WO2013131162A1

WO2013131162A1 - Method for producing bioethanol from banana pseudostem by enzymatic hydrolysis, and use of the same

Info

Publication number: WO2013131162A1
Application number: PCT/BR2013/000069
Authority: WO
Inventors: Mauro César Marghetti LARANJEIRA; Cristiane PILISSÃO; Mariane MARTINI
Original assignee: Companhia Paulista De Força E Luz - Cpfl; Mater Pesquisa E Desenvolvimento Tecnológico Ltda.
Priority date: 2012-03-08
Filing date: 2013-03-08
Publication date: 2013-09-12
Also published as: BR102012005230A2; BR102012005230B1

Abstract

The present invention relates to a method for producing bioethanol from banana pseudostem by enzymatic hydrolysis, using a pre-treatment with sodium hydroxide pure or in combination with sodium hypochlorite, acetic acid and/or vinegar in combination with hydrogen peroxide, sulphuric acid, the combination of sodium hydroxide and sulphuric acid, the combination of vapour explosion and sodium hydroxide, followed by enzymatic hydrolysis of the biomass pre-treated with the NS 22074 and NS 50012 cellulase enzymes in an ultra-sound bath or shaker-type bath, the best result being obtained by enzymatic hydrolysis of the biomass in a shaker for 48 hours, with 3% sodium hydroxide (0.2 g), a mixture of the NS 22074 enzyme (0.015 mL) and NS 50012 enzyme (0.015 mL), since only 1.2 kg of pre-treated biomass and only 180 mL of the mixture of the NS 22074 and NS 50012 enzymes are required to produce 1 kg of fermentable sugars, thus proving to be an economically viable method for producing bioethanol.

Description

Patent Descriptive Report for: "BIOETHANOL PRODUCTION PROCESS FROM BANANE PSEUDOCAULE WITH ENZYMATIC HYDROLYSIS AND USE".

Field of the Invention

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.

Background of the Invention

Prospects for depletion of oil reserves and stronger commitments to the environment since the Kyoto Protocol signed attention on alternative energy sources. There are several alternative sources of energy that, while they cannot replace oil in its entirety, can at least contribute to lowering its consumption. Among them, one of particular interest, due to its abundance and renewable nature, is plant biomass, mainly that formed by forest and agricultural residues.

However, the raw material for bioethanol production must come from inedible parts of food in order to avoid direct competition between bioethanol and food production (Sakai et al., 2007). 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. However, due to the close association of cellulose and hemicellulose with lignin in plant cell walls, 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.

Endo and colleagues described in a paper entitled "Genome-wide screening of the genes required for tolerance to vanillin, which is a potential inhibitor of bioethanol fermentation in Saccharomyces cerevisiae", published in Biotechnology for Biofuels in 2008, 1, 3, which for economic reasons. Thermochemical pretreatments have been performed, such as dilute acid hydrolysis and steam explosion, which solubilizes the hemicellulose component and increases the accessibility of cellulose.

However, not all types of lignocellulosic biomass can be pretreated in the same way. While steam blasting has been tried and tested for agroforestry such as maize straw and rice straw, acid hydrolysis is required to produce Sugar from soft wood. Thus, research is needed to determine the best pretreatment strategy. it can be the most economical, and at the same time can satisfy all the characteristics of good pretreatment as described in various prior art works.

Several 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.

Among the residues generated in agriculture is the banana biomass. The conditions of production, The industrialization and commercialization of bananas results in a large amount of plant matter, both in terms of accumulated residues and rejected fruits.

Souza and colleagues described in their work entitled "Biodegradation of lignocellulosic waste generated in banana farming and its valorization for biogas production" published in the Brazilian Journal of Agricultural and Environmental Engineering in .2010, V. 4, pags. 438-443, that bioethanol production from banana biomass becomes a very attractive process. In addition to allowing the creation of an alternative and renewable source of energy and contributing to the reduction of waste in the environment, power generation can add value to the fruit production matrix, thus reducing the risk of losses caused by fluctuations in its price. market.

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. In 2005, 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. According to data from a food company in the municipality of Garuva, one of the largest banana producers in the northeast region of the state of Santa Catarina, to. each ton of banana industrialized approximately 3 tons of _pseudostem,. 160 kg of stems, 480 kg of leaves and 440 kg. of _. Barks are generated.

According to Yoswathana. and collaborators wrote in their paper entitled "Bioethanol Production. from Rice Straw", published in the Energy Research Journal, 1; 26-31 on _. In 2010, rice straw has been treated with different chemicals (acids or alkalis) and physical methods (subcritical water, ultrasound) to convert lignocellulosic material into sugar. Sulfuric acid treatment (1-9%) at 121 ° C for 15 minutes is an effective pretreatment method for converting lignocellulose to sugar, with 21% conversion to sugars measured after acid treatment. . A good result was obtained using the combination of chemical and enzymatic pretreatment, which increased the sugar yield to 37 and 28% for the acid and alkali pretreated samples, respectively. The best result was obtained by using the combination of acidic pretreatment with ultrasound before enzymatic treatment, and the conversion to sugar increased to 44%.

