WO2021198077A1

WO2021198077A1 - Process for recovering crude palm oil

Info

Publication number: WO2021198077A1
Application number: PCT/EP2021/057954
Authority: WO
Inventors: Morten Emil Moeldrup
Original assignee: Novozymes A/S
Priority date: 2020-04-02
Filing date: 2021-03-26
Publication date: 2021-10-07

Abstract

The present invention describes a process for recovering palm oil from palm oil mill waste, the process comprising the steps of: a) mixing press fibre and palm oil mill liquid waste; b) optionally incubating the mixture from step (a); c) optionally further mixing the mixture from step (a); d) pressing the mixture from step (a), (b) or (c) to obtain an oil-enriched liquid phase and an oil-depleted press fibre, and e) recovering the crude palm oil from the oil-enriched liquid phase from step (d).

Description

PROCESS FOR RECOVERING CRUDE PALM OIL

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

FIELD OF INVENTION

The present invention generally relates to palm oil milling process, and more particularly, to a process for recovering crude palm oil from palm oil mill waste.

BACKGROUND OF INVENTION

Palm oil has been commercially used as cooking oil and raw material in producing other products, such as detergents, cosmetics and biodiesel. Due to several advantageous properties of palm oil, such as high productivity, low price, high thermal and oxidative stability and rich in antioxidant vitamin E, palm oil has become an important source of vegetable oil and the demand for palm oil is increasing rapidly.

Palm oil is derived from the fruits of oil palms, mainly from the Elaeis guineensis, by a series of processes in a palm oil mill. Typically, the process starts with sterilization of fresh fruit bunch (FFB) where the FFB is cooked at pressurized environment. Thereafter, the sterilised fruits are stripped off from the bunch before they are digested to free palm oil in the fruits by rupturing the oil-bearing cells. The palm oil is then extracted from the digested fruits by pressing, leaving a press liquor which contains mixture of crude palm oil, water and solid impurities, and a press cake which contains press fibre (PF) and nuts. The press cake will normally be further separated into PF and nuts for further uses, whereas the press liquor is clarified to separate crude palm oil from its entrained impurities. In a typical clarification process, the press liquor is first diluted with water to cause heavy solids to settle to the bottom while the lighter oil droplets rise to the top when heat is applied. Thereafter, the diluted mixture is screened to remove coarse fibre, followed by allowing the screened mixture to settle so that the lighter oil droplets will be separated and rise to the top. The overflow oil is then collected for further processing to produce crude palm oil product, while residual oil in the underflow is recovered through a series of operations comprising centrifugation, desanding and separation or decantation, leaving substantially de-oiled sludge, also known as the heavy phase (HP). As described above, steps have been taken in the industries to maximise the oil extraction rate (OER) in the palm oil mill process. However, there remains a substantial amount of unavoidable oil losses in the waste generated from the palm oil mill process, for example, the PF and the HP, which usually amount to about 0.8- 1.2% OER un-extracted residual oil. Conventionally, the PF will be used to produce steam for electricity generation, e.g. via biomass boilers, while the HP will be discharged together with other palm oil mill liquid waste as palm oil mill effluent (POME).

SUMMARY OF INVENTION

It is an object of the invention to provide a process for recovering palm oil from palm oil mill waste.

Accordingly, the present invention relates to a process for recovering palm oil from palm oil mill waste, the process comprising the steps of:

(a) mixing press fibre and palm oil mill liquid waste;

(b) optionally incubating the mixture from step (a);

(c) optionally further mixing the mixture from step (a);

(d) pressing the mixture from step (a), (b) or (c) to obtain an oil-enriched liquid phase and an oil-depleted press fibre, and

(e) recovering the crude palm oil from the oil-enriched liquid phase from step (d).

In one embodiment, the palm oil mill liquid waste includes at least one of heavy phase (HP) and palm oil mill effluent (POME).

In one embodiment, the incubation may be performed with or without heat, and with or without further mixing.

Preferably, PF is mixed with HP and incubated for a period of time with further mixing, particularly, for up to 48 hours at 30°C to 200°C and at rotational speed of up to 30 rpm.

In one embodiment, the mixture is pressed at 1 bar and above. In one embodiment, the mixture is pressed at 40 bar and above. In another embodiment, the mixture is pressed at 50 bar and above, particularly, 100 bar and above. In yet further embodiments, the mixture is pressed at a pressure in the range from 1 to 100 bar.

