WO2013131162A1 - Procédé de production de bioéthanol à partir du pseudo-tronc du bananier par hydrolyse enzymatique, et utilisation de celui-ci - Google Patents

Procédé de production de bioéthanol à partir du pseudo-tronc du bananier par hydrolyse enzymatique, et utilisation de celui-ci Download PDF

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Publication number
WO2013131162A1
WO2013131162A1 PCT/BR2013/000069 BR2013000069W WO2013131162A1 WO 2013131162 A1 WO2013131162 A1 WO 2013131162A1 BR 2013000069 W BR2013000069 W BR 2013000069W WO 2013131162 A1 WO2013131162 A1 WO 2013131162A1
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WIPO (PCT)
Prior art keywords
production
bioethanol
sodium hydroxide
banana
banana pseudostem
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PCT/BR2013/000069
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English (en)
Portuguese (pt)
Inventor
Mauro César Marghetti LARANJEIRA
Cristiane PILISSÃO
Mariane MARTINI
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Companhia Paulista De Força E Luz - Cpfl
Mater Pesquisa E Desenvolvimento Tecnológico Ltda.
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Publication of WO2013131162A1 publication Critical patent/WO2013131162A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a process for the production of bioethanol from fermentable sugars formed by enzymatic hydrolysis of chemically pretreated banana pseudostem biomass. More specifically, the present invention relates to enzymatic hydrolysis of lignocellulosic material present in banana pseudostem for the production of fermentable sugars for further bioethanol formation.
  • Ethanol can be produced from a range of renewable energies.
  • Lignocellulosic wastes from agribusiness contain cellulose in combination with lignin.
  • Cellulose is the predominant polymer in lignocellulosic biomass, with hemicellulose and lignin found in smaller quantities.
  • the cellulose component in these materials can be converted to ethanol in a two step process, where the cellulose is first converted to sugars (glucose) by hydrolysis; The resulting sugars in turn can be converted to ethanol by fermentation.
  • sugars sugars
  • a pretreatment of these available carbohydrates is required to facilitate enzymatic hydrolysis and fermentation, as revealed by El-Zawawy, WK and colleagues in the study. work entitled "Acid and enzyme hydrolysis to convert pretreated lignocellulosic materials into glucose for ethanol production" published in Carbohydrate Polymers in 2011, V. 84, pgs. 865-871.
  • pretreatment processes have been developed for lignocellulosic materials in the state of the art. These are performed in part by solubilization of hemicelluloses and in part by lignin degradation. Pretreatments are: grinding and grinding, pyrolysis, high energy radiation, high vapor pressure, alkali or acid, gas treatment (chlorine dioxide, nitrogen and ozone), hydrogen peroxide, organic solvent treatment, hydrothermal treatment. , steam explosion, wet oxidation and biological treatment.
  • Plant waste has aroused great interest in bioethanol production.
  • the most studied route for its conversion to fermentable sugars has been acid hydrolysis, both in isolation, as Del Campo and colleagues describe, in their paper entitled “Diluted acid hydrolysis pretreatment for agri-food wastes for biotethanol production", published in Industrial. Crops and Products in 2006, V.24, pages. 214-221, as combined with enzymatic hydrolysis, according to the article by Cara et al., Entitled “Conversion of olive tree into fermentable sugars by dilute acid pretreatment and enzymatic saccharification” published in Bioresource Technology in 2008, V. 99, pags .1869-1876.
  • the banana tree is one of the main fruit in exploitation in Brazil; However, the amount of bananas produced annually in the country is only surpassed by oranges. Banana is grown without exception in all states of the Federation.
  • the state of Santa Catarina the second largest national banana producer, produced 668,000 tons of the fruit, mainly the species Musa sapientu and Musa cavendischii, popularly known in the region as white banana and banana nanica, respectively (CEPA, 2006).
  • Data from EMBRAPA (2006) show that of every 100 kg of fruits harvested in 2006, 46 kg were not used. In addition to this waste, the banana crop generates other residues. in the countryside from its industrialization.
  • this treatment modifies the lignocellulosic structure so that it can be quickly solubilized by cellulase, even using high solids contents (10 to 30%), without affecting enzymatic activity.
  • Commercial enzymes such as Spezyme CP (Genecor international Co) and Novo 188 (Novozyrr.es) were used.
