RO134127B1 - Method for alkaline pre-treatment of lignocellulosic biomass in the presence of fatty acid methyl esters (fame) - Google Patents

Description

RO 134127 Β1

The invention relates to a simplified method of alkaline pretreatment of lignocellulosic biomass in the presence of fatty acid methyl esters (FAME). This method delineates the lignocellulosic material simultaneously with the removal of waxes and extractable substances that can act as inhibitors of subsequent processes of hydrolysis of cellulose. The lignocellulosic biomass thus pretreated shows a high availability for enzymatic hydrolysis.

Pre-treatment of lignocellulosic biomass plays a key role in obtaining materials with acceptable enzymatic digestibility and subsequent fermentability for the production of cellulosic ethanol or other biofuels such as biomass-derived butanol. Pretreatment is a necessary step in modifying the structure of lignocellulose and improving the access of enzymes during enzymatic hydrolysis (Alvira P., Tomas-Pejo E., Ballesteros M., Negro MJ, "Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review Bioresource Technology, (2010), 10: 4851-4861). Currently, procedures for the preparation and pretreatment of lignocellulosic biomass for fractionation into valuable components have been intensively investigated. Pre-treatment methods can be physical (grinding, ultrasonic or microwave irradiation), chemical (treatment with acids or bases, ozonolysis, ionic liquids) or biological (by treatment with fungi or bacteria). Although these methods have evolved, pretreatment is still one of the most expensive steps in the entire process of converting lignocellulosic biomass into valuable products. The telinico-economic analysis of lignocellulosic ethanol production processes shows that the pretreatment steps represent between 11-27% of the total costs, depending on the biomass pretreatment method used (Kucharska K., Rybarczyk P., Holowacz I., Lukajtis R. , Glinka M., Kaminski M., „Pretreatment of Lignocelluloslc Materials as Substrates for Fermentation Processes, Molecules, (2018), 23: 2937). To reduce these costs, it is advisable to replace expensive solvents, for example ionic liquids (US 2016/0017540 A1, Pretreatment and fractionation of lignocellulosic biomass) with much cheaper solvents (FAME) and also to reduce the number of process steps. An effective pre-treatment method is the action of alkaline solutions. Alkaline treatment can be used to remove lignin thus increasing the digestibility of cellulose. Compared to acid pretreatment and hydrothermal processes, alkaline pretreatment leads to a lower solubilization of hemicelluloses, the formation of smaller amounts of inhibitory compounds and has the advantage that it takes place at lower temperatures. Alkaline reagents degrade ester side coffees and glycosidic bonds that lead to structural modification of lignin, cellulose swelling, cellulose decrystallization, and hemicellulose solubilization. For enzymatic hydrolysis it is essential to eliminate lignin in order to have efficient access to reducing cellulose (Kumar AK, Sharma S., “Recent updates on differentmethods of pretreatment of lignocellulosic feedstocks: a review, Bioresourcess and Bioprocessing, (2017), 4 (7) : 1-19).

Lignocellulosic biomass consists mainly of cellulose (38-50%), hemicellulose (23-32%), lignin (15-25%), as well as small amounts of extractables. Extractables or foreign components are those substances that are removed from the walls of lignocellulosic cells by extraction with neutral solvents. These materials are extra-parietal substances that are deposited after the formation of the cell wall and are not considered essential components of the cell wall (Soltes EJ, Wood and Agriculture! Residues, Research on Use for Feed, Fuels, and Chemicals, Academic Press, 1983). The largest amounts are found in the cell lumen, but may also be present in the cell wall matrix. The quantities of tissue extractables vary according to the plant species,

RO 134127 Β1 family and tissues, and the components are extraordinarily diverse (Hillis WE 1 „Occurrence of Extractives in Wood Tissues in Biosynthesis and Biodegradation of Wood Component, Academic Press, 1985). Due to this diversity, there were no 3 generalized procedures for isolation and determination of individual compounds. Several researchers presented an in-depth discussion on chemical classification, biosynthesis, 5 isolation and quantification of extractables (Sjostrom E. „Wood Chemistry Fundamentals and Applications, ^2nd ed. Academic Press, 1993, Lewin M and Goldstein I„ WoodStructure 7 and Composition Marcel Dekker, Inc., 1991).

