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    • 专利摘要:
    Procedure for the transformation of the cactus clans of opuntia ficus-indica cactus to produce second generation bioethanol. The invention is based on the application of a series of techniques of mechanical pre-treatment, acid thermochemical pre-treatment, saccharification and simultaneous alcoholic fermentation (ssf) and distillation, with the aim of extracting the fermentable sugars from the lignocellulosic matrix at the same time of the cactus cladodes and then convert them to second generation bioethanol. The invention allows the valuation of the cactus cladodes opuntia ficus-indica as a resource of lignocellulosic non-food biomass, abundant and not exploited, for the development of second generation biofuels as renewable and clean energy, to face the remarkable depletion of resources in fossil energies. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2552603A1
    申请号:ES201400440
    申请日:2014-05-30
    公开日:2015-11-30
    发明作者:Kaouther ZAAFOURI；Moktar Hamdi；Luis Alberto FERNÁNDEZ GÜELFO；Carlos José Álvarez Gallego；Luis Isidoro Romero García；Sebastián SÁNCHEZ VILLASCLARAS；Kaoutar ABOUDI；Juana Fernández Rodríguez
    申请人:Universidad de Cadiz；Universidad de Jaen；
    IPC主号:
    专利说明:

    Procedure for the transformation of dried Opuntia ficus-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 lignocellulosic 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 biomass constitutes an adequate, safe, abundant substrate 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 (sugarcane bagasse, cotton stalk, wheat straw, rice straw, wood ... etc.), waste agro-industrial, 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 c1adodios that constitute the majority of the species in biomass are very little exploited despite its chemical composition
    rich in nutrients 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.
    S 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 in optimal conditions, ensures an annual biomass production that can exceed 30 tons of MSlhectare / year as shown in Table 1 (Hamdi, 2006).
    10 Table 1: The theoretical production of bioclimatic conditions biomass (Hamdi, 2006).Cactusinfunctionof the
    Regions
    Climate Arid Semi-arid Irrigation
    Size (m x m)
    Plant ~ Ectare
    ss
    4x4
    2.63 0.79
    Plant matter
    2 x 2
    Biomass (tons / hectare) 10.5
    3.15 2x1 5000
    6.3
    On the other hand, the Opuntia species presents the biomass production rate plus
    high of invasive plants in Tunisia. In addition, biomass production of
    lS Cactusincreases even more,todespite theincreaseatrociousand harmful of
    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 hownatural support for the capture and sequestration of
    carbon dioxide.
    twenty To date, patents related to the production of second bioethanol
    generation from lignocellulosic biomass mainly employ the
    following substrates:
    The bagasse of sugarcane (US 2010/0330638 Al .;andthe paper industry, industrial and municipal solid waste,different herbaceous plants (straw, corn, sorghum, wheat, barley,rice, soy) (WO 2011/137150 Al. /137147 Al .;Pine, oak and some perennials (WO 2011/136616 A2.Nov. 03,2011).
    DESCRIPTION OF THE INVENTION
    The main objective of the present invention is the transformation of the dry cactus claws "Opuntia ficus-indica" by applying thermochemical pre-treatments and enzymatic saccharification to extract fermentable sugars and thus produce second generation bioethanol by alcoholic fermentation . This invention allows, on the one hand, the use of available and unexploited lignocellulosic biomass resources exclusively, and on the other hand, it contributes to the development of second generation biofuel as a source of clean renewable energy.
    The renewable energy sector may benefit from the remarkable advantages of this invention in the biofuel development plan and thus favor the energy independence of fossil fuels. Thus, the notable depletion of resources in fossil energies and the requirements dictated for 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 of non-food lignocellulosic biomass, which is available and unexploited (in some cases it is used as animal feed).
    It should be noted that cactus cladodes are substrates rich in organic matter
    seca composed mainly of carbohydrate s (Nefzaoui, et al., 2002), which justifies its use to produce bioethanol.
    The invention, as detailed below, is based on the application of a series of mechanical pre-treatment techniques (stage 1), thermochemical acid pre-treatment (stage 2), simultaneous alcoholic fermentation and saccharification (SSF) (stage 3 ) and distillation (step 4), in order to extract both fermentable sugars from the lignocellulosic matrix of cactus cladodes and convert 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 1 mm. This step aims to reduce the degree of polymerization of cellulose and lignin and increase the contact surface, and therefore, the accessibility of cellulose and hemicellulose to the different reagents.
    Stage 2.-Pre-treatment thermochemical acid in an inert atmosphere in the presence of N2, at a temperature of 175 oC, a pressure of 5 bar, a concentration of sulfuric acid H2S04 of 0.06% (m / v), for a time 75 minute contact 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.
    Stage 3.-Simultaneous saccharification and fermentation (SSF) through the use of an enzymatic mixture, with the endocellulase and exocellulase (fJglucosidase) activities as well as the xylanase activities, and inoculating the yeasts Saccharomyces cerevisiae and Pachysolen tannophilus ATCC 32691 to convert the disaccharides ( sucrose) and the fermentable monosaccharides in C6 (glucose, fructose ..) and in C5 (mainly xylose) from the simultaneous enzymatic digestion of the cellulosic fraction of the thermochemical acid 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 stages of the process of the transformation of cactus fibers for the production of second generation bioethanol.
