Methods and systems for producing crude palm oil

Abstract

Provided herein are methods, compositions, and systems for producing a crude palm oil product.

Description

METHODS AND SYSTEMS FOR PRODUCING CRUDE PALM OILFIELD OF THE INVENTION

[0001] Provided herein are methods, compositions, and systems for producing a final crude palm oil product.BACKGROUND

[0002] Palm oil is a commercially important edible oil with numerous advantageous properties, including high productivity, low price, and high thermal and oxidative stability. Conventional processes for palm oil extraction, however, fail to achieve theoretical oil yields due to loss during processing steps. Furthermore, the high temperatures and water needed for operation result in high energy consumption and increased safety risks for plant operators.

[0003] Strategies to improve oil yield have typically included the use of enzymes, but such strategies have proved challenging. Thus, there is a need for new methods, compositions, and systems to improve palm oil extraction.

[0004] The methods, compositions, and systems described herein address these and other needs in the art.SUMMARY OF THE INVENTION

[0005] In an aspect is provided a method for producing a final crude palm oil product, including contacting a crude palm oil (CO) with an enzyme composition, wherein the enzyme composition includes a cellulase. In some embodiments, the contacting occurs at a temperature of or of less than about 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C. In some embodiments, the contacting occurs at a temperature of or of less than about 65°C, 60°C, 55°C, or 50°C. In some embodiments, prior to the contacting, the CO is cooled to a temperature of or of less than about 75 °C, 70°C, 65 °C, 60°C, 55°C, or 50°C. In some embodiments, prior to the contacting, the CO is cooled to a temperature of or of less than about 65 °C, 60°C, 55 °C, or 50°C. In some embodiments, the method includes incubating the CO contacted with enzyme composition for a duration of at least about 30 minutes. In some embodiments, the CO is heated to a temperature in a range of about 70°C to about 100°C after the incubating. In some embodiments, the CO is maintained at a temperature of or of less than about 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C after the incubating. In some embodiments, the CO is maintained at a temperature of or of less than about 65°C, 60°C, 55°C, or 50°C after the incubating. In some embodiments, the method includes prior to the contacting: sterilizing fresh fruit bunches (FFB) to produce sterilized FFB (SFB) and sterilizer condensate; separating the SFB to produce a mass passing to digester (MPD) and empty sterilizedFFB (EFB); optionally, pressing the EFB to produce a EFB liquor; digesting the MPD to produce a fruit mash; pressing the fruit mash to produce an undiluted crude palm oil (UDCO); and extracting the UDCO by separation. In some embodiments, the method includes diluting the UDCO to produce a diluted crude palm oil (DCO). In some embodiments, the CO is UDCO or DCO. In some embodiments, the contacting occurs in an enzyme reaction tank unit. In some embodiments, the cellulase includes a cellobiohydrolase, an endoglucanase, a beta-glucosidase, or any combination thereof. In some embodiments, the cellulase includes a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta-glucosidase (BGL), or any combination thereof. In some embodiments, the cellulase is derived from a fungus. In some embodiments, the cellulase is derived from a strain of Trichoderma. In some embodiments, the cellulase is derived from a strain of Trichoderma reesei. In some embodiments, the enzyme composition further comprises a hemicellulase. In some embodiments, the enzyme composition further includes a xylanase. In some embodiments, the method further includes clarifying the CO contacted with the enzyme composition to produce a pure oil and purifying and drying the pure oil to produce a final crude palm oil product.