In a particular embodiment, the invention relates to a process for recovering palm oil from palm oil mill waste, the process comprising the steps of:

(a) mixing press fibre, palm oil mill liquid waste and an enzyme composition;

(b) optionally incubating the mixture from step (a); (c) optionally further mixing the mixture from step (a);

The invention consists of novel features and a combination of parts hereinafter fully described and illustrated in the accompanying drawings and particularly pointed out in the appended claims; it being understood that various changes in the details may be effected by those skilled in the arts but without departing from the scope of the invention or sacrificing any of the advantages of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of facilitating an understanding of the invention, there is illustrated in the accompanying drawing the preferred embodiments from an inspection of which when considered in connection with the following description, the invention, its construction, operation, and many of its advantages would be readily understood and appreciated.

FIG. 1 illustrates an embodiment of the process for recovering palm oil from palm oil mill waste.

FIG. 2 Pressing of PF alone compared to pressing of PF after mixing with HP (see Example 1)

FIG. 3 Pressing of PF after mixing with HP and incubation, compared to no incubation (see Example 2)

FIG. 4 Effect of ratio of PF to HP (see Example 3)

FIG. 5 Pressing of PF after mixing with HP and enzyme, and incubation (see Example

4)

FIG. 6. Pressing of PF after mixing with HP and enzyme, and incubation (see Example

5)

FIG. 7 Overview of Palm Oil Milling

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the invention shall be described according to the preferred embodiments of the present invention and by referring to the accompanying description and drawings. However, it is to be understood that limiting the description to the preferred embodiments of the invention and to the drawings is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claims.

For the purpose of illustration of the invention, specific terminologies will be resorted for the sake of clarity. However, it is not intended that the invention be limited to the specific terms so selected and it is to be understood that each specific term includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.

The term “crude palm oil” described herein refers to extracted or recovered palm oil which has yet to be refined and can be of different qualities. This may include, but is not limited to, palm oil extracted from pressing of digested fruits, palm oil separated in clarification process and palm oil recovered by the process of the invention. The qualities of crude palm oil may be determined by various factors such as content of impurities.

The present invention generally relates to palm oil milling process, and more particularly, to a process for recovering crude palm oil from palm oil mill waste. The invention in one aspect relates to a process for recovering palm oil from palm oil mill waste, the process comprising the steps of:

(a) mixing press fibre and palm oil mill liquid waste;

(b) optionally incubating the mixture from step (a);

(c) optionally further mixing the mixture from step (a);

(d) pressing the mixture in step (a), (b) or (c) to obtain an oil-enriched liquid phase and an oil-depleted press fibre, and

The term palm oil mill waste refers to all fractions or side streams generated in the oil milling process, which do not form part of the final crude palm oil. The term comprises both liquid waste (e.g. Heavy phase) and solid waste (e.g. press fibre).

As described in “BACKGROUND OF INVENTION”, the term “press fibre” (PF) herein refers to fibre separated from a press cake from pressing of digested oil palm fruits, which is substantially free of nuts or kernel.

The term palm oil mill liquid waste as used herein refers to any type of liquid waste generated from a palm oil mill. The term comprises for example, condensate from sterilisation of fresh fruit bunch (FFB), mill cleaning water, palm oil mill effluent (POME), as well as side streams which may be recovered and/or further processed, such as underflow from clarifier (clarifier tank underflow, CTU), and heavy phase (HP) respectively from decanter or separator. The term palm oil mill liquid waste also comprises any combination of these. HP is however preferred over other type of palm oil mill liquid waste due to substantial amount of residual oil contained therein as well as relatively lower amounts of contaminants, especially as compared to POME. For ease of illustration, hereinafter, the invention will be described with respect to HP although the same may be replaced by any other palm oil mill liquid waste such as CTU or POME. It should be understood that there is no intent to limit the palm oil mill waste, that is, the PF and the palm oil mill liquid side stream to particular qualities, such as, their freshness, contaminants content, free fatty acid content and/or residual oil content.

Mixing in the context of this invention means the blending of at least two compositions. The mixing may for example be accomplished mean the addition of the one or more liquid or semi-liquid compositions to another, leading to a mixture comprising them. In other words, mixing does not necessitate a specific stirrer or agitator, though the use of one or several such means may be applied if deemed useful. The mixing of step (a) is typically achieved by adding PF and palm oil mill liquid waste (eg HP) into the same vessel. The further mixing of step (c) is typically achieved by use of a stirrer or agitator, see further below.

