opuntia ficus-indica species cultivated under optimal conditions, ensures annual biomass production that can exceed 30 tons of MSlhectare / year as shown in Table I (Harndi, 2006).

10 Table 1: The theoretical production of cactus biomass according to bioclimatic conditions (Harndi, 2006).

Regiooes CUma Arid Semi - Arid Irrigation

Size (m x m) Hectare plants

Cladodios Fruits

4x4

2.63 0.79

Plant matter

2 x 2

Biomass (tonnes Hectare) 10.5

3.15

2 xl 5000

21 6.3

On the other hand, the Opuntia species presents the biomass production rate plus

high of invasive plants in Tunisia. Further. biomass production of

1S

Cactus increases even more, to despite the increase atrocious and judicial of the

atmospheric C02 concentrations. In this way, the development of the cactus can

come to curb the greenhouse effect (Florian et aL, 2005). Therefore, the cladodes

of cactus can serve

how support nature for capture and kidnapping from

carbon dioxide.

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Till the date, the patents relating to the production of second bioethanol

generation

to from lignocellulosic biomass they mainly use the

following substrates:

The sugarcane bagasse (US 2010/0330638 Al. Dec. 30, 2010); Agricultural waste: hard and soft wood, forest residues, sludge from the paper industry, industrial and municipal solid waste, different herbaceous plants (straw, corn, sorghum, wheat, barley, rice, soy) (WO 2011 1 137150 Al. Nov. 03, 2011) (WO 20111 137147 Al. Nov. 03, 201 1); Pine, oak and some perennials (WO 2011 1 136616 A2. Nov. 03,2011).

DESCRIPTION OF THE INVENTION

The main objective of the present invention is the transfonation of the dry cactus clades "Opuntia jicus-indica" by applying the tennochemical pre-treatments and enzymatic saccharification to extract the fennentable sugars and thus produce second generation bioethanol by alcoholic fermentation . This invention, on the one hand, exclusively values the resources of available and unexploited lignocellulosic biomass and, on the other hand, contributes to the development of second generation biofuel as a source of clean renewable energy.

The renewable energy sector can benefit from the remarkable advantages of this invention in e! plan of! development of biofuels and thus favor the energy independence of fossil energies. Thus, the remarkable depletion of resources in fossil energies and the requirements dictated for e! Sustainable development are the main arguments in favor of this invention.

The crops of Opuntia spp., Spread throughout all Mediterranean countries, and specifically in Tunisia, represent an important renewable resource for non-food lignocellulosic biomass, which is available and nexploited (in some cases it is used as animal feed).

It should be noted that cactus cladodes are substrates rich in dry organic matter composed mainly of carbohydrates (Nefzaoui, et al, 2002), which justifies their use to produce bioethanol.

S 10

The invention, as detailed below, is based on the application of a series of techniques of mechanical pre-treatment (stage 1), thermochemical acid pre-treatment (stage 2), saccharification and simultaneous alcoholic fennentation (SSF) (stage 3 ) and distillation (step 4), with the objective of extracting at the same time the sugars of the lignocellulosic matrix of cactus cladodes and converting them to second generation bioethanol. Each stage is briefly described below:

Stage 1.-Mechanical pre-treatment of crushing, to reduce the size of the fibers to an average diameter of the particles of I mm. This stage aims to reduce the degree of polymerization of cellulose and lignin and

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increase the contact surface, and therefore, the accessibility of cellulose and hemicellulose to the different reagents.

Step 2.-Pre-thermochemical acid treatment in an inert atmosphere in the presence of N2, at a temperature of 175 oC, a pressure of 5 bar, a concentration of

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H2S04 sulfuric acid of 0.06% (mlv), during a contact time of 75 minutes. This stage aims at hydrolysis of the hemicellulosic fraction of the biomass of the dry cactus cladodes and thus make the cellulosic fraction more accessible to subsequent enzymatic digestion.

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Stage 3.-Simultaneous saccharification and fermentation (SSF) through the use of the enzymatic mixture, with the endocellulase and exocellulase (fJglucosidase) activities as well as the xylanase activities, and inoculating the yeasts Saccharomyces cerevisiae and Pachyso / in tannophi / us ATCC 32691 for convert deaccharides (sucrose) and fermentable monosaccharides into C6 (glucose, fructose ...) and C5 (mainly xylose) from simultaneous enzymatic digestion of the cellulosic fraction of the acid thermochemical pretreatment.