    Figure 2.- Represents the curve related to the adsorption isotherm 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 mglg of the Cellic®Cetc2 enzyme diluted 10 times.
    Figure 4. Photonic micrographs (3X) of cactus cladode fibers
    raw dried (a) and crushed (b).
    Figure 5. Scanning Electron Micrographs, MEB, of the fibers of the
    dried cactus cladodes (370 X) (c) and their pores (1500 X) (d).
    EMBODIMENT OF 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 that are difficult to biodegrade and less assimilable by a biological treatment process.
    The procedure for the transformation of the cladodes of dry cactus Opuntia ficus-indica by means of mechanical and thermochemical acid pre-treatment, as well as saccharification and simultaneous fermentation (SSF), followed by a stage
    distillation, in order to obtain the second generation bioethanol is illustrated in Figure 1.
    The procedure applied to samples of dried cactus cladodes from the arid bioclimatic of Tunisia is detailed below.
    Test 1. 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. Electron micrographs have shown the uniformity of the porous surface of cactus fibers with a
    15 pore diameter of approximately 10 ... tm.
    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 cladodes.
    Physicochemical 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 (rnL / g) 2,649 ± 0,158
    Water retention capacity (g of water! G MS) 2,157 ± 0,769
    Oil retention capacity (sunflower oil) (mi oil / MS) 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 isotherms at 20 ° C (Figure 2).
    5 The water absorption diagram allows the monitoring of the hydration of the cactus fibers depending on the activity of the water in the environment. The maximum water content that allows to fix the cactus fibers is of the order of 15.36% (g / 100g DM).
    The composition in a-cellulose, hemicellulose, lignin, carbon orgamco total
    10 (COT), inorganic carbon (C ing), total carbon (C tot) and total nitrogen (N tot) of the dried cactus cactus are shown in Table 3.
    Table 3. Composition in a-cellulose, hemicellulose, lignin, total organic carbon (COT), inorganic carbon (C ing), total carbon (C tot) and total nitrogen (N tot) of the dry C1adodios.
    Value Component (Ofo)
    a-Cellulose fifty
    Hemicellulose 18
    Lignin 27
    COT 6.206 ± 0.001
    C ing 0.451 ± 0.008
    C tot 6,705 ± 0,173
    Ntot 1,878 ± 0,041
    In fact, the holocellulose and lignin content of dried cactus cactus confirms the lignocellulosic nature of dried cactus samples.
    Test 2. Pre-thermochemical acid treatment of cactus fibers.
    Once crushed with a hammer mill (Leshan Dongchuan Machinery Co. Ud Sichuan. China) and thoroughly characterized, the dried cactus fibers were pre-treated by an acid thermochemical pre-treatment using a
    5 pressure reactor (Parr®, 4600-4620 series) 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.
    The pressure reactor used in this pre-treatment stage is of the 'batch' type, without stirring. 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.
    The conditions used for the acidic tennochemical pretreatment of the dried cactus fibers used were a pressure of 5 bar, 175 ° C temperature, with the addition of the sulfuric acid H2S04 at a concentration of
    15 0.06% And a reaction time of 75 minutes.
    The analysis of the composition of the cactus fibers after thermochemical pre-treatment is presented in Table 4.
    Table 4. Analysis of the composition of cactus fibers before and after tennochemical pre-treatment.
    Component Before pretreatmentAfter pretreatment
    Total soluble carbon (%) 6,70511.85
    Xylose (%) 0.5775,350
    Glucose (%) OR0.360
    Fructose (%) OR0.539
    Sucrose (%) OR0.107
    Total volatile acidity (%) OR0.50
    Hydroxy-methyl-furfural (%) OROR
    a-Cellulose (%) 50.0055.17
    Hemicellulose (%) 18.001.82
    Lignin (%) 27.0015.36
    Ashes (%) 8.247.90
    The total soluble carbon content (organic and inorganic) was stopped by automatic carbon and nitrogen equipment (multi N / C3100 Analytic Lena analyzer) 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 100g of sample) has been determined using a Shimadzu GC2010 gas chromatograph. The determined short chain volatile fatty acids are acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic, caproic and heptanoic. The hydroxymethyl-furfural (HMF) content was stopped by gas chromatography.
    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 Novozymes brand enzyme preparation, with a total protein concentration of 217 giL, a filter paper activity of 120.5 FPU / mL and a ~ -glucosidase activity of 2731 U / mL.
    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 19.10% ± 0.530 was achieved. and a residual dry matter 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, determined by gas chromatography.