[0006] In an aspect is provided a system for producing a final crude palm oil product, comprising a buffer tank unit, a cooling unit, and an enzyme reaction tank unit, wherein the units are in operable contact, and wherein the buffer tank unit is in operable contact with a screening unit of an existing palm oil extraction system and receives crude palm oil (CO) from the screening unit, and the enzyme reaction tank unit is in operable contact with a holding tank unit of the existing palm oil extraction system, and the holding tank unit receives a CO contacted with one or more enzymes or an enzyme composition from the enzyme reaction tank unit. In an aspect is provided a system for producing a final crude palm oil product, including a sterilizing unit, a stripping unit, a digester unit, a pressing unit, a screening unit, a buffer tank unit, a cooling unit, an enzyme reaction tank unit, a holding tank unit, a clarifying unit, a pure oil tank unit, a purifying unit, and a drying unit, wherein the units are in operable contact. In some embodiments, the cooling unit receives crude palm oil (CO) from the buffer tank unit and decreases a temperature of the CO prior to the CO entering the enzyme reaction tank unit. In some embodiments, the buffer tank unit and the cooling unit are in operable contact by a pump. In some embodiments, the pump is located between the buffer tank unit and the cooling unit. In some embodiments, the enzyme reaction tank unit includes an inlet for dosing one or more enzymes or an enzyme composition. In some embodiments, the one or more enzymes or the enzyme composition is dosed to the enzyme reaction tank unit by inline dosing and / or direct injection. In some embodiments, the one or more enzymes or the enzyme composition is dosed using a pump. In some embodiments, the pump is acontinuous pump, optionally comprising a flow meter. In some embodiments, the enzyme reaction tank unit includes temperature and / or pH sensors. In some embodiments, the enzyme reaction tank unit includes a water jacket. In some embodiments, the enzyme reaction tank unit includes an agitator mechanism and / or is a continuous stirred tank reactor. In some embodiments, the system includes one or more sampling points for performing mass balance analysis. In some embodiments, the one or more sampling points include a flow meter.

[0007] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 shows the percentage of oil, water, and sludge after fractionation of undiluted crude palm oil (UDCO) treated with a Trichoderma Whole Cellulase I, a Trichoderma Engineered Whole Cellulase, a Trichoderma Whole Cellulase II, or untreated (Control).

[0009] FIG. 2 shows the percentage and structure of carbohydrates of non-oil solids in sludge from UDCO treated with a Trichoderma Whole Cellulase I, a Trichoderma Engineered Whole Cellulase, or untreated (Control).

[0010] FIGS. 3 A and 3B show the moisture content (FIG. 3 A) and composition of dried sludge (FIG. 3B) after fractionation of UDCO treated with a Trichoderma Whole Cellulase I, a Trichoderma Engineered Whole Cellulase, a Trichoderma Whole Cellulase II, or untreated (Control).

[0011] FIG. 4 shows a conventional palm oil extraction system.

[0012] FIG. 5 shows an exemplary modified palm oil extraction system.

[0013] FIG. 6 shows the relative mass of dried sludge obtained following treatment of UDCO with a Trichoderma Whole Cellulase I at different doses (0.1% or 0.2% wt / wt) incubated for a duration of 4 hours at 60°C followed by incubation at 70°C for 4 hours compared to DCO Control (no enzyme, incubated at 96°C for 5 hours total) dried sludge mass (DCO Control dried sludge mass equal to 100%).

[0014] FIG. 7 shows the moisture content of the dried sludge (percentage of sludge) obtained following UDCO treatment with different doses of a Trichoderma Whole Cellulase I (0.1% or 0.2% wt / wt) and incubation for a duration of 4 hours at 60°C followed by incubation at 70°C for 4 hours and no enzyme treatment, incubated at 96°C for 5 hours (DCO Control).

[0015] FIG. 8 shows the percentage of oil in the dried sludge following UDCO treatment with different doses of a Trichoderma Whole Cellulase I (0.1% or 0.2% wt / wt) and incubation for a duration of 4 hours at 60°C followed by incubation at 70°C for 4 hours and no enzyme, incubated at 96°C for 5 hours (DCO Control).

[0016] FIG. 9 shows the relative oil loss reduction in sludge obtained after treatment of UDCO with a Trichoderma Whole Cellulase I at different doses (0.1 % or 0.2% wt / wt) and incubation for a duration of 4 hours at 60°C followed by incubation at 70°C for 4 hours compared to DCO Control sludge oil (no enzyme, incubated at 96°C for 5 hours; oil loss reduction is equal to 0%).DETAILED DESCRIPTION

[0017] Palm oil is the highest produced vegetable oil commercially. It has been a prominent fat and oil resource for the food industry due to several advantageous properties, such as high productivity, low price, high thermal and oxidative stability, and plasticity at room temperature. In addition, compared with other vegetable oils, palm oil is a rich source of the antioxidant vitamin E.