Stage 4.-Distillation (liquid / vapor separation) to increase the alcohol content of the must obtained.

DESCRIPTION OF THE CONTENT OF THE FIGURES

Figure 1.- Represents a detailed diagram of the steps of the process of transfusion of cactus fibers for the production of second generation bioethanol.

Figure 2.- Represents the curve related to the isotenna of adsorption of dry cactus fibers, allowing the study of the behavior of these fibers with respect to water, calculating the maximum amount of water adsorption by cactus fibers.

Figure 3.- Represents the kinetics of saccharification of the 10% cactus fiber suspension in dry matter in the presence of 100 rnglg of the CeIlic®Cetc2 enzyme diluted 10 times.

Figure 4. Photonic micrographs (3X) of the cladodes fibers of raw dried cacti (a) and crushed (b).

Figure 5. Scanning Electron Micrographs, MEB, of the fibers of the dried cactus cladodes (370 X) (e) and their pores (1500 X) (d).

MODE OF CARRYING OUT THE INVENTION The experimental strategy adopted in this invention aims to operate in optimal and favorable conditions, with the application of pre-treatments that favor the hydrolysis of lignocellulosic fractions difficult to biodegrade and less assimilable by a biological treatment process. .

The procedure for the transfonnación of the cladodes of dry cactus Opuntia ficus-indica by means of mechanical and tennochemical acid pre-treatment, as well as saccharification and simultaneous fennentation (SSF), followed by a distillation stage, in order to obtain the Second generation bioethanol is illustrated in Figure 1.

The procedure applied to the samples of dried cactus cactus from the arid biocytics of Tunisia is detailed below.

Essay t. Mechanical pre-treatment and physicochemical and structural characterization of cactus fibers.

The mechanical pre-treatment used consisted of crushing, using a hammer mill type (Leshan Dongchuan Machinery Co. Ud Sichuan, China).

10 The structural study of raw lignocellulosic fibers of plant biomass, previously crushed, was carried out by means of photonic and electronically scanning microscopic observation MEB. Figures 4 and 5 show the fibrous structure of dried cactus cladodes. Electronic rnographs have demonstrated the uniformity of the porous surface of cactus fibers with a

15 pore diameter of approximately 10 Jlm.

The physicochemical characterization of the dry cactus cladodes has allowed to determine the dry matter contained in the residues, the density of the fiber, the volume of inflation of the fibers within the water, as well as the water and oil retention capacity. These characteristics are shown in Table 2.

20 Table 2. Physico-chemical characteristics of cactus cactus.

FIslcD-9ufmicos characteristics

Values

Dry material (%)

96,122 ± 1,482

Ash content (%)

8.568 ± 0.326

Contained in water (%)

3,878 ± 1,482

Density of fiber particles with respect to iso-butanol

2,688 ± 0,056

Volume of swelling of the fibers in the water (mUg)

2,649 ± 0,158

Water retention capacity (g of water! G MS)

2,157 ± O, 769

Oil retention capacity (sunflower oil) (mi from aceitelMS)

4,978 ± 0,117

Protein content (%)

2,155 ± 0,158

The study of the behavior of dried cactus samples with respect to water was carried out by establishing water vapor adsorption isotennes at 20 ° C (Figure 2).

5 The water absorption diagram follows the hydration of the cactus fibers according to the activity of the water in the environment. The maximum water content to fix the cactus fibers is of the order of 15.36% (glloog MS).

The composition in a-cellulose, hemice Julose, lignin. total organic carbon

10 (COT), inorganic carbon (C ing), total carbon (C tot) and total nitrogen (N tot) of dried cactus cladodes are shown in Table 3.

Table 3. Composition in a-cellulose, hemicellulose, Jignin, total organic carbon (COT), inorganic carbon (C ing), total cathode (C tot) and total nitrogen (N tot) of dry cJadodios.

Value Component ('lo)

a-Cellulose

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Hemicellulose

18

Lignin

27

COT

6.206 ± 0.001

e ng

0.451 ± 0.008

e tot

6,705 ± 0,173

N tot

1,878 ± 0,041

In fact, the holocellulose and lignin content of dried cactus cladodes confines the lignocellulosic nature of dried cactus samples.

