FIELD OF INVENTION

The invention discloses an integrated process for fractionation of oil palm empty fruit bunch (EFB) to obtain cellulosic solid, hemicellulosic sugars and high-purity lignin, and conversion of the cellulosic solid to ethanol by enzymatic hydrolysis and microbial fermentation. The end-products of the disclosed integrated process comprises cellulosic solid, hemicellulosic sugars, high-purity lignin and cellulosic ethanol.

BACKGROUND ART

Lignocellulose is one of the most abundant organic materials on the planet and has long been recognized as a potential feedstock for producing fuels, chemicals and materials. Currently, biorefining of lignocellulosic biomass to produce multi-products has attracted great interest all over the world due to the shortage of fossil-based oil and increasing environmental pollution. Lignocellulose is composed of carbohydrate polymers (cellulose and hemicelluloses) and an aromatic polymer (lignin), all of which show promising uses in a biorefinery platform.

Cellulose is a polysaccharide consisting of a linear chain of several hundred to over ten thousand β (1→4) linked D-glucose units. It can be used in many industrial applications, such as to make paper or other pulp-derived products. The cellulose can also be converted into glucose which can be further used as carbon source for producing many fuels and chemicals, such as ethanol. Hemicellulose is a diverse group of short-chain branched, substituted polymer of sugars with degree of polymerization ~ 70 to 200. After hydrolysis, the obtained constituent sugars (referred to herein as "hemicellulosic sugar") can also be converted into fuels and chemicals by either biological or chemical ways, or a combination of both. Lignin obtained from lignocellulosic biomass has value as a solid fuel and also as an energy feedstock to produce liquid fuels, syn-gas or hydrogen. It is also useful as an intermediate to make a variety of polymeric compounds and many other lignin-derived chemicals, such as vanillin, syringic acid, ferulic acid, etc.

It will be beneficial to fractionate lignocellulosic biomass and further utilize the resulting components respectively. However, a major disadvantage of previous biomass fractionation technologies is that the process is usually conducted using high temperatures with associated high pressures. For example, the ethanol organosolv delignification process is usually conducted at higher than 160 °C in order to effectively remove lignin (Zhao et al., Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis"; Applied Microbiology and Biotechnology 82 (5): 815-827; 2009). Another disadvantage associated with fractionation at high temperature relates to the degradation of fermentable sugars to other compounds such as furfural (Li et al., "Fractionating pretreatment of sugarcane bagasse by aqueous formic acid with direct recycle of spent liquor to increase cellulose digestibility - the Formiline process"; Bioresour Technol. 117:23 - 32; 2012). Traditional biomass pulping (to produce paper and related goods) is also a biomass fractionation process, in which cellulose is recovered in high yields, but lignin is primarily consumed by combustion for heat recovery. Moreover, the lignin products are always not of high purity which limits its utilization.

International Patent Publication No. WO 201 1/002330 A1 (hereinafter referred to as '330 Publication) disclosed a method for utilization of lignocellulose waste of palm oil production, first of all, empty fruit bunches (EFB) after separation of oil-bearing fruit. The method for utilization is based on peculiarities of the structure of cell tissue of bunches and provides obtaining soluble sugars useful for ethanol production and obtaining solid fuel enriched in lignine. '330 Publication disclosed a method which consists of several stages including conversion of polysaccharides (cellulose and hemicellulose) of lignocellulose feedstock into soluble sugars, biotechnological conversion of soluble sugars in methanol by microorganisms. '330 Publication acknowledges the fact that the presence of lignine in the reaction mixture is the factor that hinders hydrolysis of carbohydrides in lignocellulose feedstock. Lignine inhibits action of enzymes as it covers cellulose fibers hindering access of enzymes to the substrate. Therefore the first task is efficient shredding of the plant substrate consisting mainly of cell walls, and creation of defects in the form of disordered and amorphous sites of cellulose. Chemical composition of the bunches shows that they contain a large amount of water- and acid-soluble compounds. Their removal from the bunches should affect the structure, and, therefore, mechanical strength of the material.