[0018] Crude palm oil is extracted from fresh fruit bunches (FFB) conventionally at high temperatures (~90°C). The process includes sterilizing the FFB to produce sterilized FFB (SFB), followed by separating palm fruitlets from the SFB by stripping, and subsequently digesting the palm fruitlets to soften the exocarp of the palm fruit. The digested fruitlets are then pressed to separate the palm kernel from the oil-water-fiber matrix. The oil-water-fiber matrix is subsequently passed through a screening unit, e.g., a vibrating screen, to further separate the insoluble fibers from the liquid phase (oil, water). The crude palm oil obtained at this step represents a mixture of oil and water, including solids not separated by the screen. The oil phase is subsequently isolated by clarification and further processed to yield the final crude palm oil product which is stored for shipment.

[0019] Crude palm oil discharged from the pressing unit is highly viscous. Thus, separation of the oil from the solids and water is difficult without the addition of dilution water. Hot water is therefore added to the pressed liquid to dilute it prior to or during clarification. This typically occurs at temperatures of 80-95°C. The dilution provides a barrier causing the heavy solids to settle to the bottom of the clarification tank while the lighter oil droplets rise through the sludge phase to the top when heat is applied. In practice it has been found that dilution with water such that 30% to 40% of the mixture is water is best for good separation in the clarification tank. Any remaining available oil post clarification is removed by decanting or centrifugation. Heavy Phase remaining after oil removal is a viscous liquid containing water, up to 1.0% oil loss wet basis (OLWB) post-decanter unit or up to 2% OLWB post-centrifugation unit, and for each ton of FFB processed typically 450 Kg of Heavy Phase is produced. Considering the massive amount of Heavy Phase generated per FFB processed, recovering remaining oil in Heavy Phase may improve OER and reduce the environmental impact of Palm Oil Mill Effluent (POME).

[0020] Conventional processes appear to achieve oil extraction rates (OER) between 15-24% (w / w of FFB) subject to seasonal variation, fruit quality, and mill equipment process control, among other variables. One of the strategies to improve palm oil extraction has been the use of enzymes; however, enzyme use has been challenging. Difficulties may exist because the conventional process runs at high temperatures, and there is poor homogeneity across oil-liquid- solid phases. As such, typical dosing strategies do not provide a conducive environment with sufficient retention time for enzyme performance. Furthermore, the conventional process is highly energy consuming. The use of steam to maintain high temperatures and water to reduce crude palm oil viscosity contributes to the process’s high energy needs. The ability to decrease energy consumption and improve safety conditions for operators at the plant are highly desirable. Thus, there remains a need for improved methods for extracting palm oil.

[0021] The methods, compositions, and systems described herein modify the existing palm milling process allowing plants to improve OER while operating at lower temperatures such that there is effective enzyme hydrolysis, a reduction in overall steam consumption, and improvement in effluent quality. The methods, compositions, and systems provided herein also reduce the quantity of effluent compared to conventional palm milling processes. As shown in the Examples, it was surprisingly found that the addition of enzyme compositions including cellulases to undiluted crude palm oil (UDCO) improved phase separation (oil from water and insolubles) and resulted in a higher oil recovery compared to untreated UDCO. This effect was observed even at low temperatures (e.g., temperatures below those used in conventional processes). Similar results were also observed when comparing oil recovery from enzyme contacted UDCO to oil recovery from non-enzyme contacted diluted crude palm oil. See, e.g., Section V. Furthermore, enzyme treatment may reduce UDCO viscosity. Thus, the methods, compositions, and systems provided herein are advantageous in that they increase oil extraction yield and may improve energy consumption and safety by allowing the process to run at lower temperatures and reducing or eliminating the need for water to decrease crude palm oil viscosity. In addition, use of enzymes according to the methods provided herein reduces or eliminates the need to dilute UDCO, e.g., by decreasing viscosity and / or improving dewatering, which in turn decreases effluent volume. Further, the crude palm oil quality may be higher due to the lower temperatures.

[0022] Additionally, current key performance indicators of palm oil milling are based on concentration, which is effective if variability in phase distribution and flowrates are limited. However, when enzymes are used, it changes phase separation and modifies the composition of streams and flowrates in the system. For example, enzymes may reduce the mass of non-oil solids in the Heavy Phase. As such, even though oil concentration may increase, due to mass reduction,overall oil loss is reduced. Therefore, the methods and systems provided herein, in some embodiments, use mass balance to assess the quantity and quality of outputs, e.g., crude palm oil, sludge, decanter cake, enzyme contacted crude palm oil (CO), recovered oil, Heavy Phase, POME, final crude palm oil product. In some embodiments, the mass balance analysis is used for optimizing the methods, compositions, and systems described herein.