Test 2. Pre-thermochemical acid treatment of cactus fibers.

S

Once crushed with a hammer mill (Leshan Dongchuan Machinery Co. Ltd Sichuan. China) and thoroughly characterized, the dried cactus fibers were pre-treated by an acidic tennochemical pre-treatment using a pressure reactor (Pan®, 4600 series -4620) of a liter of useful volume, equipped with pressure and temperature control valves; Pressure regulation is done by a pneumatic valve with an auxiliary compressor.

10

The pressure reactor used in this pre-treatment stage is of the 'batch' type. without agitation This reactor was filled to 10% of its total capacity with a 10% fiber suspension in total solids. To generate an inert environment inside the reactor, nitrogen was used at a fixed pressure.

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The conditions used for the acid thermochemical pretreatment of the dried cactus fibers used were of a pressure of 5 bar, 1750C temperature, with the addition of H2S04 sulfuric acid at a concentration of 0.06% and a reaction time of 75 minutes .

The analysis of the composition of the cactus fibers after the tennochemical pre-treatment is presented in Table 4.

Table 4. Analysis of the composition of cactus fibers before and after thermochemical pre-treatment.

Component

Before pre-treatment After pre-trQtQmiento

Total soluble carbon (%)

6,705 11.85

Xylose (%)

0.577 5,350

Glucose(%)

OR 0.360

Fructose ('lo)

OR 0.539

Sucrose (%)

OR 0.107

Total volatile acidity (%)

OR 0.50

Hydroxy-methyl-furfural (%)

OR OR

a-Cellulose (%)

50.00 55.17

Hernicellulose ('lo)

18.00 1.82

Lignin (%)

27.00 15.36

Ashes ('lo)

8.24 7.90

The total soluble carbon content (organic and inorganic) was stopped by an automatic carbon and nitrogen equipment (multi N / e3100 analyzer from Analytic Jena) using a mobile phase of 10% H3P04. The sugar content has been determined by an enzymatic kit. Total volatile acidity (expressed in grams of acetic acid per sample IOOg) has been determined by a Shimadzu GC201O gas chromatograph. The delenninated short chain volatile fatty acids are acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic, caproic and heptanoic. The hydroxy methyl furfural (HMF) content was stopped by chromatography

soda.

Test 3. Enzymatic saccharification of pre-treated cactus fibers.

A study of the enzymatic saccharification of pre-treated dried cactus fibers was then carried out. This enzymatic hydrolysis was performed by preparing suspensions of 10% cactus fibers in dry matter with a concentration of the commercial CelliC®Cetc2 enzymes of 100 mg / g, diluted 10 times at a pH of 4.8 and at a temperature of 50 ± 1 ° C with a stirring speed of 150 rpm by microincubators. The duration of enzymatic saccharification was 72 hours.

CelliC®Cetc2 is an enzymatic preparation of the Novozymes brand, with a total protein concentration of 217 giL. a filter paper activity of 120.5 FPUlrnL and a ~ -glucosidase activity of 2731 U / rnL.

The analysis of sugars by gas chromatography allowed studying the kinetics of enzymatic saccharification, likewise, the glucose content has been determined with an enzymatic technique. Thus, Figure 3 shows in detail the kinetics of enzymatic saccharification of pre-treated cactus fibers.

With this saccharification test, a glucose yield of 19 ~ 1 O% ± 0.530 was achieved. and a residual dry matter J of 5.50% ± 0.36.

The sugar composition of the liquid fractions of the hydrolyzed matter carried out by gas chromatography is presented in Table 5.

Table 5. Composition in sugars released during enzymatic saccharification

of the pre-treated fibers of the dried cactus, arrested by chromatography

soda.

Eatíu hydrolysis, 10% dry matter and 100 mg / g enzyme

EIp.

CelobiOla Loct ... GlueOl XiIos. Ga ....... Arabinosa M'ROIA

24 h pattern

0.093

Pattern 48 b

0.319 0.079 0.082

Pattern 12 b

0.47 0.144

24 hour sample

10,027 0.681 9,237 2,602

Sample 48 b

8,233 8.3 l 8 2,268

Sample 12 h

8.07 0.825 8,936 2,458

· The sugar contents are expressed in gIL. The concentrations are determined after 72 hours of hydrolysis. Test 4. Saccharification and simultaneous fermentation (SSF) of the fibers of

Cactus pre-treated thermochemically in acidic medium with 10 Saccharomyces cerevisiae yeast and the enzyme Cellic®Cetc2.