Similarly, Malaysian Patent No. MY-142233-A (hereinafter referred to as '233 Patent) disclosed a process of producing ethanol from a fibrous lignocellulosic raw material. However, it must be taken into account that the importance of process of simultaneous saccharification and fermentation (SSF) process by adding cellulose enzyme and microorganisms as highlighted in the present invention was not disclosed in '233 Patent.

One of the most challenging technical obstacles in biomass fractionation processes is that the recovered cellulose is often recalcitrant to subsequent hydrolysis to form glucose. The hydrolysis is often done enzymatically, and the resistance of the cellulose to hydrolysis is often compensated for by using high enzyme loading. For conventional pulping process, the objective of delignification is to remove lignin and maintain fiber strength and degree of polymerization (DP) of cellulose. However, during biomass pretreatment for increasing the enzymatic digestibility of cellulose, decreasing cellulose DP is beneficial for its enzymatic hydrolysis. Traditional biomass pretreatment processes achieve a high cellulose digestibility by chemical or hydrothermal treatment to deconstruct the compact structure of lignocellulose; however, these processes do not pertain to a fractionation of the feedstock to obtain the individual components. The lignin products obtained are often highly condensed and not suitable for further modification and application. A disadvantage of the prior art pretreatment processes is that lignin is not sufficiently removed from the lignocellulosic biomass. The lignin that remains present in the cellulosic solid has a negative effect on an enzymatic hydrolysis, and can give higher handing costs in downstream processing. In particular the presence of residual lignin can irreversibly adsorb cellulase enzymes and thus increasing enzyme loading for highly effective saccharification of cellulosic substrate.

Oil palm empty fruit bunch (EFB) is a by-product of palm oil extraction and milling process. EFB has become an attractive biomass for biorefinery since a large amount of EFB is produced along with palm oil production. Malaysia has a ready source of biomass in EFB conveniently collected and available for exploitation in all palm oil mills. As a lignocellulosic biomass, EFB similarly shows many applications in various fields (Sumathi et al., "Utilization of oil palm as a source of renewable energy in Malaysia"; Renewable and Sustainable Energy Reviews 12(9):2404 - 2421 ; 2008). However, if EFB is fractionated into its primary components such as cellulose, hemicellulosic sugars and lignin, it would be used more easily in potentially distinct downstream process with more revenues.

Present invention provides solution to the above problems by utilizing Organosolv pretreatment which has been recognized as a promising biomass fractionation due to the following advantages:

(1 ) organic solvents are always easy to recover by distillation and recycled for pretreatment;

(2) the chemical recovery in organosolv pretreatment can isolate lignin as a solid material and hemicellulose as syrup, both of which show promise as chemical feedstocks;

(3) the isolated lignin are of high purity, which can be used for polymer substitution and production of other high-value-added products. SUMMARY OF INVENTION

One aspect of the present invention is to provide an integrated process for fractionation of oil palm empty fruit bunch (EFB) into cellulosic solid.

A further aspect of the present invention is to provide an integrated process for converting the said cellulosic solid into ethanol and specifically employing simultaneous saccharification and fermentation (SSF) process. In another aspect of the present invention is to provide a process for utilizing the produced spent liquor comprises lignin product and hemicellulosic sugars in more beneficial way via biological or chemical procedures which may include production of polymer substitutes or derivatives like surfactant. The present invention consists of features and a combination of parts hereinafter fully described and illustrated in the accompanying drawings, it being understood that various changes in the details may be made without departing from the scope of the invention or sacrificing any of the advantages of the present invention.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

To further clarify various aspects of some embodiments of the present invention, a more particular description of the invention will be rendered by references to specific embodiments thereof, which are illustrated, in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail through the accompanying drawing in which:

FIG. 1 is the flow sheet of the integrated process for fractionation of EFB into cellulosic solid (pulp), hemicellulosic sugars and lignin

FIG. 2 is the graph showing enzymatic hydrolysis of cellulosic solid at different solid consistency characterized in the form of (a) Sugar concentration; (b) Enzymatic glycan conversion

FIG. 3 is the graph showing Simultaneous Saccharification and Fermentation (SSF) of cellulosic solid for ethanol production characterized independently in the form of (a) Glucose concentration during SSF; (b) Ethanol concentration during SSF

FIG. 4 is the flow sheet of the general mass balance of fractionation of EFB for ethanol, lignin and hemicellulosic syrup production DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to an integrated process for fractionation of oil palm empty fruit bunch (EFB) to obtain cellulosic solid, hemicellulosic sugars and high-purity lignin, and conversion of the cellulosic solid to ethanol by enzymatic hydrolysis and microbial fermentation i.e. simultaneous saccharification and fermentation (SSF).