  • each lignocellulosic material behaves differently with the pretreatments due to its lignin, cellulose and hemicellulose content. Therefore, in the present invention different pretreatments were performed with a single substance or in combinations, for example: sodium hydroxide (5%) under reflux of 2 hours; sulfuric acid (5%) at reflux for 2 hours; combination of sodium hydroxide and sulfuric acid; combination of sodium hydroxide with sodium hypochlorite and hydrogen peroxide; combination of sodium hydroxide with vinegar or acetic acid and hydrogen peroxide. This may include 3% sodium hydroxide and pure steam explosion or in combination with sodium hydroxide.
  • the yield of glucose with NaOH (3%) at 2 hours of reflux and with 0.03mL of the enzymes NS 2207 (cellulosic complex ⁇ -glucosidase, xylanase) and NS 50012 (cellulosic complex arabinase, hemicellulase, cellulase, pectinase and xylanase) for each 0.2 g of pretreated material was 85% within 48 hours of shaker reaction at 50 ° C.
  • the process of the invention provides a process comprising the steps of: (a) pre-treating the lignocellulosic plant biomass with an alkaline hydrogen peroxide solution; and b) Enzymatic hydrolysis of the pretreated biomass using mixtures comprising cellulases, glycosidases, hemicellulases, or combinations thereof.
  • said process further comprises a fermentation step of the carbohydrates produced in the previous steps, providing the production of ethanol and / or other alcohols.
  • the present invention uses only sodium hydroxide (3%) in 2h reaction whereas Brazilian patent application PI 0802559-2 uses 7.35% hydrogen peroxide plus the base amount to reach pH 11.5. , the concentration required to carry out this process was not cited in said document PI 0802559-2.
  • the glucose yield obtained by the present invention was 85% within 48 hours of reaction with only 0.03 ml of the NS 22074 and NS 50012 enzyme mix for each 0.2 grams of substrate.
  • Brazilian patent application PI 0802559-2 obtained 84% glucose yield.
  • the present invention employs a banana pseudostem lignocellulosic material, while Brazilian patent application PI 0802559-2 employs sugarcane bagasse and / or straw.
  • the present invention utilizes sodium hydroxide (3%) in 2h reaction after steam explosion reaction, which assists in the removal of any interferent that forms during steam explosion which is undesirable for fermentation and afterwards. obtaining ethanol.
  • the pretreatments disclosed in the present invention resulted in increased glucose yield from 51% to 71.5% with only 0.03 ml of enzyme, and enzymes NS 22074 (cellulosic complex-p-glucosidase) were used.
  • NS 22074 cellulosic complex-p-glucosidase
  • NS 50012 cellulosic complex arabinase, ⁇ - glucanase, hemicellulase, cellulase, pectinase and xylanase.
  • the present invention will provide significant advantages over bioethanol production processes using chemical pretreatment of banana pseudostem followed by enzymatic hydrolysis with commercial enzymes, enabling an increased performance and cost effectiveness. / most favorable benefit.
  • the process of the present invention is based on the chemical treatment of banana pseudostem followed by enzymatic hydrolysis using commercial enzymes such as NS 22074 and NS 50012 to obtain fermentable sugars for further bioethanol formation.
  • each lignocellulosic material behaves differently with the pretreatments, since the composition of biomass varies according to its plant origin (lignin content, cellulose and hemicellulose) and the way they are associated.
  • the process of the present invention is quite versatile as it provides the selection of the best pretreatment to be applied to banana pseudostem.
  • the processes employed in the present invention have shown by HPLC analysis that the main fermentable sugar formed is sucrose and there is no generation of compounds that are inhibitory in the later stages.
  • the present invention describes a method of improving the degradation of lignocellulosic material using chemical pretreatment, as well as increasing the yield of fermentable sugars in enzymatic hydrolysis with the mixing of commercial cellulases NS 22074 and NS 50012 for further ethanol production.
  • the present invention relates to the evaluation of the percentage conversion to fermentable sugars and the amount of biomass required to produce fermentable sugars for ethanol production.
  • Another advantage of the present invention is that it employs the main enzyme for use in the hydrolysis of lignocelluloses to NS 22074 (cellulosic complex ⁇ -glucosidase, xylanase) and the effect of this enzyme is enhanced by the addition of the enzyme NS 50012. (cellulosic complex - arabinase, hemicellulase, cellulase, pectinase and xylanase).