El-Zawawya and colleagues wrote in their paper entitled: "Acid and enzyme hydrolysis to convert pretreated lignocellulosic materials into glucose for ethanol production", published in Carbohydrate Polymers, 84 865-871 in 2011, Acid and enzymatic hydrolysis for glucose production. from rice straw, pre-treated banana waste and corn cob as lignocellulosic materials. Enzymatic hydrolysis of cellulose to glucose is performed by cellulase enzyme. The hydrolysis was performed under mild conditions (eg pH 4.5-5.0 and temperature 40-50 ° C). In this case we had no major ^problems' corrosion, showed low power consumption and low toxicity of the hydrolyzate as the main advantages of this process compared to acid hydrolysis carried out with dilute acid at a higher temperature. When performing the enzymatic hydrolysis with the banana residue, the best result was obtained by using the steam explosion pretreated biomass, and within 24 hours of reaction 9 g / L of glucose was formed.

International patent application WO 2008/095098, filed January 31, 2008 on behalf of the Board of Supervisors of Louisiana State University & Agricultural & Mechanical College describes a process for obtaining sugars from lignocellulosic biomass in which a Hot alkaline pretreatment of the material with a mixture of calcium hydroxide and water at a temperature of 80 ° C to 140 ° C for about 30 min to 3 hours. After treatment, the bagasse is pressed; The liquid contains mainly soluble lignin components in addition to lime (which can be recovered) and the fibrous solid material is subjected to hydrolysis by cellulosic enzymes. According to the authors, 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.

Brazilian patent application PI 0700481-8, filed on March 2, 2007, entitled "Pre- sugarcane bagasse treatment for ethanol production "on behalf of the University of Caxias do Sul Foundation describes alkaline pretreatment processes for sugarcane bagasse and further hydrolysis with the Penicillíum echinulatum enzyme for This invention employed enzyme amounts of 10 to 15 FPU per gram of dry biomass at 40-60 ° C for 24-48 hours.The yields of fermentable sugars were 50-90%.

International patent application WO 2006/110901 filed April 12, 2006 in the name of E. Du Pont de Nemours and Company, entitled "Treatment of biomass to obtain fermentable sugars", reports the pretreatment of biomass using low concentrations of aqueous ammonia at high biomass concentrations. The pretreated biomass is hydrolyzed with enzymes.

International Patent Application WO 2006/026863, filed September 9, 2005 on behalf of Iogen Energy Corporation and entitled "Process for producing a pretreated feedstock" discloses a method for treating lignocellulosic biomass - grass, cereal straw and cobs or combinations of these materials. By this method, the waste is ground and pretreatment is carried out with the addition of dilute acid to the waste. This mixture is kept at a temperature of 160 ° C to 280 ° C.

International patent application WO 2005/078140, filed February 8, 2005 on behalf of JGC Corporation and entitled "Process for producing monosaccharide from biomass and monosaccharide production apparatus" discloses a process comprising a first step, where biomass is treated. with sulfuric acid in concentration of 65 to 85% at temperatures of 30 to 70 ° C and, in the second stage, saccharification of the product obtained in the first stage occurs. This saccharification is performed with sulfuric acid at a concentration of 20 to 60% at temperatures of 40 to 100 ° C.

Reyes and colleagues described in the paper, intilulated enzymatic hydrolysis of rice husk using cellulases. The effects of chemical and photochemical treatments, "published in Quim. 1998 New, 21.2, enzymatic _hydrolysis. Rice husks using a commercial cellulase preparation. The results showed that pre-treatment with sodium chlorite and light inhibits the process of enzymatic hydrolysis, while with hydrogen peroxide and ozone favored the enzymatic production of reducing sugars, being 5.9 and 54.9%, respectively.

Brazilian patent application PI 9200100-9 filed January 15, 1992 in the name of the US Department of Energy entitled "Process for Simultaneous Saccharification and Fermentation" describes a process for producing ethanol from biomass wherein the substrate includes a hydrolyzate. of cellulose, hemicellulose and starch to produce fermentable sugars of six carbons. Fermentation uses a genetically modified yeast strain (Brettanomyces custersii CBS 5512) that produces the enzyme B-glucosity, which makes this yeast capable of fermenting both glucose and cellobiose.

Brazilian patent application PI 0408165-0, filed March 8, 2004 in the name of Athenix Corporation, entitled "Processes for Improving Lignocellulose Degradation Enzyme Activity" describes a method for hydrolyzing lignocellulosic materials to obtain sugars, further utilizes a pretreatment:. chemical with temperature (10-90 ° C) and pressure (2 atm) conditions. In this invention the pretreatment is carried out with hydrogen peroxide, calcium hypochlorite gives corn husk biomass. By using the hydrogen peroxide pretreated biomass and T. longibrachiatum cellulase (25mg) for 24h at 65 ° C, the yield was 47%. In the mentioned document PI 0408165-0, when using the enzyme Spezyme (0.3mL to 0.2g of biomass) the obtained yield was 71.9% in 24 hours of reaction, and without previous pretreatment the yield was 32 , 8%. Furthermore, the authors describe that from hydrolyzed material they can generally produce a fuel, a drug, an organic acid, a lactic acid, an industrial enzyme and an amino acid. In the present invention all these products can be obtained, however the formation of ethanol fuel has been emphasized. Furthermore, in the present invention different chemical pretreatments were performed.