[0023] The headings provided herein are not limitations of the various aspects or embodiments of this disclosure which can be had by reference to the specification as a whole. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. The reader will appreciate that statements made in one section may apply to other sections. Any terms defined may be more fully defined by reference to the specification as a whole.

[0024] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications, and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.Definitions

[0025] Definitions of terms may appear throughout the specification. It is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0026] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” include “at least one” and “one or more.”

[0027] The terms "comprising", "comprises," and "comprised of” as used herein are synonymous with "including," "includes," "containing," "contains," and grammatical variants thereof, and are inclusive or open-ended and do not exclude additional, non-recited members, elements, or method steps. The terms "comprising," "comprises," "comprised of,” "including," "includes," "containing," "contains," and grammatical variants thereof also include the term "consisting of’.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0029] As used herein, the term “Enzyme Commission” Number, abbreviated “EC", refers to enzyme nomenclature recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), as generally known to one skilled in the art (e.g., see enzyme nomenclature from NC-IUBMB, 1992 (Academic Press, San Diego, California), including supplements 1-5 published in 1994 (Eur. I. Biochem., 223: 1-5), 1995 (Eur. I. Biochem. 232: 1-6); 1996 (Eur. J. Biochem, 237: 1-5), 1997 (Eur. J. Biochem. 250: 1-6) and 1999 (Eur. J. Biochem. 264: 610-650), respectively. Likewise, the nomenclature is regularly supplemented and updated (see, e.g., chem.qmul.ac.uk / iubmb / enzyme / mdex.html).

[0030] As used herein, the term “cellulase” means enzymes that hydrolyze cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. The two basic approaches for measuring cellulolytic activity include: (1) measuring the total cellulolytic activity, and (2) measuring the individual cellulolytic activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., Outlook for cellulase improvement: Screening and selection strategies, 2006, Biotechnology Advances 24: 452-481. Total cellulolytic activity is usually measured using insoluble substrates, including Whatman Nol 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 Nol filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (1987, Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

[0031] As used herein, the term “beta-glucosidase” means a beta-D-glucoside glucohydrolase (EC 3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues with the release of beta-D-glucose.

[0032] As used herein, the term “cellobiohydrolase” includes 1,4-p-D-glucan glucohydrolases (EC 3.2.1.74) as well as 1,4-p-D-glucan cellobiohydrolase (EC 3.2.1.91). Cellobiohydrolases typically cleave cellulose strands to produce cellobiose. In some embodiments, the cellobiohydrolase is a cellobiohydrolase I (CBHI). In some embodiments, the cellobiohydrolase is a cellobiohydrolase II (CBHII).

[0033] As used herein, the term “endoglucanase” means an endo-l,4-(l,3;l,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 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components. An endoglucanase also refers to anenzyme classified according to EC 3.2.1.6. In some embodiments, the endoglucanase is an endoglucanase I (EGI). In some embodiments, the endoglucanase is an endoglucanase II (EGII).

[0034] The term “hemicellulase” means enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom, D. and Shoham, Y. Microbial hemicellulases. Current Opinion In Microbiology, 2003, 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 pectate lyase, a xylanase, and a xylosidase. The substrates of these enzymes, the 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, e.g., 50°C, 55°C, or 60°C, and pH, e.g., 5.0 or 5.5.

[0035] As used herein, the term “protease” includes any enzyme belonging to the EC 3.4 enzyme group (including each of the eighteen subclasses thereof). As described herein, proteins (polypeptides) having protease activity (i.e., proteases), are also known in the art as peptidases, proteinases, peptide hydrolases, and proteolytic enzymes.

[0036] As used herein, protease activity means proteolytic activity (EC 3.4). Protease activity can generally be measured using any assay, in which a substrate is employed, that includes peptide bonds relevant for the specificity of the protease in question. Assays for pH and assays for temperature are likewise to be adapted to the protease in question. Examples of assay pH-values are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 and examples of assay temperatures are 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 80°C, 90°C, or 95°C. Examples of general protease substrates are casein, bovine serum albumin (BSA) and hemoglobin.