It has been well known that biomass pretreatment is a prerequisite step for effective saccharification of cellulose for producing glucose. The objective of pretreatment is to increase cellulose accessibility and thus cellulase enzymes can contact cellulose to perform the hydrolysis process. Conventional biomass pretreatment such as steam explosion, dilute acid pretreatment, hydrothermal pretreatment showed some merits to increase cellulose digestibility. However, in these processes, lignin is often of low quality and only can use as a fuel.

It has been known that removing lignin from lignocellulose is more important than removing hemicellulose for biomass fractionation, since hemicellulose can be easily hydrolyzed. Organosolv pretreatment has been recognized as a promising biomass fractionation due to the following advantages: (1 ) organic solvents are always easy to recover by distillation and recycled for pretreatment; (2) the chemical recovery in organosolv pretreatment can isolate lignin as a solid material and hemicellulose as syrup, both of which show promise as chemical feedstocks; (3) the isolated lignin are of high purity, which can be used for polymer substitution and production of other high-value-added products. In recent years, ethanol organosolv pretreatment has been well investigated for biomass pretreatment under acid catalysis or autocatalysis (Zhao et al., Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis"; Applied Microbiology and Biotechnology 82 (5): 815-827; 2009). However, ethanol actually does not have a high solubility to lignin, so that a relatively high liquid-to-solid ratio is often used for an effective delignification. However, even so there is still a high residual lignin content in the cellulosic pulp. It has been known that a good solvent for lignin should have a solubility parameter (δ value) around 1 1 cal ^1/2 /cm ^{"3 2} . Therefore, actually organic acids, such as formic acid and acetic acid with δ values of 12.1 and 10.1 cal ^1/2 /cm ^{" 3 2} respectively, are better solvents for lignin than ethanol.

Accordingly, the present invention provides a process for fractionation of oil palm empty fruit bunch (EFB) for producing cellulosic solid (pulp), hemicellulosic sugars and lignin as illustrated in FIG. 1 , and comprises the following steps:

a) mixing EFB with aqueous organic acid solution containing at least 50-90% by weight of organic acid in a digester at a temperature in the range of 50-160°C, thereby producing a first mixture (1 );

b) separating the said first mixture (1 ) into a first solid phase (2) and a first liquid stream (3) comprising dissolved lignin and hemicellulosic sugars;

c) washing the said solid phase (2) with fresh aqueous organic acid solution to obtain a second mixture (4), and separating the said second mixture (4) into a second solid phase

(5) and a second liquid stream (6);

d) heating the said second solid phase (5) to recover organic acid under vacuum to obtain a evaporated solid phase (7) and gas phase, and condensing the said gas phase to obtain a third liquid stream (8);

e) washing the evaporated solid phase (7) with water to obtain a cellulosic solid and a liquid stream (12).

f) mixing the first liquid stream (3) and second liquid stream (6) in a spent liquor collector and obtain a fourth liquid stream (9);

g) distillating the liquid stream (9) for recovering organic acid to obtain a top liquid stream (10) and a concentrated spent liquor stream (1 1 );

h) recycling the top liquid stream (10) and the third liquid stream (8) for EFB cooking and washing the said first solid phase (2); i) precipitating lignin from the said concentrated spent liquor stream (1 1 ) by adding the said liquid stream (12) to obtain a mixture stream (13);

j) separating the said mixture stream (13) to obtain lignin product and hemicellulosic sugars.