Several chemical or physical pretreatments are well described in the prior art, but 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.

Importantly, in the present invention the yield of glucose with NaOH (3%) at 2 hours of reflux and with 0.03mL of the enzymes NS 2207 (cellulosic complex β-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. When performing hydrolysis of the steam blast sample the yield was 51%, and when combining with sodium hydroxide and steam blast the yield was 71.5% whereas in the invention present in PI 0408165- 0 0.3 mL of Spezyme enzyme was required to obtain 71.9% glucose yield. It is also described in the prior art that to hydrolyze the lignocellulosic material it is necessary to use cellulase. Therefore it is noted that the present invention is different from that described in Brazilian patent application PI 0408165-0 which uses the following enzymes Spezyme (0.3mL) and T. Longibhachiatum (25mg). In addition to being different enzymes the amount used in Brazilian patent application PI 0408165-0 was greater than the amount used in the present invention. Moreover, pretreatment with sodium hydroxide proved to be better than that of sodium hypochlorite, and in the case of the present invention it was not necessary to use high pressure and temperatures when performing the reaction with sodium hypochlorite, were lower than in the optimal condition obtained. in Brazilian patent application PI 0408165-0 and, finally, it is noted that the Banana pseudostem substrate of the present invention is different from the above. lignoeelulosic materials employed in the document. of Brazilian patent application PI 0408165-0.

_. Brazilian patent application PI 0802559-2, filed on 07. de. July 2008 on behalf of Campinas State University - Unicamp and entitled "Lignocellulosic Plant Biomass Pre-Treatment and Hydrolysis Process, and Product for Industrial Production of Alcohols" describes pre-treatment and hydrolysis processes of vegetable biomass. The product obtained by said process is substantially useful as an input in the industrial production of ethanol and / or other alcohols. In a preferred aspect, 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. Optionally, 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. While Brazilian patent application PI 0802559-2 obtained 84% glucose yield. In addition, the present invention employs a banana pseudostem lignocellulosic material, while Brazilian patent application PI 0802559-2 employs sugarcane bagasse and / or straw. The enzymes used in Brazilian patent application PI 0802559-2 have not been defined, let alone which ones have been used, but which are a cellulase, β-glycosidase, hemicellulase or combination thereof and the amount is much higher than that used in the present. invention.

Brazilian patent application PI 0608369-2, filed March 14, 2006 on behalf of Novozymes North America, INC entitled "Process for the Production of a Fermentation Product from Lignocellulosic Material" provides a process for the production of a a fermentation product comprising the steps of i) pretreating the lignocellulosic material for the release or separation of cellulose, hemicellulose and / or lignin, ii) subjecting the pretreated material to a cellulase, iii) fermenting in the presence of an organism fermentation, wherein xylose isomerase is added in step ii) and / or step iii). 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. In addition, 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. xylanase) and NS 50012 (cellulosic complex arabinase, β- glucanase, hemicellulase, cellulase, pectinase and xylanase). Meanwhile, Brazilian patent application PI 0608369-2 uses the enzymes Novozym 188. and celullast 1.5L or viscozyme and uses approximately 0.5 ml enzyme. In addition, the use of acid is already well described which forms interferers such as HMF that will make the fermentation stage difficult, thus decreasing the yield.

As already described in the prior art, a large group of researchers have been studying ways to produce ethanol from lignocellulosic materials. Thus, for this process to be possible, it is necessary that the physical ^'or chemical pretreatment is economically feasible as well as the enzyme to be used as a catalyst. Currently, different enzymes (cellulases, hemicellulases) are being studied and the search for new catalysts that provide hydrolysis of lignocellulosic material with higher yields and lower cost.

As can be seen no prior art document describes or suggests an enzymatic hydrolysis process. of banana pseudostem with the mixture of enzymes NS 22074 and NS 50012 and different pretreatments for bioethanol production.

Summary of the Invention

To solve the above mentioned problems, 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.

Several chemical or physical pretreatments are well described in the prior art, but 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.

Although prior art documents disclose pre-treatments of lignocellulosic biomass and ethanol production from lignocellulosic material and / or different culture media, the method or process used in the present invention is novel because none of the processes cited in state of the art, describes the pre-treatment technology of banana pseudostem employing sodium hydroxide (5%) under 2 hour reflux or sodium hydroxide (3%) under 2 hour reflux; 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 and steam explosion for 8 minutes at 200 ° C followed by 3% sodium hydroxide for 2 hours at reflux.

The process of the present invention is quite versatile as it provides the selection of the best pretreatment to be applied to banana pseudostem. In addition, 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.

Detailed Description of the Invention

While the present invention may be susceptible to different embodiments, it is shown in the tables and the following detailed discussion, a preferred embodiment with the understanding that the present embodiment should be considered an exemplification of the principles of the invention and is not intended to limit the present invention to what has been illustrated and described here.

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. .