The objective of this stage is to improve the efficiency of the procedure, particularly with respect to the variable reaction time and the final concentration in ethanol. For this purpose, a suspension of 14% cactus fibers in dry matter prepared with 50 mM sodium acetate buffer at a pH of 4.82 with 3% of

The 0.2% chloramphenicol solution prepared in the acetate buffer to avoid bacterial contamination throughout the reaction.

In the same way, a suspension of the Cellic®Cetc2 enzyme at 100 mglg was added to the SSF medium. diluted 10 times at a rate of 0.70 vlv with respect to the total volume of the reaction. The reaction medium was inoculated at 10% with Saccharomyces cerevisiae yeast from a synthetic liquid culture medium of 20 giL of sugars at pH 4.2. The composition of this culture medium consists of yeast extract (4.00 giL), casein peptone (3.60 giL), (N \ -4), SO, (3.00 giL). MgSO, 7H, O (2.05 giL), KH, PO, (2.00 giL), xylose (5 giL); glucose (10 giL), fructose (2.5 giL), and sucrose (2.5 giL). To favor the growth of Saccharomyces cerevisiae during the SSF, a medium of

S

Synthetic liquid culture concentrated 25 times with a concentration of 0.025 v / v with respect to the total volume. This concentrated culture medium has the following composition: Yeast extract (50 giL), casein peptone (45 giL), (NH, hSO, (37.5 giL), MgSO, 7H, O (25.625 giL), and KH , PO, (25 giL); all reagents were adjusted to a pH of 4.28.

The SSF was developed in a micro-incubator operating at 35 ± 1 ° C with fixed agitation at 150 rpm for 120 hours (5 days).

Temperature of

10

The ethanol production obtained at the end of the SSF process was analyzed by gas chromatography (OC) and using an enzymatic kit (Boehringer Manheim, Cat. No. 176290) that allows calculating the final yield of the conversion of glucose to ethanol. The yield obtained was 75% with respect to the theoretical conversion yield. The final concentration obtained in ethanol has been 0.5 giL.

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Test 5. Simultaneous saccharification and fermentation (SSF) of thermochemically pre-treated cactus fibers in an acid medium by means of a culture of two yeasts (Saccharomyces cerevisiae and Pachysolen tannophilus ATCC 32691) and the enzyme CelliC®Cetc2

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In this test the possibility of converting the different sugars into C6 (glucose and fructose) yene (xi slab) was tested. as well as the disaccharides (sucrose) produced during enzymatic digestion. This objective was achieved through the use of a mixed culture of two yeasts: Saccharomyces cerevisiae and Pachysolen tannophi / us during the SSF.

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To achieve the objective, an experimental protocol was established for the establishment of the SSF similar to that previously described in example 4, on which the following modifications were applied: the reaction medium was inoculated at 10% with the mixed culture of the two yeasts Pachysolen tannophilus and Saccharomyces cerevisiae with a proportions of 46% and 54% respectively from different precultures.

The final concentration of the ethanol produced was stopped by gas chromatography (OC) and with the enzyme kit adapted, this concentration being close to 0.7 giL.

S Test 6. Distillation

The objective of this last distillation stage is to increase the concentration of ethanol in the final product of the fencing. The distillation stage consists of a liquid-vapor separation of the components of the mixture obtained in the previous stage by means of a succession of vaporization and condensation carrying the

10 boiling mixture (or vaporization) of ethanol, whose boiling point at 1 atm is 78.2 oc.

INDUSTRIAL APPLICATION

The industrial sectors that can benefit from this invention are:

15 • Companies operating in the field of biofuels, renewable energy or in general any biotechnology company.

•

Refiners that want to develop a new and clean energy line.

•

All industries that treat ncos waste in fractions

20 lignocellulosic, cellulosic fractions (pastry-paper industry) or polysaccharide fractions (starch industry).

• Any company that collects agricultural, forestry, or urban solid waste, etc ...