The process advantageously produces cellulose pulp from EFB which can be further used for paper production. In addition, the produced celluose is suitable for enzymatic hydrolysis into sugars, which can be further fermented into alcohols such as ethanol and/or butanol. Preferably, the said organic acid is selected from formic acid, acetic acid or propanoic acid; more preferably, the said organic acid is formic acid. Since organic acid, in particular formic acid and acetic acid have δ value around 1 1 cal ^1/2 /cm ^{"3 2} , they are good solvents for lignin fragmentation. Moreover, the carboxyl group of organic acid can easily form hydrogen bond with lignin molecule, thus aqueous organic acids have high lignin solubility. Therefore, during fractionation of EFB, the required liquid-to-solid ratio could be reduced compared with other organosolv processes. On the other hand, the FT dissociated from organic acid can play as a catalyst to accelerate the formation of lignin fragments and hydrolysis of hemicellulose. Thus an exogenous acid such as sulfuric acid or hydrochloric acid is unnecessary. More preferably, the used aqueous organic acid should have an organic acid concentration of 50-90%. Too low organic acid concentration is negative to lignin solubilization; however, too high organic acid concentration can lead to high acylation of cellulose, which can further affect the utilization of cellulose. More preferably, the organic acid concentration should not be higher than 90% by weight in order to control the acyl group content of lower than 5% by weight. However, the organic acid concentration should not be less than 50% by weight, in order to obtain a high degree of delignification. After the delignification process, the obtained slurry is further separated. Preferably, the said separation process is performed by squeezing, filtration or centrifugation; more preferably, after separation the said solid phase contains dry solid mater of 20-50% by weight. It should be careful to control the liquid content of the obtained solid phase. In a preferable embodiment of this invention, after squeezing the liquid content should not be less than 50% (corresponding dry solid matter content higher than 50%); otherwise the mechanical pressing power would collapse the pore structure of the cellulosic solid, which whereby could decrease the cellulose digestibility.

In order to further remove the residual lignin fragments remaining in the said solid phase (2) obtained in step b), the solid phase (2) must be washed with fresh organic acid as shown in step c). Preferably, the said organic acid in step c) is selected from formic acid, acetic acid or propanoic acid; more preferably, the said organic acid is the same as that used for cooking in both organic acid type and concentration. The said solid phase (2) cannot be directly washed with water in order to avoid the precipitation of dissolved lignin onto cellulosic fiber which will affect the subsequent enzymatic hydrolysis of cellulose. The washing step can be conducted by either batch or continuous flow. Preferably, the solid phase (2) is washed with fresh medium comprising the same organic acid as that used in step a) in a counter flow or cross flow manner. In another preferable embodiment of this invention, after washing the solid phase (2), a separating step is performed as that used in step b) to obtain a solid phase (5). The said solid phase (5) is wet with some residual organic acid that must be recovered.

Preferably, in step d) the said solid phase (5) is heated under vacuum to recover the residual organic acid by evaporation and obtained an evaporated solid phase (7). The evaporation temperature should be well controlled. Too high a temperature might cause significant hornification of the said cellulosic solid, and whereby decreases the subsequent enzymatic hydrolysis. In a preferable embodiment of this invention, the evaporation is conducted at 20- 80 °C under pressure of 1 -13 kPa; more preferably, the recovery condition meets the demand that the said evaporated solid phase (7) has a liquid content of less than 5% by weight. By this evaporation process, more than 95% of the residual organic acid can be recovered. The evaporated solid phase (7) still contains some residual organic acid, which might inhibit the microorganism growth in the subsequent fermentation process. Therefore, the said evaporated solid phase (7) is further washed with water to remove the residual organic acid. Preferably, the said washing process in step e) is conducted at 30-60 °C with water-to-solid ratio of 20-5:1 by weight. More preferably, the said washing process is conducted in a counter flow or cross flow manner.

This invention also comprises a step for recovering the organic acid from the spent liquor obtained in step b) and c). The spent liquor contains dissolved lignin and sugars that mainly come from hemicellulose hydrolysis. In a preferable embodiment of this invention, the said spent liquor is evaporated under vacuum to recover organic acid. Preferably, the spent liquor is distillated under vacuum to obtain the recovered fresh aqueous organic acid at the top as the top liquid stream (10) and a concentrated spent liquor stream (1 1 ). The said top liquid stream (10) is further recycled to step a) for delignification of EFB and step c) for washing the said solid phase 2). More preferably, 90% of the organic acid in spent liquor should be recovered by distillation.