  • the present invention has several advantages over the state of the art, such as practicality, high enzymatic hydrolysis of banana pseudostem, providing good yields of reducing sugars and thus enabling good yields in the production of. ethanol.
  • the process disclosed by the present invention utilizes a highly sustainable substrate because it is a tailings, which enables the production of ethanol from this lignocellulosic material.
  • the present invention discloses different pretreatments to evaluate, which one is most suitable for banana pseudostem biomass, where the treatment with refluxing sodium hydroxide (3%) is better than those described in the state of the art, even better than Sodium hypochlorite.
  • Another aspect of the present invention is that the reaction condition described in the process of the present invention has not been reported in the prior art. None of the pretreatment methods described in the prior art utilize the same combinations of reagents and equipment as the ultrasonic bath used in the process of the present invention. Considering that, in the state of the art, the studied biomass is sugarcane bagasse or straw, the present invention has relevance when using banana pseudostem as an alternative lignocellulosic biomass, abundantly generated in the agricultural sector, for the production of second generation bioethanol. .
  • the present invention has obtained 85% yield in fermentable sugars with a minimal amount of enzyme, much lower than that described in the prior art, and another important aspect is that the biomass of the present invention is free of interfering affecting fermentation due to the sodium hydroxide treatment that was used, in addition to the absence of xylose and the presence of sucrose as the main sugar reducer formed.
  • the enzymes used in the process of the present invention are different from those described in the prior art, although: they are cellulase, ⁇ -glycosidase hemicellulase, their origin is distinct
  • banana caturra (fresh) pseudostem was washed with running water about 3 times to remove the dirt and the resin, then placed in an oven at 60 ° C for drying. After drying the banana pseudostem was ground in a Willye model MA680 mill with a 30 mesh sieve and stored in plastic bags for later chemical treatments and enzymatic hydrolysis (this washed material is called from this biomass step ), described in Brazilian patent application PI 1102362-7 and incorporated herein by reference in its entirety.
  • Pretreatment with sodium hydroxide was performed by adding 10g of the banana pseudostem biomass in a round bottom flask and 200 mL of a (5% w / v) or (3% w / v) sodium hydroxide solution, this mixture was refluxed for 2 hours. At the end of this period the solution was filtered and the remaining solid was washed with distilled water. The collected solid was placed in the steam oven until its drying at a temperature of approximately 60 ° C. After drying the material was ground and enzymatic hydrolysis was performed.
  • Treatment with sodium hydroxide followed by the mixture of acetic acid and hydrogen peroxide was performed by adding 10g of the banana pseudostem biomass in an erlenmeyer and 100 ml of a sodium hydroxide solution (0.1M), this mixture was stirred on a shaker. magnetic for 1 hour at 70 ° C or in a cold bath. ultrasound at a fixed temperature of 60 ° C.
  • reaction mixture was filtered and washed with distilled water until the pH was adjusted to 4-5. After the collected solid was placed in the steam oven until its drying at a temperature of approximately 60 ° C. Subsequently, the dried material was ground and stored for later enzymatic hydrolysis.
  • Sulfuric acid treatment was performed by adding 10 g of biomass in a round bottom flask and 200 mL of a 5% (v / v) sulfuric acid solution, the reaction mixture was kept for 2 hours under reflux. At the end of this. The reaction mixture was then filtered and the solid was washed with distilled water, the collected solid material was placed in a steam oven until drying at approximately 60 ° C. Subsequently, the dried material was ground and stored in flasks for further enzymatic hydrolysis.
  • Table 1 shows the percentage of reducing sugars released from the 0.1 lb suspensions of banana pseudostem with different chemical pretreatments with the mixture of NS 22074 (0.015mL) and NS 50012 (0.015mL) cellulas directly assisted in the ultra bath. only at a fixed temperature of 60 ° C for 4 hours and the amount of biomass required to produce 1 kg of reducing sugars.
