(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual Property

Organization
International Bureau
(10) International Publication Number
(43) International Publication Date WO 2015/108409 Al
23 July 2015 (23.07.2015) W P O PCT

(51) International Patent Classification: Bangi, Kajang, Selangor, 43000 (MY). CHOO, Yuen
D21C 3/02 (2006.01) D21C 11/00 (2006.01) May; c/o MALAYSIAN PALM OIL BOARD, No. 6, Per
D21C 3/04 (2006.01) CIOL 1/02 (2006.01) siaran Institusi, Bandar Baru Bangi, Kajang, Selangor,
D21C 5/00 (2006.01) 43000 (MY). XUEBING, Zhao; Department of Chemical
Engineering, Institute of Nuclear and New Energy Techno
(21) International Application Number:
logy, Tsinghua University, Haidian District, Beijing,
PCT/MY20 15/050002
100084 (CN). DEHUA, Liu; Department of Chemical En
(22) International Filing Date: gineering, Institute of Nuclear and New Energy Techno
19 January 2015 (19.01 .2015) logy, Tsinghua University, Haidian District, Beijing,
100084 (CN).
(25) Filing Language: English
(74) Agent: MIRANDAH ASIA (MALAYSIA) SDN BHD;
(26) Publication Language: English Suite 3B-19-3, Plaza Sentral, Jalan Stesen Sentral 5, 50470
(30) Priority Data: Kuala Lumpur (MY).
PI2014000163 20 January 2014 (20.01.2014) MY (81) Designated States (unless otherwise indicated, for every
(71) Applicants: MALAYSIAN PALM OIL BOARD kind of national protection available): AE, AG, AL, AM,
[MY/MY]; No. 6, Persiaran Institusi, Bandar Baru Bangi, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
Kajang, Selangor, 43000 (MY). TSINGHUA UNIVER¬ BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
SITY [CN/CN]; Haidian District, Beijing, 100084 (CN). DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
(72) Inventors: LOH, Soh Kheang; c/o MALAYSIAN PALM KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG,
OIL BOARD, No. 6, Persiaran Institusi, Bandar Baru MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM,

[Continued on nextpage]

(54) Title: AN INTEGRATED PROCESS FOR FRACTIONATION OF OIL PALM EMPTY FRUIT BUNCH AND CONVER
SION OF THE CELLULOSIC SOLID TO ETHANOL
(57) Abstract: The invention relates to an integrated process
for fractionation of oil palm empty fruit 5 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 at
least a cellulosic solid, hemicellulosic sugars, high-purity
lignin and cellulosic ethanol. 10
 w o 2015/108409 Ai III III II II III I I11 III I 11III I I I llll III I
II II

PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC,
SD, SE, SG, SK, SL, S , ST, SV, SY, TH, TJ, TM, TN,
TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
(84) Designated States (unless otherwise indicated, for every
kind of regional protection available): ARIPO (BW, GH,
GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ,
TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,
TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,
DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT,
LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE,
SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA,
GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG).
Declarations under Rule 4.17:
— as to applicant's entitlement to apply for and be granted
a patent (Rule 4.1 7( ))
— of inventorship (Rule 4. 7(iv))
Published:
— with international search report (Art. 21(3))
— before the expiration of the time limit for amending the
claims and to be republished in the event of receipt of
amendments (Rule 48.2(h))
 AN INTEGRATED PROCESS FOR FRACTIONATION OF OIL PALM EMPTY FRUIT

BUNCH AND CONVERSION OF THE CELLULOSIC SOLID TO ETHANOL

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. 7:23 - 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. W O 20 1/002330 A 1 (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): 8 1 5-827; 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 11 cal 2/cm 2. Therefore,

actually organic acids, such as formic acid and acetic acid with δ values of 12.1 and 10.1

cal 2/cm 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 50-1 60°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 ( 1 2).

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 ( 1 0) and a concentrated spent liquor stream ( 1 1);

h) recycling the top liquid stream ( 1 0) 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 ( 1 3) 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 11 cal 2/cm 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- 1 3 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 ( 1 0) 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 no n

productive 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 : 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-51 0-4261 8 , 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 m l 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

vti ti

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 m l 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 3 1 .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 m l 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 gni Forrnyl

Glucan {%) Xylan {%
concentration (%) | ( ) roup (%)

58 49.4±1 .2 73.7+0.4 10.1 +0.1 9.6+0.3 2.65+0.01

68 43.1 10.9 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+0.1 5 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 m l 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:

· lucose ^cellubiose ylose

glycan conversion (/ )\ = — - x 100%
W

where w u se , wbeiiobiose and x iose are the weight of glucose, cellobiose and xylose produced

by enzymatic hydrolysis; |G i 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 m l 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 m l liquid medium. The yeast

used was Saccharomices cerevisiae CICC 31014. The liquid medium contained 2 g/L

(NH 4)2S0 4, 5 g/L KH2P0 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 12 1 °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.
CLAIMS