Extractable wood substances are a heterogeneous group of compounds that can be extracted with polar or non-polar solvents. They consist mainly of terpenes, fats, proteins, waxes, resins and phenolic compounds and although they are present in small quantities, they are 11 essential for further biomass processing (Thammasouk K., Tandjo D., Penner MH, "Influence of Extractivity on the Analyaia of Herbaceoua Biomass, Journal of 13 Agriculture! and Food Chemistry, (1997), 45 (2): 437-443). Most lignocellulose inhibitors are formed during pretreatment when the extractable substances, hemicelluloses and / or lignin are solubilized and degraded. During alkaline pretreatment the extractable substances may be transformed into phenolic compounds or fatty extracts which may precipitate and act as inhibitors in subsequent enzymatic saccharification processes (Jonsson U., Martin C., “Pretreatment of lignocellulose: Formation of inhibitory by products and 19 strategiesforminimizingtheireffects, Bioresource Technology, (2016), 199: 103-112). In order to avoid the formation of inhibitory compounds, before the alkaline pretreatment of lignocellulosic biomass 21, the extractable materials are removed by reflux treatment for 4-6 hours with various solvents (acetone, dichloromethane, toluene, ethanol) and subsequent treatment with hot water. 2. 3

These methods reduce the economic efficiency of industrial processes, both due to the consumption of solvents and the energy consumption during treatment. In addition, the processes require 25 additional equipments to perform these steps (Call RC, Tomkinson J., “Characterization of hemicelluloses obtained by classical and ultraaonically assisted 27 extraction from wheat straw, Carbohydrate Polymers, (2002) 50: 263-271; Liu CF , Ren JL, Xu F., Liu JJ, Sun JX, Sun RC, Laolation and Characterization of 29 Celluloae Obtained from Ultraaonic Irradiated Sugarcane Bagasse, J. Agric. Food Chem. (2006) 54: 5742-5748). 31

The technical problem solved by the invention consists in establishing a simplified process for pretreatment of lignocellulosic biomass so as to determine the obtaining of a high concentration of sugars in the enzymatic hydrolysis process.

The alkaline pretreatment method of lignocellulosic biomass in the presence of methyl esters of fatty acids, in order to simultaneously remove the extractable substances and lignin, according to the invention, performs the treatment of 2 g of sawdust, with a size of 0.315 ... 0.5 mm, 37 for 20 min, with 200 mL NaOH solution with a concentration of 0,1 ... 1 N, in which 0,1 ... 0,5% of the total reaction volume is introduced, methyl esters of fatty acids, at a temperature of 39 ° C, with stirring at 600 rpm, followed by separation of the residue and washing with distilled water to neutral pH and with a known aqueous solution of 0,1% surfactant, filtration 41 of the pretreated residue and its enzymatic hydrolysis in a mixture with a citric acid-disodium phosphate buffer at a pretreated sawdust ratio: a buffer solution of 1:25 weight / volume 43 solid / liquid, using as a biocatalyst - cellulase-type enzyme in a ratio of 0.7 mL / g _sawdust for 72 hours at 50 ° C and speed of agitation 120 rpm, obtaining 45 a sawdust delignification by up to 24.5% and an increase in the concentration of reducing sugars by up to 21.8% compared to those obtained by enzymatic hydrolysis of pretreated sawdust 47 in the absence of methyl esters of acids fat.

RO 134127 Β1

The invention has the following advantages:

- reducing the number of stages of the lignocellulosic biomass delignification process by removing waxes and extractable substances simultaneously with biomass delignification;

- an increase of approximately 22% in the content of reducing sugars in alkaline pretreated biomass in the presence of FAME subject to enzymatic hydrolysis compared to conventional methods.