The term incubation is interchangeable with retention time or resting time, and refers the period after admixing and before recovery of the oil.

There is no particular limit to the ratio of PF to palm oil mill liquid side stream, however, it is preferred that the weight ratio of PF to palm oil mill liquid waste is at least 1:1 to ensure sufficient wetting of the PF by the palm oil mill liquid waste. For example, the ratio may be in the range from 1 :1 to 1:12. Specific embodiments relate to wherein the ratio of PF to palm oil liquid side stream is the range from 1.1 to 1:10, or e.g. 1:1 to 1:8, or e.g. 1 :1 to 1 :6. In some embodiments, the ratio of PF to palm oil mill liquid side stream is 1:2, 1:3, 1 ;4 , 1:5 , 1:6 , 1:7 or 1 :8

In some embodiments, the process comprises mixing of palm oil mill liquid waste (such as HP) with PF, and subsequent incubation of the mixture.

The incubation may be performed with or without further mixing, although the former is preferred in order to achieve a homogenous mixture and sufficient wetting of the PF. The further mixing may be performed by any suitable means, the selection of which is in the purview of the skilled person. Typically, the further mixing is performed by stirring. For this reason, PF and palm oil mill liquid waste may be incubated with stirring at rotational speed suitable to achieve a homogenous mixture and wetting. The selection of appropriate speed is within the purview of the skilled person and will depend for example on the size of the tank.

Thus, the stirring may be performed at a rotational speed of up to 40 rpm. Larger tanks will use lower speeds such as in the range of from 1 to 19 rpm, while smaller tanks may use higher speeds, such as in the range of from 20 to 40 rpm. This is well known in the art. Thus, in some embodiments the stirring speed is in the range of from 1 to 10 rpm, such as 2 to 8 rpm, such as 3 to 5 rpm. In other embodiments, the stirring speed is from 11 to 19 rpm, such as 12 to 18 rpm, such as 14 to 16 rpm. In yet other embodiments, the stirring speed is in the range from 20 to 40 rpm, such as 20 to 35 rpm, 20 to 30 rpm, such as 22 to 26 rpm.

The incubation may be performed at a temperature in the range of from 30°C to 200°C, 30°C to 100°C, 30°C to 80°C or 50°C to 80°C.

In some embodiments, the incubation is performed at a temperature in the range of from 20°C to 55°C. Incubation at these temperatures have the advantage of lower heating costs.

In other embodiments the incubation is performed at a temperature in the range of from 56 to 120°C, such as for example 30°C to 95°C, 50°C to 90°C. Incubation at these temperatures has the advantage of enhancing wetting of the PF and mobilisation of the residual oil contained in the mixture.

The means of achieving the incubation temperature may be by any suitable means, and it is within the purview of the skilled person to select such means. The palm oil liquid waste mixed with the PF may have elevated temperatures due to previous processing steps. E.g, the vertical clarifier tank is typically kept at 90-95 °C, and thus underflow from this tank will typically have a temperature in this vicinity.

Thus, in the case the palm oil side stream already has a suitable temperature, the incubation may be performed without further heating. Alternatively, if a higher incubation temperature is desired, a heating means may be provided in order to heat the mixture to the selected incubation temperature. Although there is no intent to limit the incubation time, it is preferred that the incubation be performed for at least a reasonable period of time to achieve a homogenous mixture, sufficient wetting of the PF and mobilisation of the residual oil contained in the mixture. Such reasonable period of time may include, but not limited to, ranges of 1 min to 48 hours, 1 min to 36 hours,

1 min to 24 hours, 1 min to 12 hours, 1 min to 6 hours, 1 min to 3 hours, 1 min to 1 hour, 6 hour to 48 hours, 6 hour to 36 hours, 6 hour to 25 hours, 6 hour to 12 hours, 12 hours to 48 hours, 12 hours to 36 hours and 12 hours to 24 hours.

The shorter incubation times have the advantage of being less disruptive of palm oil milling process, avoiding delays or stops in processing. Thus, particular embodiments relate to wherein the incubation time is less than 5 hours, such as less than 4 hours, less than 3 hours, or less than 2 hours.

In further preferred embodiments, the incubation time in the range of 0 min to 1.5 hrs,

0 min to 1 hr, 0 min to 30 mins, 0 min to 25 mins, 0 min to 20 mins, or for example from 1 min to 2 hrs, from 5 min to 1.5 hrs, from 5 min to 1 hr, from 1 min to 30 mins, 2 mins to 25 mins, 2 mins to 20 mins, 1- 15 mins, or 1 to 12 mins.