1. A process for fractionation of oil palm empty fruit bunch (EFB) comprising the steps

of:

a) mixing EFB with aqueous organic acid solution containing at least 50-90% by

weight of organic acid in a digester at a temperature of 50-1 60°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 ( 1 2).

f) mixing the first liquid stream (3) and second liquid stream (6) in a spent liquor

collector to obtain a fourth liquid stream (9);

g) distilling the liquid stream (9) for recovering organic acid to obtain a top liquid

stream ( 1 0) and a concentrated spent liquor stream ( 1 1);

h) recycling the top liquid stream ( 1 0) 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 ( 1 3) to obtain lignin product and hemicellulosic

sugars.
2. The process according to Claim 1, wherein the said mixing EFB with aqueous

organic acid solution is performed at a solid-to-liquid ratio of 1:3-1 0 by weight.

3. The process according to Claim 1 and 2 , wherein the said organic acid are selected

from formic acid, acetic acid or propanoic acid; preferably, the said organic acid is formic

acid.

4. The process according to Claim 1, wherein the said separating the mixture in step b)

and c) is performed by squeezing, filtration or centrifugation; preferably, after separation the

said solid phase contains dry solid mater of 20-50% by weight.

5. The process according to Claim 1, wherein in step c) the said organic acid is selected

from formic acid, acetic acid or propanoic acid; preferably, the said organic acid is the same

as that used for cooking.

6. The process according to Claim 1, wherein the said heating the said second solid

phase (5) to recover organic acid in step d) is conducted at 20-80°C under pressure of 1-13

kPa; preferably, the recovery condition meets the demand that the said evaporated solid

phase (7) has a liquid content of less than 5% by weight.

7. The process according to Claim 1, wherein the said washing the evaporated solid

phase (7) with water in step e) is conducted at 30-60 °C with water-to-solid ratio of 20-5:1 .

8. The process according to Claim 1, wherein the said hemicellulosic sugars is

converted to furfural and 5-(Hydroxymethyl)-furfural (5-HMF); more preferably, the said

conversion of the hemicellulosic sugars to furfural and 5-HMF is under the catalysis of the

residual organic acid and no additional mineral acids need to be added.

9. The process according to Claim 1, wherein the said lignin products is used as

polymer substitutes and/or is further modified for the production of derivatives such as

surfactant.

10. The process according to Claim 9 , wherein the said lignin products used as polymer

substitutes is further modified for the production of derivatives such as surfactant.

11. 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 and 20-1 20 °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 yeast by a simultaneous saccharification and fermentation (SSF) process.

12. The process according to Claim 11, wherein the said alkali is selected from sodium

hydroxide, potassium hydroxide, lime or ammonia.

13. The process according to Claim 11 and 12 , wherein the said alkali is used at a

loading of 0.1-10% by weight based on the dry cellulosic solid.

14. The process according to Claim 11, wherein the said SSF is conducted with 5-30

FPU/g solid of cellulase loading, at 30-40 °C and 5-20% solid consistency.

 International application No

PCT/MY2015/05Q002
A . CLASSIFICATION O F SUBJECT MATTER
INV. D21C3/Q2 D21C3/04 D21C5/O0 D21C11/00 ClOLl/02
ADD.

According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols)
D21C CIOL

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)

EPO-Internal , WPI Data

C . DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

EP 2 573 259 Al (CHEMPOLIS OY [FI] ) 1-10

27 March 2013 (2013-03-27)
the whole document

WO 2011/002330 Al (ARTER TECHNOLOGY LTD; 1-10

P0LIT0V ANAT0LY ALEKSANDROVICH [RU] ;
G0LYAZIM0VA) 6 January 2011 (2011-01-06)
c i ted i n the appl i cati on
the whole document

US 2010/167351 Al (EYAL AHARON [ I L] ET AL) 1-10

1 July 2010 (2010-07-01)
the whole document

EP 2 336 195 Al (SHELL INT RESEARCH [NL] ) 1-10

22 June 2011 (2011-06-22)
the whole document

/ -

X Further documents are listed in the continuation of Box C . See patent family annex.