In addition, 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, β-glycosidase hemicellulase, their origin is distinct

Initially, the 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.

Subsequently different chemical treatments were carried out, such as: with pure sodium hydroxide or in combination with sodium hypochlorite and hydrogen peroxide, with sodium hydroxide in combination with acetic acid or vinegar and hydrogen peroxide; and the combination of sodium hydroxide and sulfuric acid.

In addition to these pre-treatments, a steam explosion was performed in combination with sodium hydroxide; The results of this invention were compared to the results obtained with the combination of sodium hydroxide and peracetic acid, pretreatment 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.

After this time a solution of acetic acid and hydrogen peroxide (30%, 40% of acetic acid (12mL) in hydrogen peroxide (18mL)) (100mL) was added to the reaction mixture. distilled water. This reaction mixture was stirred for 6 hours at 70 ° C or in another experiment in the fixed temperature ultrasound bath at 60 ° C. At the end of this period the reaction mixture was filtered and the pH adjusted to 4-5, washing with distilled water, the collected solid material was placed in the steam oven until drying. Subsequently, the dried macerated material is stored in vials for further enzymatic hydrolysis.

Treatment with sodium hydroxide followed by the vinegar and hydrogen peroxide mixture was performed by adding 10g of the banana pseudostem biomass in an erlenmeyer and 100 ml NaOH solution (0.1 M), the reaction mixture was stirred for one hour on a magnetic stirrer at -70 ° C. After this one. To this period, a solution of apple vinegar (Rosina brand) and hydrogen peroxide (30%, 40% vinegar (12 ml) in hydrogen peroxide (18 ml)) (100 ml) was added to the reaction mixture. supplemented with distilled water. This reaction mixture was stirred on a magnetic stirrer for 7 hours at 70 ° C. At the end of this period the 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.

Steam blast treatment was carried out at 200Â ° C for 8 minutes, then weighed 10g of steam blast pretreated biomass in a round bottom flask and 200 ml of sodium hydroxide solution (3 %) (v / v), the reaction mixture was kept for 2 hours under reflux. At the end of this period the reaction mixture was filtered and the solid was washed with In distilled water, the collected solid material was placed in the steam oven until it was dried at a temperature of approximately 60 ° C. Subsequently, the dried material was ground and stored in flasks for further enzymatic hydrolysis.

To perform the hydrolysis, enzymatic was used 0.1 l of pretreated biomass, 0.03 mL of the mixture of enzymes NS 22074 and NS 50012, 5 mL of sodium acetate buffer pH = 4.8. The mixture was left in a fixed temperature ultrasound bath at 60 ° C for 4 hours. The percentage results of fermentable sugars and the amount of biomass _. needed to produce 1 kg of fermentable sugars are shown in Table 1.

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.

Table 1 - Assisted enzymatic hydrolysis in the ultrasound bath of the chemically pretreated banana pseudostem with the mixture of the enzymes NS 22074 and NS 50012. Chemical treatment Yield (%) Biomass required to produce 1 kg of reducing sugars

Without treatment 19 5.3 kg

NaOH / Acid, peracetic ^" 47 2.1 kg

NaOH / Acid. acetic acid / H ₂ 0 ₂ ^~ 45 2.2 Kg NaOH / Acid. 33 3.0 kg

Acetic / H ₂ 0 ₂ / US

NaOH / vinegar / H ₂ 0 ₂ 47 2.2 Kg

45 NaOH / hypochlorite 2.2 kg

sodium / H ₂ 0 ₂

NaOH 51 2.0 kg

H ₂ S0 ₄ 35 3.0 Kg

NaOH / H ₂ S0 ₄ 47 2.1 Kg

Reaction conditions: biomass (3.3 grams of biomass per mL of NS 22074 and NS 50012 enzyme mixture, 1:50 biomass / volume of sodium acetate buffer pH = 4.8), 4 hour ultrasound reaction at ¹ temperature reaction in

60 ° C.

Where :

NaOH / AC. peracetic: Banana pseudostem treated with NaOH (0.1 M) solution for 1 hour at 70 ° C followed by treatment with peracetic acid (30%) at 70 ° C for 7 hours.

NaOH / Acid. 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 / Acid. acetic acid / H ₂ 0 ₂ / US: Sodium hydroxide-treated banana pseudostem for 1 hour at 60 ° C in the ultrasound bath, followed by treatment with the mixture acetic acid and hydrogen peroxide for 6h at 60 ° C in the ultrasound bath

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 .

> NaOH: Banana pseudostem treated with sodium hydroxide (5%) for 2 hours under reflux.

H2SO4: Banana pseudostem treated with sulfuric acid (5%) for 2 hours under reflux.

aOH / H ₂ SO ₄ : Banana pseudostem treated with sodium hydroxide (5%) for 2 hours under reflux followed by treatment with sulfuric acid (5%) for 2 hours under reflux.

From the results obtained in Table 1, 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.

In this case, when using sodium hydroxide pretreatment, the amounts of enzyme and biomass needed to produce 1 kg of fermentable sugars were lower than when using sodium hydroxide pretreated biomass followed by peracetic acid.