The mixture may be pressed at any suitable pressure. The pressure may be in the range of from 30 bar to 200 bar, or for example from 40 to 70 bar. The pressure which is conventionally adopted in the palm oil mill for pressing digested fruits is typically in the range of from 50 to 65 bar and may be used here. However, the mixture may be pressed at 50 bar and above, and more particularly, pressed at a pressure in the range of from 50 to 200 bar, such as from 60 to 200 bar, for example from 70 to 200 bar, from 80 to 200 bar, or 90 to 200, or 100 to 200 bar, as the mixture is now substantially free of nuts or kernel and thus one need not worry that they may be cracked under high pressure.

The recovery in step (e) may be by any suitable means and selection of such means is within the purview of the skilled person. Typically, recovery of oil from a liquid phase is done by exploiting the difference in density between the oil and the aqueous phase in which it is suspended. For example, recovery of oil may be done by centrifugation, such as by decanter and/or separator; or by gravity/ settling.

Thus, in one embodiment, the invention relates to a process according to the invention comprising a step e) of recovering the crude palm oil from the oil-enriched liquid phase from step (d) by decanter and/or separator.

The oil in the palm fruit is present as oil droplets within the mesocarp cells. The inventors have surprisingly found that the combination of press fibre, palm oil mill liquid waste synergistically releases the oil remaining in the press fibres. Thus, the invention in one aspect relates to a process for recovering palm oil from palm oil mill waste, the process comprising the steps of: (a) mixing press fibre, palm oil mill liquid waste and an enzyme composition;

(b) optionally incubating the mixture from step (a);

(c) optionally further mixing the mixture from step (a);

In a particular embodiment, the invention relates to a process for recovering palm oil from palm oil mill waste, the process comprising the steps of: a) mixing press fibre, palm oil mill liquid waste and an enzyme composition comprising at least one hemicellulase and/or cellulase; b) optionally incubating the mixture from step (a); c) optionally further mixing the mixture from step (a); d) pressing the mixture from step (a), (b) or (c) to obtain an oil-enriched liquid phase and an oil-depleted press fibre, and e) recovering the crude palm oil from the oil-enriched liquid phase from step (d). Hemicellulases

The term "hemicellulolytic enzyme" or "hemicellulase" means one or more (e.g., several) enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom and Shoham, 2003, Current Opinion In Microbiology 6(3): 219-228). Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase. The substrates for these enzymes, hemicelluloses, are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation. The catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups. These catalytic modules, based on homology of their primary sequence, can be assigned into GH and CE families. Some families, with an overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A). A most informative and updated classification of these and other carbohydrate active enzymes is available in the Carbohydrate- Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitable temperature such as 40°C-80°C, e.g., 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C, and a suitable pH such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0.

Xylanases

In one embodiment of the process according to the invention, the at least one hemicellulase comprises or consists of one or more xylanases.

The term "xylanase" means a 1 ,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1 .8) that catalyzes the endohydrolysis of 1 ,4-beta-D-xylosidic linkages in xylanase. Xylanase activity can be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01 % TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1 .0 mb of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6. Alternatively, xylanase activity may be determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, MO, USA) by xylan-degrading enzyme(s) under the following typical conditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50°C, 24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47: 273-279.

Xylanases suitable for the present invention have been described in WO2019038418 with earliest priority date 24 August 2017. In further embodiments, the one or more xylanases is selected from the group consisting of the xylanases described in WO2019038418, which xylanases are hereby incorporated by reference.

Particular embodiments relate to the process according to the invention wherein at least one hemicellulase comprises or consists of GH 10 xylanase, for example one or more of the following GH 10 xylanases described in WO2019038418, which are hereby incorporated by reference:

GH10 xylanase derived from Talaromyces leycettanus (SEQ ID NO: 1 , or mature polypeptide SEQ ID NO: 2 in attached sequence listing), GH10 xylanase derived from Rasamsonia byssochlamydoides (SEQ ID NO: 3 in attached sequence listing)

GH10 xylanase derived from Aspergillus niger (SEQ ID NO: 4 in attached sequence listing),

GH10 xylanase derived from Aspergillus fumigatus (SEQ ID NO: 5 in attached sequence listing),

Other particular embodiment of the present invention relates to wherein the at least one hemicellulase comprises or consists of xylanase as described in WO 2019/068850, in particular one or more GH 10 Xylanase as set forth in SEQ ID NO 1, SEQ ID NO:2 or SEQ ID NO:4 of WO 2019/068850 (hereby incorporated by reference).