  • acetic acid / H 2 0 2 Banana pseudostem treated with sodium hydroxide for 1 hour at 70 ° C, followed by treatment with the mixture acetic acid and hydrogen peroxide for 6h at 70 ° C.
  • NaOH / vinegar / H 2 0 2 banana pseudostem treated with sodium hydroxide for 1 hour at 70 ° C, followed by treatment with mixing vinegar and hydrogen peroxide for 6h at 70 ° C.
  • NaOH / sodium hypochlorite / H 2 0 2 Banana pseudostem treated with sodium hydroxide for 1 hour at 70 ° C, followed by treatment with the mixture of sodium hypochlorite and hydrogen peroxide for 6h at 70 ° C .
  • H2SO4 Banana pseudostem treated with sulfuric acid (5%) for 2 hours under reflux.
  • the present invention shows that when using pretreatment with sodium hydroxide (5%), the conversion to fermentable sugars was 51%, this result was better than using pre-treated biomass. treated with sodium hydroxide followed by peracetic acid (this pretreatment was developed in patent application PI 1102362-7 and incorporated herein by reference in its entirety), however, to compare pretreatments, it performed enzymatic hydrolysis on the same reaction conditions as described in this invention.
  • the best result obtained was using sodium hydroxide pretreated biomass (5%) and performing assisted enzymatic hydrolysis in an ultrasound bath.
  • Table 2 shows the percentage of reducing sugars released from suspensions of 0.1 g of sodium hydroxide pretreated banana pseudostem (5%) under reflux for 2 hours or pre-treated biomass.
  • sodium hydroxide followed by pretreatment with peracetic acid with the mixture of cellulase NS 22074 (0.015mL) and NS 50012 (0.015mL) in the shaker bath at 50 ° C and rpm 150 for 4 and 24 hours and the amount of biomass needed to produce 1 kg of reducing sugars
  • Table 2 also shows that sodium hydroxide pretreatment is better than sodium hydroxide treatment followed by peracetic acid, since the conversion to fermentable sugars was about 40% higher when using the sodium hydroxide pretreated biomass. sodium.
  • the present invention shows that depending on the chemical pretreatment, the enzyme and the reaction conditions used the enzymatic hydrolysis may be more efficient with one method or another.
  • Table 3 The results of the percentage of fermentable sugars and the amount of biomass required to produce 1 kg of fermentable sugars are presented in Table 3.
  • Table 3 shows the percentage of reducing sugars released from 0.2g suspensions of sodium hydroxide pretreated banana pseudostem (5%) under reflux for 2 hours or pretreatment with sodium hydroxide followed by peracetic acid with mix the NS 22074 (0.015mL) and NS 50012 (0.015mL) cellulases in the shaker bath, the temperature of 50 ° C and rpm of 150 for 4, 24 and 48 hours and the amount of biomass required to produce IKg of reducing sugars. . .
  • the results of the percentage of fermentable sugars and the amount of biomass required to produce 1 kg of fermentable sugars are presented in Table 4.
  • Table 4 shows the percentage of reducing sugars released from 0.2g suspensions of sodium hydroxide pretreated banana pseudostem (3%) under reflux for 2 hours or steam blast pretreatment followed by sodium hydroxide (3%) for 2 hours under reflux with the mixture of NS 22074 (0.015mL) and NS 50012 (0.015mL) cellulases in the shaker bath at a temperature of 50 ° C and rpm of 150 by 4, 24 and 48 hours and the amount of biomass needed to produce 1 kg of reducing sugars.
  • Table 5 HPLC chromatography analyzes of the sodium hydroxide-treated banana pseudostem (3%) samples at 2 hours of reflux.
  • Table 6 shows that for steam blast treated samples followed by pretreatment with 2 hours reflux sodium hydroxide (3%), the main reducing sugar is sucrose, the second is. glucose and xylose is absent. In this treatment, the presence of very small amount of hydroxymethylfurfural in the samples is confirmed.
  • Table 6 HPLC chromatography analyzes of steam-blown banana pseudostem samples followed by pre-treatment with sodium hydroxide (3%) with 2 hours of reflux.
  • An important aspect of the present invention is that enzymatic hydrolysis using the mixture of sodium hydroxide-treated banana pseudostem enzymes NS 22074 and NS 50012 (3%) with 2 hours reflux and treated with steam explosion followed by pretreatment with sodium hydroxide resulted in the formation of sucrose as the main fermentable sugar which can be easily and efficiently converted to ethanol by the microorganism Saccharomyces cereviseae under industrial conditions.
  • the influence of the mixture of the enzymes NS 22074 and NS 50012 in comparison with the pure cellulase NS 22074 is emphasized.
  • the use of the enzyme NS 50012 enhances the enzymatic hydrolysis and thereby reduces the enzymatic cost.