In addition, the cost of chemical pretreatment with sodium hydroxide is lower compared to using mixing sodium hydroxide and peracetic acid, showing that pretreatment with sodium hydroxide is better and more economically viable and efficient.

It should be noted in the present invention that pretreatment with sodium hydroxide followed by the mixture of vinegar and hydrogen peroxide and the mixture of sodium hydroxide and sulfuric acid, the conversion to fermentable sugars was approximately 47%, the same percentage. obtained by performing hydrolysis. with biomass pretreated with sodium hydroxide followed by peracetic acid under the same reaction conditions. However, these pretreatments have the advantage of being more economically viable than using peracetic acid.

The results shown so far indicate that the conversion to fermentable sugars was 60% higher compared to the untreated sample using the sodium hydroxide pretreated sample, so this process becomes viable for production. of fermentable sugars.

As described in the present invention, the best result obtained was using sodium hydroxide pretreated biomass (5%) and performing assisted enzymatic hydrolysis in an ultrasound bath.

Another aspect of the present invention is to perform enzymatic hydrolysis using pre-treated sodium hydroxide (5%) (0.1g) or sodium hydroxide biomass followed by pretreatment peracetic acid described in Brazilian patent application PI 1102362 -7 and incorporated herein by reference in its entirety with the mixture of the enzymes NS 22074 and NS 50012 (0.03mL), 5 ml buffer ethyl acetate sodium pH = 4.8, stirring this mixture in a horizontal shaker - type stirrer to 50 ° C, 150 _rpm,. for 4 and 24 hours.

In this present invention also evaluated the. enzymatic hydrolysis in a shaker bath with sodium hydroxide pretreated banana pseudostem biomass (5%) and these results were compared with those obtained with sodium hydroxide pretreated biomass followed by peracetic acid. These results are presented in Table 2.

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 - Enzymatic hydrolysis of pre-treated biomass with sodium hydroxide (5%) or sodium hydroxide followed by peracetic acid with the mixture of enzymes NS 22074 and NS

50012 in horizontal shaker.

NaOH NaOH / Acid. Peracetic

Time Biomass Yield Biomass Yield

(h) (%) required (%) required

for to produce produce lKg lKg of sugars reducing sugars 4 72 1.5 kg 43 2.3 kg

24 96 ^: 1.1 kg 98 1.0 kg

Reaction conditions: chemically pretreated biomass 3.3g / mL enzyme), 1:50 biomass / volume sodium acetate buffer pH = 4.8) shaker bath temperature 50 ° C, 150 rpm.- From the results obtained Table 2 shows that when performing enzymatic hydrolysis in a horizontal type shaker with sodium hydroxide pretreated biomass, the conversion to fermentable sugars was 72% in only 4 hours, ie, it presented a higher yield in comparison using ultrasound assisted hydrolysis (described in Table 1). Moreover, this percentage is higher than that described in the literature with different lignocellulosic substrates and in shorter time.

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.

Fits. It should be noted here that when using sodium hydroxide pretreated biomass followed by peracetic acid, the conversion results were better when the reaction was performed in the ultrasound bath by about 10% compared to that performed in shaker, which confirms the results obtained in patent application PI 1102362-7 and incorporated herein by reference in its entirety.

Therefore, 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. The third aspect of the present invention is to realize the; enzymatic hydrolysis using sodium hydroxide pretreated biomass (5%) (0.2g) and compare the results with sodium hydroxide pretreated biomass followed by peracetic acid (0.2g) (pretreatment described in Patent Application PI 1102362-7 and incorporated herein by reference in its entirety), with the mixture of enzymes NS 22074 and NS 50012 (0.03mL), 10 ml buffer. Sodium acetate pH = 4.8, stir this mixture on a horizontal shaker shaker at 50 ° C, rpm 150 for 4, 24 and 48 hours. 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.

In this present invention it should also be noted that by doubling the amount of biomass pretreated with sodium hydroxide using the same amount of enzyme. The results were significant as they observed a reduction in the enzyme required for ethanol production, these data were compared with the results obtained with sodium hydroxide pretreated biomass followed by hydrogen peroxide and 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. . _.

Table 3 - Enzymatic hydrolysis of pretreated biomass chemically with sodium hydroxide (5%) or sodium hydroxide followed by peracetic acid with the mixture of enzymes NS 22074 and NS 50012 in horizontal shaker.

Reaction conditions: pretreated biomass (6.7g / mL

5 enzyme); 1:50 biomass / volume of sodium acetate buffer pH = 4.8) shaker bath 50 ° C, 150 rpm.

From the results obtained in Table 3, it is observed that by performing enzymatic hydrolysis with a larger mass and the same amount of enzyme, it formed a larger amount of fermentable sugars, that is, the cost of the process became economically lower. feasible as a lower amount of enzyme is required.

In addition, the conversion results obtained using sodium hydroxide pretreated biomass are better than those obtained with sodium hydroxide pretreated biomass followed by peracetic acid.