Cellulases

The term "cellulolytic enzyme" or "cellulase" means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. The two basic approaches for measuring cellulolytic enzyme activity include: (1 ) measuring the total cellulolytic enzyme activity, and (2) measuring the individual cellulolytic enzyme activities (endoglucanases, cellobiohydrolases, and beta- glucosidases) as reviewed in Zhang et al., 2006, Biotechnology Advances 24: 452- 481. Total cellulolytic enzyme activity can be measured using insoluble substrates, including Whatman N°1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common total cellulolytic activity assay is the filter paper assay using Whatman N°1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (lUPAC) (Ghose, 1987, Pure Appl. Chem. 59: 257-68).

Cellulolytic enzyme activity can be determined by measuring the increase in production/release of sugars during hydrolysis of a cellulosic material by cellulolytic enzyme(s) under the following conditions: 1 -50 mg of cellulolytic enzyme protein/g of cellulose in pretreated corn stover (PCS) (or other pretreated cellulosic material) for 3- 7 days at a suitable temperature such as 40°C-80°C, e.g., 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C, and a suitable pH, such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0, compared to a control hydrolysis without addition of cellulolytic enzyme protein. Typical conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodium acetate pH 5, 1 mM MnS04, 50°C, 55°C, or60°C, 72 hours, sugar analysis by AM IN EX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cellulosic material: The term "cellulosic material" means any material containing cellulose. The predominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemicellulose, and the third is pectin. The secondary cell wall, produced after the cell has stopped growing, also contains polysaccharides and is strengthened by polymeric lignin covalently cross-linked to hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1 -4)-D-glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents. Although generally polymorphous, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicelluloses usually hydrogen bond to cellulose, as well as to other hemicelluloses, which help stabilize the cell wall matrix.

The term "cellobiohydrolase" means a 1 ,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176) that catalyzes the hydrolysis of 1 ,4-beta-D- glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1 ,4-linked glucose containing polymer, releasing cellobiose from the reducing end (cellobiohydrolase I) or non reducing end (cellobiohydrolase II) of the chain (Teeri, 1997, Trends in Biotechnology 15: 160-167; Teeri et al., 1998, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity can be determined according to the procedures described by Leveret al., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581.

The term "endoglucanase" means a 4-(1 ,3; 1 ,4)-beta-D-glucan 4- glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1 ,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3-1 ,4 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components. Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452- 481). Endoglucanase activity can also be determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40°C.

The term "beta-glucosidase" means a beta-D-glucoside glucohydrolase (E.C. 3.2.1 .21) that catalyzes the hydrolysis of terminal non-reducing beta-D- glucose residues with the release of beta-D-glucose. Beta-glucosidase activity can be determined using p-nitrophenyl-beta-D-glucopyranoside as substrate according to the procedure of Venturi et al., 2002, J. Basic Microbiol. 42: 55-66. One unit of beta- glucosidase is defined as 1 .0 pmol of p-nitrophenolate anion produced per minute at 25°C, pH 4.8 from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodium citrate containing 0.01 % TWEEN® 20.

In one embodiment the enzyme composition may be or comprised and/or cellulase, wherein the at least one cellulase comprises one or more of cellobiohydrolase, endoglucanase and beta-glucosidase.

In one embodiment the enzyme composition may be or comprised Examples of suitable cellulases include a crude or purified extract of a Trichoderma reseei fermentate, a crude or purified extract of Humicola insolens.

In one embodiment the could be or comprise LAMINEX ® BG2, LAMINEX Super 3G ®, Laminex C2K (available from DuPont Biosciences), Celluclast (available from Novozymes).

Further enzymes

Further embodiments relate to the process according to the invention, wherein one or more further enzymes are added in step a. Said one or more further enzymes may be provided by addition of further enzyme compositions. The one or more further enzymes may be for example one or more pectinases, mannanases, amylases or combinations thereof.

Specific embodiments relate to wherein no mannanase is added.

Certain embodiments of the invention relate the process according to the invention wherein at least one of the least one hemicellulase and/or at least one cellulase is thermostable.