The aim of this invention is to simplify the process of pretreatment of lignocellulosic biomass by using a cheap solvent - methyl esters of fatty acids (FAME) added in small amounts during alkaline pretreatment and which is able to simplify the process (removable substances are removed in the same stage with delignification of biomass) and at the same time to make it more efficient (allows to obtain high concentrations of sugars in the stage of enzymatic hydrolysis of the material thus pretreated).

FAME has two roles in alkaline pretreatment, on the one hand, it acts as a solvent for extractable materials and compounds resulting from delignification of lignocellulosic material, and on the other hand it can partially react with sodium hydroxide and form soaps (sodium salts of acids fats) which due to their superficially active properties can emulsify waxes and extractable substances.

Description of the alkaline pretreatment method of lignocellulosic biomass in the presence of fatty acid methyl esters (FAME)

The technical problem solved by the invention is the efficiency of the pretreatment of lignocellulosic materials by a simplified method of alkaline pretreatment of lignocellulosic biomass in the presence of fatty acid methyl esters (FAME). This method delineates the lignocellulosic material simultaneously with the removal of waxes and extractable substances that can act as inhibitors of subsequent processes of hydrolysis of cellulose.

Alkaline pretreatment was performed at room temperature in the presence of a small amount (0.1-0.5% of the total reaction volume) of methyl esters of fatty acids (FAME with specifications according to EN 14214 - European Committee for Standardization, Liquid Petroleum products - Fatty acid methyl esters (FAME) for use in diesel engines and heating applications - Requirements and test methods BS EN 14214: 2012 + A2: 2019), using sodium hydroxide solutions of different concentrations (0.1-1 N) . The role of esters is solvent for extractable materials and to react during the process with sodium hydroxide with the formation of soap to emulsify and entrain the extractables from the raw sawdust. After the alkaline pretreatment, the mixture is filtered and the liquid phase is subjected to spectrophotometric analysis to determine the lignin content (Sluiter A., Hames B., Ruiz R., Scarlata C., Sluiter J., Templeton D., Crocker D., Determination of Structural Carbohydrates and Lignin in Biomass, Laboratory Analytical Procedure (LAP), NREL, (2005). can act as inhibitors in the process of enzymatic hydrolysis, followed by washing with distilled water to pH-neutral. The pretreated sawdust is subjected to enzymatic hydrolysis for 72 hours to obtain sugars. pH = 5), using as biocatalysts - cellulases, the commercial assortment Celluclast 1.5 L.

After enzymatic hydrolysis, the reaction mixture is heated to 100 ° C to deactivate the enzyme, cooled and centrifuged to separate the solid residue. The liquid phase was subjected to spectrophotometric analysis to determine the reducing sugars using the spectrophotometric method by treatment with DNS reagent (3,5-dinitrosalicylic acid) and the

RO 134127 rea1 absorbance at a wavelength of 575 nm (Miller GL, Use of dinitrosalicylic acid 1 reagent for determination of reducing sugar ”, Anal. Chem. (1959), 31 (3), 426-428 and TAPPI (1979) Official Test Methods and Provisional Test Methods (Technical 3 Association of the Pulp and Paper Industries).

The following are 3 examples of alkaline pretreatment in the presence of 5 methyl fatty acid esters which illustrate the invention without limitation. The examples are also given in connection with the flow chart of operations of the invention. 1. The following examples show the efficiency of the pretreatment method depending on the modification of the main reaction parameters, as well as the efficiency of this method in the enzymatic hydrolysis processes of the pretreated lignocellulosic materials. 11

Example 1

To study the effect of the addition of FAME on the delignification of sawdust and later on the enzymatic hydrolysis of pretreated sawdust, the first example refers to a comparison between alkaline pretreatment of different types of sawdust (extracted sawdust, 15 raw sawdust and raw sawdust with FAME).