The fourth aspect of the present invention is to perform enzymatic hydrolysis using sodium hydroxide pretreated biomass (3%) (0.2g) and compare the results with? 0 the steam explosion pretreated biomass followed by treatment with sodium hydroxide (3%) (0.2g) with the mixture of the enzymes NS 22074 and NS .50012 (0.03mL), 10 ml sodium acetate buffer pH = 4.8, stir this mixture on a shaker shaker horizontal at 50 ° C, rpm 150 for 4, 24 and 48 hours. 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.

To further lower the cost of the process, enzymatic hydrolysis of the sodium hydroxide (3%) pre-treated biomass at reflux for 2 hours and steam blast pretreated biomass followed by pre-treatment was carried out in the present invention. with sodium hydroxide (3%).

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 4 - Enzymatic hydrolysis of biomass pretreated chemically with sodium hydroxide (3%) or with steam explosion followed by sodium hydroxide (3%) with the mixture of enzymes NS 22074 and NS 50012 in horizontal shaker.

NaOH Steam Burst

/ NaOH Time Biomass Yield Biomass Yield

(H) , . (%) ^■ required (%) required to produce for

1 kg producing 1 kg reducing sugars reducing sugars

4 25 4.0 kg 21.5 4.7 kg

24 62.5 1.6 kg 37.5 2.7 kg

48 85 1.2 kg 71.5 1.4 kg

Reaction conditions: pretreated biomass (6.7g / mL enzyme); 1:50 biomass / volume of sodium acetate buffer pH = 4.8) shaker bath 50 ° C, 150 rpm.

From the results obtained in Table 4, it can be seen that by performing enzymatic hydrolysis of sodium hydroxide pretreated biomass (3%) or steam blast pretreated biomass followed by sodium hydroxide treatment ( 3%), a higher amount of fermentable sugars was formed, ie the cost of the process became more economically viable as a lower amount of enzyme is required.

Therefore, the best result obtained is to perform enzymatic hydrolysis in shaker during 48 hours of reaction of sodium hydroxide pretreated biomass (3%), because to produce lkg of fermentable sugars only 1.2 kg of pretreated biomass is required. and only 180mL of the mixture of enzymes NS 22074 and NS 50012, proving to be economically viable. Furthermore, another advantage of using sodium hydroxide pretreatment under these reaction conditions is that this treatment does not form inhibitors, thus showing a further major advantage. The results of HPLC chromatography analyzes of the 2-hour refluxed steam-blown sodium hydroxide (3%) banana pseudostem samples followed by pretreatment with sodium hydroxide are presented in Tables 5 and 6, respectively. Table 5 shows the absence of glucose and xylose and the presence of sucrose as the main reducing sugar contrary to expectations. In addition, Table 5 shows only very small amounts of organic acids, and the absence of the main interfering fermentation process, hydroxymethylfurfural.

Table 5 - HPLC chromatography analyzes of the sodium hydroxide-treated banana pseudostem (3%) samples at 2 hours of reflux.

NaoH pretreated banana pseudostem (3%) with 2 hours reflux

UNIT RESULT PARAMETER

Glucose g / 100 mL ND

Fructose g / 100 mL ND

Xylose g / 100 mL ND

Sucrose g / 100 mL 1,53

Galactose g / 100 mL 0.14

Mannose g / 100 mL ND

Ribose g / 100 mL ND

Mannitol g / 100 mL 0.38

Sorbitol g / 100 mL 0.04

Arabinosis g / 100 mL 0.04

Maltose g / 100 mL ND

Maltotriose g / 100 mL 0.01

Acetic acid mg / L 124.6

Lactic acid mg / L 5, 95 Propionic acid mg / L 16.4

Citric acid mg / L 4.79

HMF mg / L ND

(hydroxymethylfurfural)

ND - Not Detected

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.

NaoH pretreated banana pseudostem (3%) with 2 hours of reflux

UNIT RESULT PARAMETER

Glucose g / 100 mL 0.81

Fructose g / 100 mL ND

Xylose g / 100 mL ND

Sucrose g / 100 mL 1.96

Galactose g / 100 mL 0.07

Mannose g / 100 mL ND

Ribose g / 100 mL ND

Mannitol g / 100 mL 0.24

Sorbitol g / 100 mL ND

Arabinosis g / 100 mL 0.06

Maltose g / 100 mL ND

Maltotriose g / 100 mL 0.02 Acetic acid mg / L 151.4

Lactic acid mg / L 8.05

Propionic acid mg / L ND

Citric acid mg / L ND

HMF mg / l 53.1

(hydroxymethylfurfural)

ND - Not Detected

The reaction conditions of the chemical treatment and subsequent enzymatic hydrolysis described in Table 4, besides resulting in the best conversion of reducing sugars, presents a very favorable condition for the fermentation of these sugars for bioethanol production. This is justified by the challenge of obtaining ethanol from cellulose, which is the fermentation of pentoses. Hemicelluloses are rich in pentoses such as xylose and arabinosis. Saccharomyces cereviseae, a microorganism commonly used in the production of alcohol from sucrose, is very inefficient in pentose conversion. The presence of pentoses actually inhibits the fermentation of hexoses. Cellulosic ethanol yields will be increased if virtually all types of sugars in cellulose and hemicellulose are converted to ethanol. 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. In the present invention, 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. enzymatic hydrolysis with sodium hydroxide (5%) (0.1g) pretreated biomass with NS 22074 (0.03 mL) and NS 22074 (0.015 mL) and 50.012 ( 0.015 mL), 5 mL sodium acetate buffer pH = 4.8, horizontal shaker shaker at 50 ° C, 150 rpm for 4 and 24 hours. The results obtained are presented in Table 7.