The concentrated spent liquor stream (1 1 ) is further treated for lignin and hemicellulosic sugar recovery. Lignin can be easily precipitated from the concentrated spent liquor as a solid phase while hemicellulosic sugars are recovered as a liquid syrup. Preferably, the volume of the water added to the said concentrated spent liquor should be at least five times that of the concentrated spent liquor. More preferably, the water added to the concentrated spent liquor is the liquid stream (12) obtained in step e). This invention also provides a process for converting the said cellulosic solid to ethanol comprising the steps of:

a) treating the said cellulosic solid with alkali solution or suspension at liquid-to-solid ratio of 20-3:1 by weight and 20-120 °C to obtain a first slurry mixture;

b) adjusting the pH value of the said first slurry mixture obtained in step a) to 4.5-6.5 to obtain a second slurry mixture;

c) converting the said second slurry mixture to ethanol by adding cellulase enzyme and microorganisms by a simultaneous saccharification and fermentation (SSF) process. During delignification, cellulose can be acylated to a certain extent, which can limit the binding of cellulase enzyme onto cellulose. Therefore, the cellulosic solid should be further treated to remove acyl group. In a preferable embodiment of this invention, alkali is used to catalyze the deacylation process. Preferably, the said alkali is selected from sodium hydroxide, potassium hydroxide, lime or ammonia. More preferably, the said alkali is selected from lime or ammonia, since lime is cheap and ammonia can be used as nitrogen source for yeast growth.

The alkali loading is important to deacylation. Preferably, the used alkali loading is 0.1 -10% by weight based on the dry cellulosic solid. However, liquid-to-solid ratio is also important since it can affect the alkali concentration for deacylation. In a preferable embodiment of this invention, the liquid-to-solid ratio is 20-3:1 . More preferably, when alkali loading is low, a low liquid-to-solid ratio should be used, but the mixing might not be homogenous at a low liquid- to-solid ratio. Deacylation temperature also significantly affects the degree of deacylation. Preferably, the temperature should be in the range of 20-120 °C. Most preferably, the deacylation condition should meet the requirement i.e. the treated cellulosic solid has an acyl group content of less than 1 % by weight based on dry matter. The obtained slurry mixture showed excellent enzymatic digestibility for ethanol production by a SSF process. However, the slurry mixture is preferably neutralized and its pH adjusted in the range of 4.5-6.5. More preferably, in most cases, since the released organic acid by deacylation process can react with the excess alkali, no other acid is required to adjust the pH value of the slurry. The neutralized slurry is thus suitable for ethanol production. Preferably, the said SSF is conducted with 5-30 FPU/g solid of cellulase loading, at 30-40 °C and 5-20% solid consistency.

The celulosic solid obtained by this invention showed very good enzymatic digestibility, and can obtain a high ethanol concentration. Removing most of the lignin by organic acid delignification not only eliminates the physical barrier of lignin, but also decreases the nonproductive adsorption of cellulase enzymes onto the residual lignin matrix. Moreover, the high cellulose content in the obtained cellulosic solid would increase glucose concentration in the enzymatic hydrolyzate, which whereby would increase the subsequent ethanol concentration. Increasing ethanol concentration has important significance for decreasing the energy consumption in ethanol recovery. A significant increase in energy demand is observed at an ethanol concentration below 4%. However, with the process provided by this invention, ethanol concentration can be higher than 5%, which is important to decrease the energy consumption for ethanol recovery.

In another embodiment of this invention, the said hemicellulosic sugars obtained in the process provided are further utilized for the production of corresponding products by biological or chemical ways; more preferably, the said hemicellulosic sugars is converted to furfural and 5-(Hydroxymethyl)-furfural (5-HMF); most preferably, the said conversion of the hemicellulosic sugars to furfural and 5-HMF is employed under the catalysis of the residual organic acid and no additional mineral acids need to be added. The obtained lignin product is of high purity and can be used for polymer substitution. The present invention will be further illustrated by the following, non-limiting examples.

Example 1 : fractionation with aqueous formic acid

The oil palm EFB's main chemical components were determined according to Laboratory Analytical Procedure (LAP) of National Renewable Energy Laboratory (Sluiter et al., Determination of Structural Carbohydrates and Lignin in Biomass, Technical Report NREL/TP-510-42618, 5 April, 2008). The results are shown in Table 1 .