Thermostable

Preferably the one or more hemicellulases, one or more cellulases, one or more pectinases or amylases, are thermostable to such an extent that at least 15% of the enzyme activity (i.e. the cellulase, hemicellulose, amylase and/or pectinase activity) is retained after incubation at 70°C for 20 minutes, to such an extent that at least 20% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 25% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 30% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 35% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 40% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 50% of the enzyme activity is retained after incubation at 70°C for 20 minutes, to such an extent that at least 60% of the enzyme activity is retained after incubation at 70°C for 20 minutes, or to such an extent that at least 70% of the enzyme activity is retained after incubation at 70°C for 20 minutes. The thermostability may in particular be determined by incubation at 70°C for 20 minutes in a 0.1 M Na-OAc buffer pH 5.0, followed by transfer to ice and determination of residual enzyme activity (i.e. residual cellulase, hemicellulase, amylase and/or pectinase activity) on Konelab by a method comprising: hydrolyzing substrate (e.g. carboxymethyl cellulose (CMC) form reducing carbohydrate; stopping the hydrolyzation by an alkaline reagent containing PAH BAH and Bismuth, which that forms complexes with reducing sugar; and measuring color production by complex formation at 405 nm in a spectrophotometer.

The invention will now be described in greater details, with reference to the accompanying drawings.

As illustrated in FIG. 1, PF and HP are mixed in a tank (10), followed by pressing (20) of the mixture to recover crude palm oil from the mixture. Although it may not always be necessary, the incubation is performed with stirring for a reasonable period of time with heat to encourage homogeneous mixing, wetting of the PF and mobilisation of residual oil contained in the mixture.

The resultant mixture is subsequently pressed (20) to obtain crude palm oil and dried fibre (24). Various means of pressing may be employed, such as, screw press, filter press, hydraulic press or any other suitable type of presses.

In some embodiments, lighter oil droplets may rise to the top of the mixture in the tank (10), forming a layer of crude palm oil at the top (14). This top layer of crude palm oil (14) may be skimmed off before the remaining mixture (12) is pressed (20).

The crude palm oil obtained from pressing the mixture of PF and HP (22) or skimming off the top layer of the mixture (1M oil obtained from pressing the mixture of PF and HP (22) or skimming off the top layer of the mixture (14) may be further processed or treated. For example, as shown in FIG. 1 , the crude palm oil (14, 22) may be treated with a decanter (30) to obtain substantially impurities-free crude palm oil (34) and an effluent (32). Apart from decanter or decantation, it should be understood that other means of processing or treatment of the crude palm oil may be employed. This includes, but not limited to, separator, clarification tank, centrifugation, purifier or crude oil tank. EXAMPLES

Materials and Methods

Press fibre (PF) and Heavy phase (HP) samples from a commercial Palm Oil Mill were mixed in a 1:4 ratio (50g PF + 200 g HP).

Samples were mixed thoroughly with a spoon before incubation in a heated water bath at temperatures between 70-90°C as indicated.

At the end of incubation, the mixture was passed through a screw press, with the liquid and solid phases are collected separately. Oil content of the respective fractions was then determined using Near infrared spectroscopy (NIR) and/or Soxhlet extraction.

NIR was performed using NIRFlex N-500 available from BOCHI Labortechnik AG, and using the bundled software (NIRWare or NIRCal). NIR methodology is described e.g. by Chung and Manaf in Journal of Oil Palm Research Vol. 26(1) March 2014 p 84-95.

Soxhlet extraction was performed using the SOXTHERM® system, available from Gerhardt Lab Instruments.

The percentage of oil remaining in press fibres after this process was calculated by comparing the amount of oil present in the wet material both before mixing with the HP, and after passing through the screw press (oil loss wet basis, OLWB).

Example 1. HP improves the extraction of oil from press fibres PF was pressed without the addition of any liquid. The amount of oil remaining in PF was compared to the sample where PF was mixed with HP and pressed again, without incubation.

Pressing PF without addition of any liquid led to a decrease in the amount of oil remaining in the fibres (about 80 % oil remaining in fibres, see table 1). Mixing HP with PF and then pressing led to a substantial decrease in the amount of oil remaining in fibres (ca 40%). This demonstrates that addition of HP facilitates extraction of residual oil from press fibres (see Table 1 , and Figure 2).

Table 1:

Example 2: HP is more efficient than water

PF was incubated with either distilled water (Fig 3, white columns) or HP (black columns), and were either pressed immediately, or incubated for 2 hours and then pressed.