    Hydrolysis of 10% Cactus in dry matter and 100 mglg of enzyme
    Exp. JealousLactoseGlucoseXilosaGalactoseArabinosaManosa
    24 h pattern 0.093
    It was 48 hours 0.3190.0790.082
    72 h pattern 0.470.144
    Sample 24h 10,0270.6819,2372,602
    Sample 48 h 8,2338,3182,268
    Sample 71. h 8.070.8258,9362,458
    * 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 process, 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 v / v with respect to the total volume of the reaction. The reaction medium was inoculated at 10% with the 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), (N1it) 2S04 (3.00 giL), MgS04 7H20 (2.05 gIL), KH2P04 (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
    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), (NH4) 2S04 (37.5 giL), MgS04 7H20 (25.625 giL), and KH2P04 (25 giL) ; All reagents were adjusted to a pH of 4.28.
    The SSF was developed in a microincubator operating at a temperature of 35 ± I ° C with fixed agitation at 150 rpm for 120 hours (5 days).
    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.
    Test 5. Simultaneous saccharification and fermentation (SSF) of the thermochemically pre-treated cactus fibers in an acid medium by a culture of two yeasts (Saccharomyces cerevisiae and Pachysolen tannophilus ATCC 32691) and the enzyme Cellic®Cetc2
    In this test the possibility of converting the different sugars into C6 (glucose and fructose) and C5 (xylose), as well as the disaccharides (sucrose) produced during enzymatic digestion were tested. This objective was achieved through the use of a mixed culture of two yeasts: Saccharomyces cerevisiae and Pachysolen tannophilus during the SSF.
    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 determination of the final concentration of the ethanol produced was determined by gas chromatography (OC) and with the enzyme kit adapted, this concentration being close to 0.7 giL.
    Test 6. Distillation
    The objective of this last distillation stage is to increase the concentration of ethanol in the final fermentation product. 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 bringing the mixture to a boil (or vaporization) of ethanol, whose boiling point at 1 atm is of 78.2 oC.
    INDUSTRIAL APPLICATION
    The industrial sectors that can benefit from this invention are:
    • Companies that operate 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 residues rich in lignocellulosic fractions, cellulose fractions (pastry-paper industry) or polysaccharide fractions (starch industry).
    • Any company that collects agricultural, forestry, or municipal solid waste, etc ...
    权利要求:
    Claims (8)
    [1]
    1. Procedure for the transfonnation of dried Opuntia ficus-indica cactus cladodes 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 diameter of 1 mm.
    b) Acid tennochemical pretreatment carried out in an inert atmosphere in the presence of N2, at a temperature of 175 oC, a pressure of 5 bar, a sulfuric acid concentration of 0.06% (m / v) and during a contact time 75 minutes
    c) Simultaneous saccharification and fennentation (SSF) by using an enzymatic mixture, with endocellulase and exocellulase (B-glucosidase) activities as well as xylanase activities, and inoculating Saccharomyces cerevisiae and Pachysolentannophilus ATCC 32691 yeasts to convert disaccharides (sucrose) ) and the C6 (glucose, fructose ..) and C5 (mainly xylose) phenentable monosaccharides from the simultaneous enzymatic digestion of the cellulosic fraction of the acid tennochemical pretreatment (step b).
    d) Distillation (liquid / vapor separation) to increase the alcohol content of the must obtained.
    [2]
    2. Procedure for the transfonnation of dried Opuntia ficus-indica cactus cladodes to produce second-generation bioethanol, according to claim 1, characterized in that the acidic tennochemical pre-treatment is carried out in a full-pressure, batch reactor without agitation, filled 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, with the use of 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]
    3. Process for the transformation of the dried Opuntia ficus-indica cactus cactus 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 50 Mm sodium acetate buffer at pH 4.82 with 3% of a 0.2% c 1 -ranfenicol solution prepared in the acetate buffer.
    [4]
    Four. Process for the transformation of the cactus cactus 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 100 mglg, diluted 10 times at a rate of 0.70 v / v with respect to the total volume of the reaction.
    [5]
    5. Process for the transformation 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 Pachysolen tannophilus ATCC 32691 and Saccharomyces cerevisiae at a pH of 4.2.
    [6]
    6. Process for the transformation of the cactus cladodes Opuntia ficus indica dry 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 tannophilus ATCC 32691 is of an extract of yeast (4.00 giL), casein peptone (3.60 gIL), (~) 2S04 (3.00 giL); MgS04 7H20 (2.05 giL), KH2P04 (2.00 gIL), xylose (5 giL), glucose (10 gIL), fructose (2.5 giL), and sucrose (2.5 gIL).
    [7]
    7. Process for the transformation of the cactus cladodes Opuntia ficus indica dry 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 gIL), casein peptone (45 gIL), (~) 2S04 (37.5 giL) , MgS04 7H20 (25,625 giL), and KH2P04 (25 gIL); The medium is adjusted to a pH of 4.82.
    [8]
    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).
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    WO2009102609A1|2008-02-12|2009-08-20|Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College|Thermochemical treatment of lignocellulosics for the production of ethanol|WO2020256535A1|2019-06-21|2020-12-24|Université Sidi Mohamed Ben Abdellah|Bioprocess for preparing a culture medium from cactus fruit of the genus opuntia for producing yeast biomass, biofuels, proteins, and renewable chemicals|
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