a) Alkaline pretreatment stage 17

The raw material is sawdust - the residue from the manufacture of furniture that contains hardwood (hardwood - beech) and softwood (fir), ground to a size between 19 0.315 and 0.5 mm. Two types of sawdust were used for pretreatment:

1. Extract sawdust - the sawdust used was ground and the sieve was separated by fraction 21 with dimensions between 0.315 and 0.5 mm. Prior to alkaline pretreatment, the lignocellulosic material was extracted successively with a mixture of toluene: ethanol = 1: 2 (for 6 h) and then with 23 water (for 2 h) to remove waxes and extractable materials that may act as inhibitors in the process of enzymatic hydrolysis. 25

2. Raw sawdust - the sawdust used was ground to a size between 0,315 and 0,5 mm and used in alkaline pretreatment without other extractions. 27

Alkaline hydrolysis of 2 g of sawdust (crude or extracted according to the procedure described above) was carried out by treating it with 200 mL of 1N NaOH solution for 20 min at 29 ° C with stirring at 600 rpm. The residue is separated and washed with distilled water to neutral pH. In the case of alkaline treatment in the presence of FAME, another washing step 31 with a weak solution of surfactant (0.1%) is introduced to completely remove the unconverted esters in the soap, which may act as inhibitors in the enzymatic hydrolysis process. At the end of the 33 alkaline pretreatment, samples were taken from the solution to determine the 320 nm metric spectrophot of soluble lignin. The alkaline pretreatment conditions and the amount of separated lignin are shown in Table 1. The lignocellulosic material resulting from the pretreatment is subjected to enzymatic hydrolysis. 37

Conditions of operation and results of alkaline pretreatment 39

Table 1

Nr. Material Alkaline pretreatment conditions: temperature Separate wood 41 exp. 25 ° C lignocellulosic, sawdust ratio: alkaline solution (mg / g _SU bstrat) *

1: 100, pretreatment time 20 min

Sawdust extracted NaOH solution 1 N 10.75 ± 0.34 43

Crude sawdust NaOH solution 1 N 26.22 ± 0.17

Raw sawdust 1 N NaOH solution and addition of 0.3% FAME 29.84 ± 0.63 45 * All experiments were performed in triplicate and the resulting values are presented together with the standard deviation 47

RO 134127 Β1

From the results presented in Table 1 it can be seen that the delignification of the lignocellulosic material is better in the case of alkaline pretreatment in the presence of methyl esters of fatty acids.

b) Stage of enzymatic hydrolysis in the process of enzymatic hydrolysis The delignified pretreated material (from previous experiments) is further mixed with a buffer solution of citric acid Na ₂ HPO ₄ to ensure an optimal pH = 5 for enzymes. Enzymatic hydrolysis processes were performed in 100 mL reactors containing a mixture of buffer solution and sawdust in a ratio of 1:25 (weight / volume solid / liquid). Enzyme - Celluclast 1.5 L in a ratio of 0.7 mL / g _sawdust is added to this mixture. 0.5% antibiotic is added to prevent bacterial contamination. The reactor was then placed in an orbital shaker (120 rpm). The hydrolysis time is 72 h and the reaction temperature is 50 ° C. During the reaction, hydrolysis samples were taken at 24, 48, and 72 h, which were passed into a vessel with hot water (to deactivate the enzyme) and centrifuged at 3500 rpm for 10 min. The supernatants were stored in the refrigerator for analysis of the sugars obtained. The amount of reducing sugars obtained from hydrolysis is shown in Table 2.

From the results presented in Table 2 it can be seen that by enzymatic hydrolysis of the sawdust subjected to alkaline pretreatment in the presence of methyl esters of fatty acids a higher amount of reducing sugars was obtained (representing about 30% of the total sugars in the sawdust studied). This increase in the amount of sugars shows that the use of FAME in the alkaline pretreatment of lignocellulosic materials has effectively removed some of the lignin together with the extractable materials which may be inhibitors of the enzymatic hydrolysis process.