[0037] As used herein, proteases may be of the “exo-type” (i.e., exopeptidases) that hydrolyze proteins (peptide bonds) starting at either N-terminal or C-terminal end of the protein chain, or of the “endo-type” (i.e., endopeptidases) that hydrolyze peptide bonds of the non-terminal ends of the protein chain (i.e., internal peptide bonds).

[0038] The term "pectinase" is defined as any enzyme that degrades pectic substances. Pectic substances include homogalacturonans, xylogalacturonans, and rhamnogalacturonans as well as derivatives thereof. Pectinase treatment may be achieved by one or more pectinases, such as two or more pectinases of the same type (e.g., two different pectin methylesterases) or of different types (e.g., a pectin methylesterase and an arabinanase). The pectinase may, for example, be selected from the group consisting of arabinanase (catalyses the degradation of arabinan sidechains of pectic substances), arabinofuranosidase (removes arabinosyl substituents from arabinans and arabinogalactans), galactanase (catalyses the degradation of arabinogalactan and galactan sidechains of pectic substances), pectate lyase (cleaves glycosidic bonds in polygalacturonic acid by beta-elimination), pectin acetylesterase (catalyses the removal of acetyl groups from acetylated pectin), pectin lyase (cleaves the glycosidic bonds of highly methylated pectins by beta-elimination), pectin methylesterase (catalyses the removal of methanol from pectin, resulting in the formation of pectic acid, polygalacturonic acid), polygalacturonase (hydrolyses the glycosidic linkages in the polygalacturonic acid chain), rhamnogalacturonan acetylesterase (catalyses the removal of acetyl groups from acetylated rhamnogalacturonans), and rhamnogaiacturonase and rhamnogalacturonan lyase (degrade rhamnogalacturonans).

[0039] As used herein, the term “phospholipase” refers to an enzyme that hydrolyzes phospholipids into fatty acids (saturated or unsaturated), lysophospholipids, diacylgycerols, choline phosphate and phophatidates, depending on the site of hydrolysis. Phospholipases are further classified into types A, B, C and D.

[0040] As used herein, the terms “contacting,” “admixing,” “adding,” “combining,” and the like, including grammatical variants thereof, refer to the combining of one or more ingredients and / or enzymes, where the one or more ingredients or enzymes are combined in any order and in any combination. In some embodiments, contact may relate to mixing one or more ingredients and / or enzymes simultaneously or sequentially.

[0041] Crude palm oil may refer to a pressed or extracted oil or a mixture thereof. The crude oil may refer to the output from the press of a palm oil mill; i.e. to the mixture of oil and water pressed out of the palm fruit mash, before it has been subject to clarification and separation of oil from water.

[0042] Palm oil mill effluent (POME), as referred to herein, is combination of waste including sterilizer condensate discharged from the sterilization process, waste from crude oil clarification process, waste from the depericarping step processed through a hydrocyclone, and wastewater from mill cleaning activities. POME may include heavy phase, e.g., from the separator.

[0043] The terms “recovered,” “isolated,” “extracted,” and “separated,” refer to a compound, protein (polypeptide), cell, nucleic acid, amino acid, oil, or other specified material or component that is removed from at least one other material or component. In some embodiments, the at least one other material or component is at least one other material or component with which the compound, protein (polypeptide), cell, nucleic acid, amino acid, oil, or other specified material or component is naturally associated as found in nature. In some embodiments, the at least one other material or component is at least one other material or component with which the compound, protein (polypeptide), cell, nucleic acid, amino acid, oil, or other specified material or component is associated with under experimental or production conditions and / or systems. For example, an “isolated” polypeptide includes, but is not limited to, a polypeptide removed from a culture broth containing a heterologous host cell expressing the polypeptide.

[0044] The term “enriched” refers to material (e.g., an isolated compound, polypeptide, polynucleotide, or other specified material or component) that is in about 50% pure, at least about 60% pure, at least about 70% pure, or even at least about 80% pure.

[0045] As used herein, “from” encompasses “derived from," "originated from," "obtained from," "isolated from," and the like, and grammatical variants thereof.

[0046] Oil extraction rate (OER) refers to the ratio of oil recovered and Fresh fruit bunch (FFB) times 100. According to this definition, the mathematical formula is: OER % = (weight of oil recovered / weight of FFB processed) x 100.