Abstract

La présente invention concerne un procédé de production de bioéthanol à partir du pseudo-tronc du bananier par hydrolyse enzymatique, faisant intervenir un pré-traitement avec de l'hydroxyde de sodium pur ou en combinaison avec de l'hypochlorure de sodium, de l'acide acétique et/ou du vinaigre en combinaison avec du peroxyde d'hydrogène, de l'acide sulfurique et la combinaison d'hydroxyde de sodium et d'acide sulfurique, ainsi que la combinaison d'une explosion à la vapeur et d'hydroxyde de sodium, puis l'hydrolyse enzymatique de la biomasse pré-traitée avec les enzymes cellulases NS 22074 et NS 50012, réalisée dans un bain d'ultrasons ou un bain de type shaker, le meilleur résultat obtenu étant l'hydrolyse enzymatique en shaker, pendant 48 heures, de la biomasse avec de l'hydroxyde de sodium (3%) (0,2 g) avec le mélange des enzymes NS 22074 (0,015 ml) et NS 50012 (0,015 ml), étant donné que la production de 1 kg de sucres fermentables ne requiert que 1,2 kg de biomasse pré-traitée et seulement 180 ml du mélange des enzymes NS 22074 et NS 50012, ledit procédé étant ainsi économiquement viable pour la production d'éthanol.
PCT/BR2013/000069 2012-03-08 2013-03-08 Procédé de production de bioéthanol à partir du pseudo-tronc du bananier par hydrolyse enzymatique, et utilisation de celui-ci WO2013131162A1 (fr)

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BRBR1020120052300 2012-03-08
BR102012005230-0A BR102012005230B1 (pt) 2012-03-08 2012-03-08 processo para a produção de bioetanol a partir do pseudocaule de bananeira com hidrólise enzimática e uso do mesmo

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Cited By (2)

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CN107429278A (zh) * 2015-03-04 2017-12-01 Fp创新研究中心 用以增强对预处理的木质纤维素生物质的酶促水解的新型后处理
WO2022235850A1 (fr) * 2021-05-06 2022-11-10 Rajakaruna Uppala Additif alimentaire en tant qu'exhausteur d'arôme ou modificateur de texture, et son procédé de fabrication

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107429278A (zh) * 2015-03-04 2017-12-01 Fp创新研究中心 用以增强对预处理的木质纤维素生物质的酶促水解的新型后处理
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CN107429278B (zh) * 2015-03-04 2022-08-30 Fp创新研究中心 用以增强对预处理的木质纤维素生物质的酶促水解的新型后处理
WO2022235850A1 (fr) * 2021-05-06 2022-11-10 Rajakaruna Uppala Additif alimentaire en tant qu'exhausteur d'arôme ou modificateur de texture, et son procédé de fabrication

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