Operating conditions and results of enzymatic hydrolysis

Table 2

Nr. exp. Lignocellulosic material subjected to enzymatic hydrolysis Reducing sugar content (mgGE / g _substrate ) * 1 Alkaline pretreated extract sawdust 119.17 ± 5.34 2 Alkaline pretreated raw sawdust without FAME 110.96 ± 5.97 3 Alkaline pretreated raw sawdust with FAME 120.48 ± 2.98

* All experiments were performed in triplicate and the resulting values are presented together with the standard deviation

Example 2

In order to highlight the efficiency of the addition of FAME in the alkaline pretreatment, the influence of the variation of the FAME content on the amount of separated lingin during the alkaline pretreatment and on the amount of hydrolyzed sugars in the pretreated sawdust was followed.

a) Alkaline pretreatment stage

The alkaline pretreatment procedure in the presence of FAME is the same as described in the previous example, changing the amount of FAME. Table 3 shows the alkaline pretreatment conditions and the lignin content separated from the pretreatment. The lignocellulosic material resulting from the alkaline pretreatment in the presence of different concentrations of FAME is subjected to enzymatic hydrolysis.

RO 134127 Β1

Operating conditions and results of alkaline pretreatment

Table 3

Nr. exp. Lignocellulosic material Alkaline pretreatment conditions FAME concentration (%) Separate lignin (mg / g _s substrate) * 1 Raw sawdust 1 N NaOH solution, temperature 25 ° C, sawdust ratio: 1: 100 alkaline solution, pretreatment time 20 min 0 26.22 ± 0.17 2 Raw sawdust 16 28.01 ± 0.34 3 Raw sawdust 3 29.84 ± 10.63 4 Raw sawdust 5 32.66 ± 10.37

* All experiments were performed in triplicate and the resulting values are presented together with the standard deviation

From the results presented in Table 3 it can be seen that the delignification of lignocellulosic material increases with increasing amount of FAME added to alkaline pretreatment, the amount of separated lignin being up to 24.5% higher in the presence of methyl esters of fatty acids compared to the process in absence of esters.

b) The stage of enzymatic hydrolysis

Enzymatic hydrolysis of alkaline pretreated sawdust using different concentrations of FAME was performed under the same conditions as in the previous example. The hydrolysis conditions and the reducing sugar content obtained after 72 hours of enzymatic hydrolysis are shown in Table 4.

Operating conditions and results of enzymatic hydrolysis

Table 4

Nr. exp. Pretreated raw sawdust Enzymatic hydrolysis conditions Reducing sugar content (mgGE / g _substrate ) * 1 Alkaline + 0% FAME Commercial enzyme - Celluclast 1.5L, 0.7 mL / g _sawdust , citric acid and disodium phosphate buffer to maintain pH 5, reaction temperature 50 ° C, sawdust ratio: buffer solution = 1:25, stirring speed 120 rpm, enzymatic hydrolysis time 72 h 110.96 ± 5.97 2 Alkaline + 0.16% FAME 119.61 ± 1.66 3 Alkaline + 0.3% FAME 120.48 ± 2.98 4 Alkaline + 0.5% FAME 135.14 ± 3.44

* All experiments were performed in triplicate and the resulting values are presented together with the standard deviation

From the results presented in Table 4 it can be seen that by enzymatic hydrolysis of the sawdust subjected to alkaline pretreatment in the presence of methyl esters of fatty acids a higher amount of reducing sugars was obtained with increasing amount of FAME used in alkaline pretreatment, obtaining an increase of the concentration of sugars reducing by up to 21.8% compared to those obtained by enzymatic hydrolysis of the pretreated sawdust in the absence of methyl esters of fatty acids.

Example 3 This example highlights the effectiveness of the addition of FAME in alkaline pretreatment when using different concentrations of sodium hydroxide. The influence of the variation of NaOH concentration on both the amount of separated lingin during the alkaline pretreatment and the amount of hydrolyzed sugars in the pretreated sawdust was followed.