[0047] Mass balance, as used herein, refers to a method of determining and tracking characteristics in a physical system by accounting for the material entering and leaving the system. An advantage of mass balance is the uncovering of mass flow, which provides information about the nature of material conserved in the system. In some embodiments, mass balance is used to determine the composition of an output, e.g., crude palm oil, sludge, decanter cake, enzyme contacted CO, recovered oil, Heavy Phase, POME, final crude palm oil product, as a result of the methods, compositions, and systems provided herein. In some embodiments, mass balance is used to optimize the methods, compositions, and systems described herein.

[0048] As used herein, the terms “wild-type” and “native” are used interchangeably and refer to genes, proteins, strains, or other components found in nature, or that are not intentionally modified.

[0049] The term “amino acid sequence” is synonymous with the terms “polypeptide,” “protein,” and “peptide,” and are used interchangeably. Where such amino acid sequences exhibit activity, they may be referred to as an “enzyme.” The conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino- to-carboxy terminal orientation (i.e., N— >C).

[0050] The term “nucleic acid” encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded and may contain chemical modifications. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

[0051] “Hybridization” refers to the process by which one strand of nucleic acid forms a duplex with, i.e., base pairs with, a complementary strand, as occurs during blot hybridization techniques and PCR techniques. Stringent hybridization conditions are exemplified by hybridization under the following conditions: 65°C and 0.1X SSC (where IX SSC = 0.15 M NaCl, 0.015 M Na3 citrate, pH 7.0). Hybridized, duplex nucleic acids are characterized by a melting temperature (Tm), where one half of the hybridized nucleic acids are unpaired with the complementary strand. Mismatched nucleotides within the duplex lower the Tm.

[0052] The terms “transformed,” “stably transformed,” and “transgenic,” used with reference to a cell means that the cell contains a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations.

[0053] The term “introduced” in the context of inserting a nucleic acid sequence into a cell, encompasses, but is not limited to, “transfection”, “transformation” and “transduction,” as known in the art. Exemplary methods for introducing polynucleotides or polypeptides by transformation into a host cell, include, but are not limited to, microinjection, electroporation, stable transformation methods, transient transformation methods (such as induced competence using chemical (e.g. divalent cations such as CaCh), mechanical (electroporation) means, or methods such as those described in published international applications WO 2018 / 114983 and WO 2010 / 149721, which are incorporated herein by reference in their entireties), ballistic particle acceleration (particle bombardment), direct gene transfer, viral-mediated introduction, cellpenetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery. Introducing a nucleic acid, construct, plasmid, or vector into a host cell may be carried out byconjugation, which is a specific method of natural DNA exchange requiring physical cell-to-cell contact. Introducing a nucleic acid, construct, plasmid, or vector into a host cell may be carried out by transduction, which is the introduction of DNA via a virus (e.g., phage) infection which is also a natural method of DNA exchange. Generally, such methods involve incorporating a polynucleotide within a viral DNA or RNA molecule.

[0054] A “host cell” is an organism into which an expression vector, phage, vims, or other nucleic acid sequence including a polynucleotide encoding a polypeptide of interest (e.g., an epimerase) has been introduced. Exemplary host cells are microorganism cells (e.g., bacteria, filamentous fungi, and yeast), mammalian cells, and plant cells capable of expressing the polypeptide of interest. The term “host cell” includes protoplasts created from cells.

[0055] Those of skill in the art are well aware of suitable methods for introducing polynucleotides into filamentous fungal cells e.g., Aspergillus sp., Trichoderma sp., etc.), wherein standard techniques for transformation of filamentous fungi and culturing the fungi (which are well known to one skilled in the art) are used to transform a fungal host cell of the disclosure. Thus, the introduction of a DNA construct or vector into a fungal host cell includes techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection mediated and DEAE-Dextrin mediated transfection), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, gene gun or biolistic transformation, protoplast fusion and the like. General transformation techniques are known in the art (see, e.g., Ausubel et al., 1987, Sambrook et al., 2001 and 2012, and Campbell et al., 1989). Also of use is the Agrobacterium-mediated transfection method such as the one described in U.S. Patent No. 6,255,115. The expression of heterologous proteins in Trichoderma has been described, for example, in U.S. Patent Nos. 6,022,725; 6,268,328; Harkki et al., 1991 and Harkki et al., 1989. Reference is also made to Cao et al. (2000), for transformation of Aspergillus strains.