Table 7 - Enzymatic hydrolysis of biomass pretreated chemically with sodium hydroxide (5%) with the mixture of the enzymes NS 22074 and NS 50012 or with the enzyme NS 22074 in horizontal shaker.

Reaction conditions: chemically pretreated biomass (3.3 g / mL enzyme), 1:50 biomass / volume of sodium acetate buffer pH = 4.8) shaker bath temperature 50 ° C, 150 rpm.

From the results obtained in Table 7, it is observed that when performing the enzymatic hydrolysis with the mixture of the NS 22074 and NS 50012 conversion results were much better compared to using the NS 22074 enzyme, ie the conversion to fermentable sugars was approximately 80% higher compared to using only the NS 22074 enzyme in just. reaction. Therefore, it is a further advantage of this patent to use enzyme blending, which causes the enzyme cost to be significantly reduced and makes the process economically viable.

References

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Claims

KING VIDICATIONS

1. Process for the production of bioethanol from banana pseudostem with enzymatic hydrolysis characterized by the fact that it comprises the following steps:

a) Wash the banana pseudostem under running water about 3 times to remove dirt and resin, then place in oven at 60 ° C for drying.

b) grind the banana pseudostem and store for later chemical treatments and enzymatic hydrolysis.

c) pretreatment with sodium hydroxide followed by the mixture of acetic acid and hydrogen peroxide.

Process for the production of bioethanol from banana pseudostem according to claim 1, characterized in that step (c) comprises the following steps:

(a) treat the banana pseudostem with sodium hydroxide for 1 hour at 70 ° C (1:10 m / v) on a magnetic stirrer;

(b) adding the mixture of acetic acid and hydrogen peroxide (30%) and making up to volume with distilled water (1:10 m / v) for 6 hours at 70 ° C on a magnetic stirrer or ultrasound with a fixed temperature of 60 ° C.

c) filter the sample and wash with distilled water to pH: 4-5, and

d) put to dry and grind.

Process for the production of bioethanol from banana pseudostem according to Claim 2, characterized in that the mixture of acetic acid and Hydrogen peroxide comprises 40% acetic acid and 60% hydrogen peroxide.

4. Process for the production of bioethanol from banana pseudostem characterized by the fact that it comprises the following steps:

a) wash the banana pseudostem with water. about 3 times to remove dirt and resin, then place in oven at 60 ° C for drying.

c) pretreatment with sodium hydroxide followed by the mixture of vinegar and hydrogen peroxide.

Process for the production of bioethanol from banana pseudostem according to claim 4, characterized in that step (c) comprises the following steps:

(b) adding the mixture of vinegar and hydrogen peroxide (30%), the remainder being complete with distilled water (1:10 m / v) for β hours at 70 ° C;

c) filter the sample and wash with distilled water to pH: 4-5, and

d) put to dry and grind.

Process for the production of bioethanol from banana pseudostem according to claim 5, characterized in that the mixture of vinegar and hydrogen peroxide comprises 40% vinegar and 60% hydrogen peroxide.

7. Process for the production of bioethanol from Banana pseudocaule characterized by the fact that it comprises the following steps:

a) Wash with running water about 3 times to remove dirt and resin, then place in oven at 60 ° C for drying.

c) pre-treating with sodium hydroxide followed by the mixture of sodium hypochlorite and hydrogen peroxide.

(b) adding the mixture of sodium hypochlorite and hydrogen peroxide (5: 1) (1:10 v / v for 6 hours at 70 ° C;

c) filter the sample and wash with distilled water to pH: 4-5,

d) put to dry and grind.

9. Process for the production of bioethanol from banana pseudostem characterized by the fact that it comprises the following steps:

) Wash with running water about 3 times to remove dirt and resin, then place in oven at 60 ° C for drying.

) grind banana pseudostem and store for later chemical treatments and enzymatic hydrolysis. ) pre-treat with sodium hydroxide.

Process for the production of bioethanol from banana pseudostem according to claim 9, characterized in that step (c) comprises the following steps:

a) treat the banana pseudostem with sodium hydroxide. (3%) or (5%) (1:20 m / v) for 2 hours under reflux,

(b) filter the sample and wash with distilled water; dry and grind.

11. Process for the production of bioethanol from banana pseudostem characterized by the fact that it comprises the following steps:

c) pre-treatment with sulfuric acid.

Process for the production of bioethanol from banana pseudostem according to claim 11, characterized in that step (c) comprises the following steps:

(a) treat banana pseudostem with 5% sulfuric acid (1:20 m / v) for 2 hours under reflux,

b) filter the sample and wash with distilled water; c) place to dry and grind.