RO 134127 Β1

a) Alkaline pretreatment stage

The alkaline pretreatment procedure in the presence of FAME is the same as described in the previous example, changing the concentration of the alkaline NaOH solution (0,1,0,5 and 1 N). Table 5 shows the alkaline pretreatment conditions and the lignin content separated from the pretreatment. The lignocellulosic material resulting from the alkaline pretreatment is subjected to enzymatic hydrolysis.

Operating conditions and results of alkaline pretreatment

Table 5

Nr. exp. Lignocellulosic material Alkaline pretreatment conditions NaOH (N) concentration Separate lignin (mg / g _s substrate) * 1 Raw sawdust FAME 0.3%, temperature 25 C, sawdust ratio: 1: 100 alkaline solution, pretreatment time 20 min 1 22.8 ± 0.19 2 Raw sawdust 5 24.16 ± 0.28 3 Raw sawdust 1 29.84 ± 10.63

* All experiments were performed in triplicate and the resulting values are presented together with the standard deviation

From the results presented in Table 5 it can be seen that the delignification of the lignocellulosic material is better with the increase of the concentration of sodium hydroxide used for the alkaline pretreatment.

b) The stage of enzymatic hydrolysis

Enzymatic hydrolysis of alkaline pretreated sawdust using different concentrations of sodium hydroxide was performed under the same conditions as in the previous examples. The hydrolysis conditions and the reducing sugar content obtained after 72 hours of enzymatic hydrolysis are shown in Table 6.

Operating conditions and results of enzymatic hydrolysis

Table 6

Nr. exp. Pretreated raw sawdust Enzymatic hydrolysis conditions Reducing sugar content (mgGE / g _substrate ) * 1 Alkaline + FAME (0.1 N NaOH Sol.) Commercial enzyme - Celluclast 1.5 L, 0.7 mL / g _sawdust , citric acid and disodium phosphate buffer to maintain pH at 5, reaction temperature 50 ° C, sawdust ratio: solution buffer = 1:25, stirring speed 120 rpm, enzymatic hydrolysis time 72 h 50.89 ± 15.97 2 Alkaline + FAME (Sol. NaOH 0.5 N) 95.60 ± 1.66 3 Alkaline + FAME (Sol. NaOH 1N) 120.48 ± 12.98

* All experiments were performed in triplicate and the resulting values are presented together with the standard deviation

From the results presented in Table 6 it can be seen that by enzymatic hydrolysis of the sawdust subjected to alkaline pretreatment in the presence of FAME, a higher amount of reducing sugars was obtained for the sawdust pretreated with more concentrated alkaline solutions.

Claims (1)

RO 134127 Β1 Claim 1 Alkaline pretreatment method for lignocellulosic biomass in the presence of 3 methyl esters of fatty acids, for the simultaneous removal of extractable substances and lignin, characterized in that it performs the treatment of 2 g of sawdust, with a size 5 of 0.315 ... 0, 5 mm, for 20 min, with 200 mL NaOH solution with a concentration of 0,1 ... 1 N, in which 0,1 ... 0,5% of the total reaction volume is introduced, methyl esters of fatty acids, 7 at a temperature of 25 ° C, with stirring at 600 rpm, followed by separation of the residue and washing with distilled water to neutral pH and with an aqueous solution of 0.1% surfactant known per se, 9 filtration of the pretreated residue and enzymatic hydrolysis thereof mixed with a citric acid-disodium phosphate buffer at a pretreated sawdust ratio: buffer solution of 1:25 11 weight / volume solid / liquid, using as biocatalyst - cellulase-type enzyme in a ratio of 0.7 mL / g _sawdust for 72 hours at 50 ° C and speed 120 rpm, 13 yielding a sawdust delignification by up to 24.5% and an increase in the concentration of reducing sugars by up to 21.8% compared to those obtained by enzymatic hydrolysis of 15 pretreated sawdust in the absence of methyl esters of fatty acids.