The results are shown in Table 2, and in Figure 3. Table 2:

The results show that mixing PF with water (1 :4 ratio by weight) will reduce the amount of oil left in PF. However, mixing PF with HP (1:4 ratio by weight) was more efficient, as the amounts of oil remaining in the press fibres was reduced as compared to PF mixed with water, both with no incubation and with 2 hrs incubation. Thus, mixing/incubation with HP will increase extraction of oil remaining in press fibre.

Example 3: Dilution can increase efficiency PF was mixed with HP at two different ratios (1:4 and 1:8 respectively by weight, PF:HP) and were either pressed immediately or incubated for 2 hours and then pressed. The amount of oil remaining in fibre was determined by Soxhlet extraction (SOXTHERM ®).

Results are shown in Table 3 and Figure 4. Black bars show 1:4 and open bars show 1 :8.

Table 3:

The results show that increasing the amount of HP relative to PF leads to more efficient extraction of oil from PF.

Example 4: Addition of enzyme increases efficiency of oil release from PF PF was incubated with distilled water, HP or HP+Enzyme for 2 hrs and then pressed to separate the crude oil and dried fibres.

Table 4: Enzyme increases efficiency of oil release

Enzyme (Palmora) denotes an enzyme composition comprising a temperature stable xylanase (SEQ ID NO 2) provided in aqueous solution. The enzyme was added in an amount 500 ppm (the enzyme composition having 1500 FXU-S/g).

The results are shown in Fig 5 and indicate that the combination of enzyme and HP give a synergistic increase of oil release from press fibres.

Example 5: Addition of enzyme

PF was incubated with HP or HP+Enzyme for 2 hrs and then pressed to separate the crude oil and dried fibres.

Table 5: Enzyme increases efficiency of oil release from press fibres

The results are shown in Fig 6 and further support that addition of enzyme increases the efficiency of oil release from press fibres.

Example 6: Oil analysis

Samples were extracted with chloroform and methanol (1-1) in the approximate ratio 1 to 40. Extracts were subsequently mixed for 60 min followed by centrifugation 5,000 g for 5 min. The supernatants were transferred to glass vials and 15mI was injected (on a 10mI loop) onto a reversed-phase chromatography and mass spectrometer (LCMS). The LC-system is an Accela and the MS is a Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer both from Thermo Scientific. The MS was operated in positive and negative ion mode electrospray ionization (ESI+) and (ESI-). Data were collected from 80-1500 m/z (enabling detection of molecules with a molecular weight of 80Da to more than 10.000Da) The chromatographic part of the LCMS-system was set up with a CSH- peptide C18 column (1.7 pm particle, 50 mm, 2.1 mm ID, Waters) using a gradient system with Eluent A: water with 0.1 % formic acid, Eluent B: Acetonitrile with 0.1 % formic acid and Eluent C: Isopropanol with 0.1% formic acid. A flow of 0.500 mL/min was used, starting at 40 % A-eluent and 40% B-eluent for 1 minute, lowering both to 10 % with a linear gradient after an additional 14 minutes. The three eluents were then running isocratically for 3 minutes and after additionally 1 minute returning to initial conditions with a linear gradient. Finally running 4 minutes of isocratic conditions to a total runtime of 24 minutes. Identification and integration of lipids are based on the software Xcalibur (from Thermo) or Expressionist (from Genedata). The quantification is based on linear calibration curves made with solutions of pure chemical purchased in Sigma. For this heptane/isopropanol/formic acid (90:9.9:0.1) was used as solvent. Calibration curves are based on the stearic acid variant of each lipid class. The concentrations of the individual lipid classes in the samples are obtained by converting the sum peak area to concentration (w/v%) in extract via the calibration curves. Finally, the concentration of lipid in extract is converted to lipid concentration in samples (w/w%) by adjusting for extraction volume and sample weight.

PF was mixed with different liquid waste streams: HP, sterilizer condensate (SC) or palm oil mill effluent (POME), incubated for 2 hrs and then pressed. The oil remaining in PF was then measured by Soxhlet extraction (SOXTHERM ®) and the results as shown in Table 5 that neither SC nor POME were able to reduce the oil in PF to the same extent as HP. Palm oil extracted by this process was compared to analytical grade palm oil (Sigma) and crude palm Oil (Singapore). Results in Table 6 show that crude palm oil extracted by this process (CPO) had a similar profile to the Singapore crude palm oil, with lower levels of FFA and polar lipids, and enriched in TAG. Polar lipids were compared and results as shown in Table 7 show that oil was enriched in both BetaCarotene and Tocopherol.