13. Process for the production of bioethanol from banana pseudostem characterized by the fact that it comprises the following steps:

a) wash with running water about 3 times to remove dirt and resin, then put in oven at 60 ° C for drying, (b) grind the pseudostem of. , banana and store to later perform the. .chemical treatments and enzymatic hydrolysis.

c) pre-treatment with sodium hydroxide. combined with sulfuric acid.

14. Procedure. for the production of bioethanol from. Banana pseudostem according to Claim 13, characterized in that step (c) comprises the following steps:

a) treat the banana pseudostem with sodium hydroxide (5%) (1:20 m / v) for 2 hours under reflux, b) add sulfuric acid (5%) (1:10 m / v) for 2 hours under reflux,

c) filter the sample and wash with distilled water,

d) put to dry and grind.

15. Process for the production of bioethanol from banana pseudostem characterized by the fact that it comprises the following steps:

(c) pre-treat with steam explosion followed by pre-treatment with sodium hydroxide.

Process for the production of bioethanol from banana pseudostem according to claim 15, characterized in that step (c) comprises the following steps:

(a) Treat the banana blast with steam for 8 minutes at 200 ° C followed by pre-treatment. with sodium hydroxide (3%) (1:20 m / v) for 2 hours under reflux,

b) filter the sample and wash with distilled water; c) place to dry and grind.

Process for the production of bioethanol from banana pseudostem according to claims 1 to 12, characterized in that said enzymatic hydrolysis is carried out according to the following steps:

a) addition of 0.1 g pretreated biomass, 0.03 ml of NS 22074 and NS 50012 mixture, 5 ml sodium acetate buffer pH = 4.8; and

b) The mixture was left in ultrasonic bath temperature set ^to 60 ° C for 4 hours.

18. Process for the production of bioethanol from banana pseudostem characterized by the fact that it comprises the following steps:

a) Hydrolyzing the pretreated biomass as defined by claims 1-12 (3.3g / ml enzyme) directly in the ultrasound bath with the mixture of the NS 22074 and NS 50012 cellulase enzymes for 4 hours;

(b) Use 1:50 biomass / volume of sodium acetate buffer pH = 4.8.

19. Process for the production of bioethanol from banana pseudostem characterized by the fact that it comprises the following steps:

a) Hydrolyze the pretreated biomass with sodium hydroxide and sodium hydroxide followed by horizontal type shaker peracetic acid (3.3g / mL of enzymes) with the mixture of cellulase enzymes NS 22074 and NS 50012 or only with the enzyme NS 22074 for 4 and 24 hours;

b) Use 1:50 biomass / volume of sodium acetate buffer pH = 4.8;

c) Use temperature of 50 ° C and rpm of 150.

20. Process for the production of bioethanol from banana pseudostem characterized by the fact that

. comprises the following steps:

The). Hydrolyze pretreated biomass with sodium hydroxide 5 or sodium hydroxide followed by horizontal type shaker peracetic acid (6.7 g. / ML of enzymes) with the mixture of cellulase enzymes NS 22074 and NS 50012 for 4, 24 and 48 hours

b) Using 1:50 of biomass / volume of buffer 10 sodium acetate pH = 4.8; and

c) Use temperature of 50 ° C and rpm of 150.

Process for the production of bioethanol from banana pseudostem according to claims 1 to 20, characterized in that the enzyme cellulase NS

22074 is a cellulosic glucosidase and xylanase complex.

Process for the production of bioethanol from banana pseudostem according to claims 1 to 20, characterized in that the enzyme NS 50012 is a cellulosic complex - arabinase,

20 hemicellulase, cellulase, pectinase and xylanase.

Process for the production of bioethanol from banana pseudostem according to claims 1 to 20, characterized in that the banana tree is preferably of the type producing banana caturra.

A process for the production of bioethanol according to any one of claims 1 to 12, characterized in that the conversion of reducing sugars by hydrolysis was 33 to 51%.

Process for the production of bioethanol according to claim 10. 19, characterized by the fact that the conversion of reducing sugars by hydrolysis was 31-96%.

26. Process for the production of bioethanol according to claim 20, characterized in that the conversion of reducing sugars by hydrolysis was from 10 to 70%.

Process for the production of bioethanol according to claim 10, characterized in that the conversion of reducing sugars by hydrolysis with sodium hydroxide (3%) was 25-85%.

Process for the production of bioethanol according to claims 15 and 16, characterized in that the conversion of reducing sugars by hydrolysis was 21.5 to 71.5%.

Process for the production of bioethanol according to claim 10, characterized in that the enzymatic hydrolyzate obtained to produce ethanol comprises a mixture of fermentable sugars having sucrose as the main sugar, the absence of glucose and xylose and the absence of hydroxymethylfurfural.

Process for the production of bioethanol according to claims 15 and 16, characterized in that the enzymatic hydrolyzate obtained to produce ethanol comprises a mixture of fermentable sugars having sucrose as main sugar, glucose as secondary sugar, absence of xylose and the presence of hydroxymethylfurfural in very small quantity.

Use of the process as defined by claims 1 to 30 wherein reducing sugars are produced to produce ethanol.

Use according to claim 31, characterized in that the ethanol production process is carried out by fermentation with fermentable sugar yeasts.