30 gram of EFB was packed in a 1 -L three-neck flask followed by addition of 300 ml 78% (by weight) formic acid. The mixture was then heated to the normal boiling point at atmospheric pressure for 1 .5 hours. A mechanical stirring was employed to ensure a good reaction. After the reaction, the mixture was filtered to obtain a solid phase with liquid content of about 75%. The obtained solid phase was first washed by 300 ml 78% (by weight) formic acid and then filtered to remove as much liquid as possible. After further washed by tap water till neutrality, the solid was deformylated with 4% Ca(OH) ₂ based on dry pulp weight at 120 °C for 1 hour. The solid was washed with water till neutrality and dried for analysis.

The main chemical components of the dried pretreated solid are shown in Table 2. It can be calculated according to mass balance that after the treatment, 7.9% of cellulose, 89.32% of hemicellulose and 87.27% of lignin were respectively dissolved into the liquid phase, corresponding to 92.1 % of cellulose, 10.68% hemicellulose and 12.73% lignin recovered as solid phase. This result indicates that aqueous formic acid treatment can obtain a good degree of delignification and hemicellulose recovery.

Table 1 : Main chemical components of EFB Sample Solid Glucan Xylan Klason lignin Acetyl group recovtirtiU

Treated EFB 39±1 .5 87.3810.8 4.1 110.8 5.68+0.3 0.0

Table 2: Main chemical components of treated EFB by aqueous formic acid

Example 2: fractionation with aqueous acetic acid

The EFB used was the same as that in Example 1 .

30 gram of EFB was packed in a 1 -L three-neck flask followed by addition of 300 ml 80% (by weight) acetic acid and 0.3% (by weight) sulfuric acid as a catalyst. The mixture was then heated to the normal boiling point at atmospheric pressure for 1 .5 hours. A mechanical stirring was employed to ensure a good reaction. After reaction, the mixture was filtered to obtain a solid phase with liquid content of about 75%. The obtained solid phase was first washed by 300 ml 80% (by weight) acetic acid and then filtered to remove as much liquid as possible. After further washed by tap water till neutrality, the solid was deacetylated with 4% Ca(OH) ₂ based on dry pulp weight at 120 °C for 1 hour. The solid was washed with water till neutrality and dried for analysis.

The main chemical components of the dried pretreated solid are shown in Table 3. It can be calculated according to mass balance that after the treatment, 9.6% of cellulose, 73.4% of hemicellulose and 68.7% of lignin were respectively dissolved into the liquid phase, corresponding to 90.4% of cellulose, 26.6% hemicellulose and 31 .3% lignin recovered as solid phase. This example indicates that aqueous acetic acid treatment also can obtain a satisfying degree of delignification and hemicellulose recovery.

Table 3 Main chemical components of pretreated EFB by aqueous acetic acid Example 3: fractionation with aqueous formic acid of different concentrations

The EFB used was the same as that used in Example 1 .

30 gram of EFB was packed in a 1 -L three-neck flask followed by addition of 300 ml 50-88% (by weight) formic acid. The mixture was then heated to the normal boiling point at atmospheric pressure for 1 .5 hours. A mechanical stirring was employed to keep a good reaction. After the reaction, the mixture was filtered to obtain a solid phase with liquid content of about 75%. The obtained solid phase was first washed by 300 ml aqueous formic acid solution - the same as that used in delignification. The solid was washed by water till neutrality and dried for analysis.

The main chemical components of the dried pretreated solid are shown in Table 4. Generally, solid recovery, xylan and klason lignin contents decreased with increasing formic acid concentration, and vice versa for glucan contents. Moreover, degree of formylation increased significantly with increasing formic acid concentration.