Claims (8)

1. Procedure for the transformation of dried Opuntia ficus-indica cactus cactus to produce second generation bioethanol. which comprises performing the following steps: a) Mechanical pre-treatment of fiber crushing, to reduce its size to an average particle size of 1mm. b) Thermochemical acid pre-treatment carried out in an inert atmosphere in the presence of N2 • at a temperature of 175 oC, a pressure of 5 bars, a sulfuric acid concentration of 0.06% (mlv) and during a contact time of 75 minutes e) Simultaneous saccharification and fermentation (SSF) through the use of an enzymatic mixture, with endocellulase and exocellulase (ft-glucosidase) activities as well as xylanase activities, and inoculating Saccharomyces cerevisiae and Pachysolentannophilus ATCC 32691 yeasts to convert disaccharides (sucrose ) and the C6 (glucose. fmetosa ..) and en es (mainly xylose) monosaccharides derived from the simultaneous enzymatic digestion of the eelulosic fraction of the acid thermochemical pretreatment (step b). d) Distillation (liquid / vapor separation) to increase the alcohol content of the must obtained. 2. Process for the transformation of dried Opuntia ficus-indica cactus cladodes to produce second generation bioethanol, according to claim 1, characterized in that the acidic thermochemical pre-treatment is carried out in a full pressure batch reactor without agitation. at 10% of its total capacity with a 10% fiber suspension in dry matter, with the following operating conditions: 5 bar pressure, 175 ° C temperature, using H2S04 sulfuric acid at concentration of 0.06% and a reaction time of 75 minutes in inert atmosphere in the presence of nitrogen gas.

3.

Process for the transformation of dried Opunlia ficus-indica cactus cladodes to produce second generation bioethanol, according to claim 1? characterized in that the concentration of cactus fibers used in the simultaneous saccharification and fermentation stage is 14% in dry matter with the sodium acetate buffer at 50 mm at a pH 4.82 with 3% of a chloramphenicol solution at 0 , 2% prepared in acetate buffer.

Four.

Process for the transformation of the cactus cladodes Opuntia ficus indica dry to produce second generation bioethanol, according to claims number 1 and 3, characterized in that in the simultaneous saccharification and fermentation stage to the cactus fibers a suspension of the enzyme is added Cellic® Cetc2 at 10 mfg, diluted 10 times at a rate of 0.70 v / v with respect to the total volwnen of the reaction.

5.

Procedure for the trnnsfonnación of the cladodes of cactus Opuntia ficus indica dry to produce second generation bioethanol, according to claims 1, 3 and 4, characterized in that the reaction medium is inoculated at 10% with a mixed culture of two yeasts with proportions of 46% and 54%, respectively, of Pachyso / in tannophilus ATCC 32691 and Saccharomyces cerevisiae at a pH of 4.2.

6.

Procedure for the transformation of cactus cactus Opuntia ficU'J indicates dried to produce second generation bioethanol. according to claim 5, characterized in that the composition of the synthetic liquid culture medium containing the yeasts Saccharomyces cerevisiae and Pachysolen tannophi / us ATCe 32691 is of a yeast extract (4.00 YL), casein peptone (3.60 ! YL), (N! -L,), SO. (3.00 YL); MgSO 7H, O (2.05 YL), KH, PO. (2.00! YL), xylose (5 YL), glucose (10! YL), fructose (2.5! YL), and sucrose (2.5! YL).

7.

Procedure for the transformation of the cladodes of cactus OpunOa jicus indicates dried to produce second generation bioethanol, according to claim 6, characterized in that, to favor the growth of S. cerevisiae and P. tannophilus yeasts during the SSF, a medium is added of synthetic liquid culture concentrated 25 times with a concentration of 0.025 v / v with respect to the total volume with the following composition: yeast extract (50! YL), casein peptone (45! YL), (NH, hSO. (37, 5 YL), MgS04 7H, O (25,625 YL), Y KH, PO. (25! YL); the medium is adjusted to a pH of 4.82.

8.

Process for the transformation of the dried Opuntia ficus-indica cactus cladodes to produce second generation bioethanol, according to claims 1 to 7, characterized in that the simultaneous saccharification and fermentation stage is carried out in a stirrer operating at a temperature of 35 ± 1 oC with fixed stirring at 150 rpm for 120 hours (5 days).