Table 6: Oil release

Table 7: Oil Profile

Table 8: Oil components

Example 7: Industrial scale experiment

150kg of PF was mixed with either 450 kg or 600 kg of HP and incubated for indicated times at 80°C in the digestor prior to subjecting the mixture to screw press to separate liquid from solids. The PF was then subjected to Soxhlet extraction (SOXTHERM ®) to determine remaining oil. PF was mixed with HP at two different ratios (1:4 and 1:3 respectively by weight, PF:HP) and pressed after 90 minutes incubation. The amount of oil remaining in fibre was determined by Soxhlet extraction (SOXTHERM ®). Results as shown in Table 8 that mixing together PF + HP in ratio of either 1:3 to 1:4 results in an approximately 50% reduction of oil in PF. Further, incubation in the digestor for either 30 mins or 90 mins did not have a significant impact on reduction of oil in PF as shown in Table 9. This experiment supports that this process is beneficial also in large scale.

Table 9: Mixing ratios of PF and HP

Table 10: Incubation time

Although the invention has been described and illustrated in detail, it is to be understood that the same is by the way of illustration and example, and is not to be taken by way of limitation. The scope of the present invention is to be limited only by the terms of the appended claims.

Claims

1. A process for recovering palm oil from palm oil mill waste, the process comprising the steps of: a) mixing press fibre and palm oil mill liquid waste; b) optionally incubating the mixture from step (a); c) optionally further mixing the mixture from step (a); d) pressing the mixture from step (a), (b) or (c) to obtain an oil-enriched liquid phase and an oil-depleted press fibre, and e) recovering the crude palm oil from the oil-enriched liquid phase from step (d).

2. A process for recovering palm oil from palm oil mill waste according to claim 1, the process comprising the steps of:

(a) mixing press fibre, palm oil mill liquid waste and an enzyme composition;

(b) optionally incubating the mixture from step (a);

(c) optionally further mixing the mixture from step (a);

3. The process for recovering palm oil from palm oil mill waste according to claim 1 or 2, the process comprising the steps of: a. mixing press fibre, palm oil mill liquid waste and an enzyme composition comprising least one hemicellulase and/or at least one cellulase; b. optionally incubating the mixture from step (a); c. optionally further mixing the mixture from step (a); d. pressing the mixture from step (a), (b) or (c) to obtain an oil-enriched liquid phase and an oil-depleted press fibre, and e. recovering the crude palm oil from the oil-enriched liquid phase from step (d).

4. The process according to any of the preceding claims wherein the palm oil mill liquid waste is one or more of condensate from sterilisation of fresh fruit bunch (FFB), mill cleaning water, palm oil mill effluent (POME), underflow from clarifier (clarifier tank underflow, CTU), heavy phase (HP) from decanter.

5. The process according to claim 4 wherein the palm oil mill liquid waste is heavy phase (HP) from decanter.

6. The process according to any of claims 3 to 5 wherein the at least one hemicellulase is selected from comprises or consists of one or more of an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase, or combinations thereof.

7. The process according to claim 6 wherein the at least one hemicellulase comprises or consists of one or more xylanases, such as one or more GH10 xylanase.

8. The process according to claim 7 wherein the at least one hemicellulase comprises or consists of one or more of the following xylanases described in WO2019038418-

GH10 xylanase derived from Talaromyces leycettanus (SEQ ID NO: 1, or mature polypeptide SEQ ID NO: 2),

GH10 xylanase derived from Rasamsonia byssochlamydoides (SEQ ID

NO: 3)

GH10 xylanase derived from Aspergillus niger (SEQ ID NO: 4),

GH10 xylanase derived from Aspergillus fumigatus (SEQ ID NO: 5),

9. The process according to any of the preceding claims 3 to 8, wherein one or more further enzymes are added in step a.

10. The process according to claim 9 where the one or more further enzyme is selected from the group consisting of an amylase and/or a pectinase.

11. The process according to any of the preceding claims 3 to 10 wherein at least one of hemicellulase and/or at least one cellulase is thermostable.

12. The use of at least one palm oil mill liquid waste to extract residual oil from press fibre.

13. The use of at least one palm oil mill liquid waste and an enzyme composition to extract residual oil from press fibre.

14. The use of at least one palm oil mill liquid waste and an enzyme composition comprising at least one hemicellulase and/or at least one cellulase to extract residual oil from press fibre.

15. The use according to any of claims 12 to 14 wherein the palm oil liquid waste is heavy phase (HP) from decanter.