Formic acid Solid recovere d Klason ligni 1 Forrnyl

Glucan {%) Xylan {%

concentration (%) | (%) group (%) 58 49.4±1 .2 73.7+0.4 10.1 +0.1 9.6+0.3 2.65+0.01

68 43.110.9 82.010.5 7.9+0.04 4.3+0.06 3.88+0.12

78 39.611 .9 82.4+0.6 5.6+0.02 4.4+0.2 4.53+0.42

88 37.812.0 85.0+0.15 4.0+0.06 2.46+0.2 6.59+0.01

Table 4 Main chemical components of pretreated EFB by aqueous formic acid of different concentrations

Example 4: Enzymatic hydrolysis of cellulosic solid

The EFB used was the same as that used in Example 1 . 30 gram of EFB was packed in a 1 -L three-neck flask followed by addition of 300 ml 78% (by weight) formic acid. The mixture was then heated to the normal boiling point at atmospheric pressure for 1 .5 hours. A mechanical stirring was employed to ensure a good reaction. After the reaction, the mixture was filtered to obtain a solid phase with liquid content of about 75%. The obtained solid phase was first washed by 300 ml 78% (by weight) formic acid and then filtered to remove as much liquid as possible. After further washed by tap water till neutrality, the solid was deformylated with 4% Ca(OH) ₂ based on dry pulp weight at 120 °C for 1 hour. The solid was washed with water until neutrality and stored at 4°C in a fridge prior to enzymatic hydrolysis.

The cellulosic solid was further digested by cellulase loading of 15 FPU/g solid at 50±0.5°C and pH 4.8 (0.1 M sodium acetate buffer) in an air-bath shaker at 130 rpm for 5 days. The solid consistency was 2.5-10%. The enzymatic digestibility of the cellulosic solid was described as enzymatic sugar concentrations and glycan conversion, which is respectively defined as follows:

1 · _{/ \} ^slucose ^cellubiose ^ ylose ^ _{1 Ar /W} glycan conversion (%) =— - x 100% where w _giucose , w eiiobiose and w _xy iose are the weight of glucose, cellobiose and xylose produced by enzymatic hydrolysis; W| _G i _y is the weight of initial total glycan (glucan+xylan) of the solid. The results are illustrated in FIG. 2, which indicate that the cellulosic solid could be very well converted to sugars by enzymatic hydrolysis.

Example 5: Simultaneous Saccharafication and Fermentation (SSF) of cellulosic solid for ethanol production

The EFB used was the same as that used in Example 1 .

30 gram of EFB was packed in a 1 -L three-neck flask followed by addition of 300 ml 78% (by weight) formic acid. The mixture was then heated to the normal boiling point at atmospheric pressure for 1 .5 hours. A mechanical stirring was employed to ensure a good reaction. After the reaction, the mixture was filtered to obtain a solid phase with liquid content of about 75%. The obtained solid phase was first washed by 300 ml 78% (by weight) formic acid and then filtered to remove as much liquid as possible. After further washed by tap water till neutrality, the solid was deformylated with 4% Ca(OH) ₂ based on dry pulp weight at 120 °C for 1 hour. The solid was washed with water until neutrality and stored at 4°C in a fridge prior to SSF. The SSF was performed in 150-ml Erienmeyer flasks with 50 ml liquid medium. The yeast used was Saccharomices cerevisiae CICC 31014. The liquid medium contained 2 g/L (NH ₄ ) ₂ S0 ₄ , 5 g/L KH ₂ P0 ₄ , 5 g/L yeast extracts, 1 g/L MgS0 ₄ and 0.2 g/L CaCI ₂ . The cellulosic solid was mixed with the liquid medium until the solid consistency was 15% (w/v) followed by sterilization at 121 °C for 20 min. Cellulase of 20 FPU/g solid plus 10 CBU/g solid of /3-glucansidase were then added to the mixture followed by inoculation with 10% (v/v) yeast inocula. The SSF process was conducted at 37-38°C and 130 rpm in an air-bath shaker. The pH value was not controlled during SSF. The experimental results are illustrated in FIG. 3.

Example 6: General mass balance of the process

The EFB used was the same as that used in Example 1 .

A general mass balance of fractionation of EFB for ethanol, lignin and hemicellulosic syrup production is illustrated in FIG. 4. The present invention may be embodied in other specific forms without departing from its essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore indicated by the appended claims rather than by the foregoing description. All changes, which come within the meaning and range of equivalency of the claims, are to be embraced within their scope.