* Special categories of cited documents :

"A" document defining the general state of the art which is not considered
to be of particular relevance
"E" earlier application or patent but published o n or after the international
filing date
"L" document which may throw doubts on priority claim(s) orwhich is
cited to establish the publication date of another citation or other
special reason (as specified)
"O" document referring to a n oral disclosure, use, exhibition or other
means
"P" document published prior to the international filing date but later than
the priority date claimed
"T" later document published after the international filing date or priority
date and not in conflict with the application but cited to understand
the principle or theory underlying the invention
"X" document of particular relevance; the claimed invention cannot be
considered novel or cannot be considered to involve an inventive
step when the document is taken alone
" document of particular relevance; the claimed invention cannot be
considered to involve an inventive step when the document is
combined with one or more other such documents, such combination
being obvious to a person skilled in the art
"&" document member of the same patent family

Date of the actual completion of the international search Date of mailing of the international search report

29 May 2015 10/06/2015

Name and mailing address of the ISA/ Authorized officer
European Patent Office, P.B. 5818 Patentlaan 2
NL - 2280 HV Rijswijk
Tel. (+31-70) 340-2040,
Fax: (+31-70) 340-3016 Karl sson, Lennart
 International application No

PCT/MY2015/05Q002
C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category * Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

US 2012/064574 Al (TOKUYASU KEN [JP] ET 11

AL) 15 March 2012 (2012-03-15)
c l aims 1-20

SUMATHI S ET AL: "Uti l i zation o f o i l palm 1-14

a s a source o f renewable energy i n
Malaysia" ,
RENEWABLE AND SUSTAINABLE ENERGY REVI EWS,
ELSEVI ERS SCI ENCE, NEW YORK, NY, US,
vol . 12, no. 9 ,
1 December 2008 (2008-12-01) , pages
2404-2421 , XP025426570,
ISSN : 1364-0321, D0I :
10. 1016/J . RSER.2007.06.006
[retri eved on 2008-09-12]
c i ted i n the appl i cati on
the whole document

EP 2 546 352 Al (NESTE OI L 0YJ [FI] ) 1-10

16 January 2013 (2013-01-16)
the whole document

W0 2011/044378 Al (AMERICAN PROCESS INC 1-10

[US] ; RETSINA THEODORA [US] ; PYLKKANEN
VESA [US] ) 14 Apri l 2011 (2011-04-14)
the whole document

W0 02/053829 Al (CHEMPOLIS 0 Y [FI] ; ROUSU 1-10

ESA [FI] ; ROUSU PASI [FI] ; ANTTI LA JUHA
[FI] ; ) 1 1 July 2002 (2002-07-11)
the whole document

W0 2007/111605 Al (VI RGINIA TECH INTELL 1-10

PROP [US] ; ZHANG PERCIVAL Y H [US] )
4 October 2007 (2007-10-04)
the whole document
 International application No
Information on patent family members
PCT/MY2015/05Q002
Patent document Publication Patent family Publication
cited in search report date member(s) date

EP 2573259 Al 27-03-2013 B R 102012023867 A2 24-06-2014

CA 2789562 Al 23-03-2013
CN 103014185 A 03-04-2013
EP 2573259 Al 27-03-2013
U 2012139480 A 27-03-2014
US 2013078695 Al 28-03-2013

O 2011002330 Al 06 -01- -2011 NONE

US 2010167351 Al 1 - 7 -2010 MY 149457 A 30--08--2013

US 2010167351 Al 01--07--2010

EP 2336195 Al 22 - 6 -2011 NONE

US 2012064574 Al 15 -03- -2012 P 5633839 B2 03--12--2014

P 2011004730 A 13--01--2011
US 2012064574 Al 15--03--2012
WO 2010134455 Al 25--11--2010

EP 2546352 Al 16 -01- -2013 NONE

O 2011044378 Al 14 .04. -2011 US 2012202253 Al 09--08--2012

WO 2011044378 Al 14--04--2011

W0 02053829 Al 11-07-2002 AT 472011 T 15--07·-2010

CN 1481462 A 10--03·-2004
DK 1368531 T3 11--10·-2010
EP 1368531 Al 10--12·-2003
ES 2346851 T3 21--10·-2010
FI 20002886 A 30--06·-2002
US 2004040830 Al 04--03·-2004
WO 02053829 Al 11--07·-2002

W0 2007111605 Al 04-10-2007 AT 497058 T 15- 02·-2011

AU 2006340913 Al 04- 10·-2007
BR PI0621525 A2 13- 12·-2011
CA 2647516 Al 04- 10·-2007
CA 2856978 Al 04- 10·-2007
CN 101449001 A 03- 06·-2009
DK 2007945 T3 09- 05·-2011
EP 2007945 Al 31- 12·-2008
ES 2360828 T3 09- 06·-2011
P 5209597 B2 12- 06·-2013
P 2009531424 A 03- 09·-2009
PT 2007945 E 29- .0 .-2011
SI 2007945 Tl 31- 05·-2011
US 2010170504 Al 08- 07·-2010
US 2014190471 Al 10- 07·-2014
WO 2007111605 Al 04- 10·-2007