[0056] Transformation of Trichoderma sp. cells generally use protoplasts or cells that have been subjected to a permeability treatment, typically at a density of 105to 107 / mL, particularly 2xlO6 / mL. A volume of 100 pL of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol and 50 mM CaCh) is mixed with the desired DNA. Generally, a high concentration of polyethylene glycol (PEG) is added to the uptake solution. Additives, such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like, may also be added to the uptake solution to facilitate transformation. Similar procedures are available for other fungal host cells.

[0057] Nucleic acid sequences (i.e. polynucleotides) or proteins (i.e. polypeptides) may be native or heterologous to the genome of the host cell. “Native,” “homologous,” or “endogenous” withrespect to a host cell, means that the nucleic acid sequence does naturally occur in the genome of the host cell or that the protein is naturally produced by that cell. The terms “native,” “homologous,” and “endogenous” are used interchangeably herein.

[0058] The term “heterologous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell. “Heterologous” also includes a native coding region, or portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native gene, e.g., not in its natural location in the organism’s genome. A heterologous gene may include a native coding region that is a portion of a chimeric gene including non-native regulatory regions that is reintroduced into the native host. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.

[0059] The term “endogenous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.

[0060] The term “expression” refers to the process by which a polypeptide is produced based on a nucleic acid sequence. The process includes both transcription and translation.

[0061] A “vector” refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.

[0062] An “expression vector” refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

[0063] The terms “operably linked,” “operable contact,” “operably connected,” and the like mean that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner.

[0064] A “signal sequence” is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.

[0065] The term “specific activity” refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specific conditions. Specific activity is generally expressed as units (U) / mg of protein.

[0066] “A cultured cell material” or similar language, refers to a cell lysate or supernatant (including media) that includes a protein of interest as a component. The cell material may befrom a cell or host cell that is grown in culture for the purpose of producing the protein. In some embodiments, the enzyme composition, e.g., first enzyme composition, second enzyme composition, is a cultured cell material. For example, the cellulases may be contained in a cultured cell material, such as a whole broth.

[0067] As used herein, “clarified,” when used in reference to cultured cell material, e.g., a whole broth, means a cultured cell material which has been subjected to at least one clarification process to remove cell debris and / or other insoluble components. Clarification processes, as understood in the art include, but are not limited to, centrifugation techniques, cross-flow membrane filtration techniques, solid / liquid filtration techniques, and the like.

[0068] “Percent sequence identity” means that a particular sequence has at least a certain percentage of amino acid residues or nucleotides identical to those in a specified reference sequence, when aligned using e.g., the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithm are:Gap opening penalty: 10.0Gap extension penalty: 0.05Protein weight matrix: BLOSUM seriesDNA weight matrix: IUBDelay divergent sequences %: 40Gap separation distance: 8DNA transitions weight: 0.50List hydrophilic residues: GPSNDQEKRUse negative matrix: OFFToggle Residue specific penalties: ONToggle hydrophilic penalties: ONToggle end gap separation penalty OFF.

[0069] Deletions are counted as non-identical residues, compared to a reference sequence. Deletions occurring at either terminus are included.

[0070] The term “increased” as used herein can refer to a quantity or activity that is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 50%, 100%, or 200% more than the quantity or activity for which the increased quantity or activity is being compared. The terms “increased,” “elevated,” “enhanced,” “improved,” and the like are used interchangeably herein.

[0071] The term “decreased” as used herein can refer to a quantity or activity that is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 50%, 100%, or 200% less than the quantity or activity for which the decreased quantity or activity is being compared. The terms “decreased,” “lowered,” “reduced,” and the like are used interchangeably herein.

[0072] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0073] Numerical values and ranges may be presented herein with the numerical value being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number can be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. For example, in connection with a numerical value, the term “about” refers to a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in context. All values and ranges implicitly include the term “about” unless the context clearly dictates otherwise.I. METHODS AND COMPOSITIONS FOR PRODUCING A FINAL CRUDE PALM OIL PRODUCT

[0074] Provided herein are methods useful for improving the production of crude palm oil, including the use of one or more enzymes or an enzyme composition. In some embodiments, the methods include the use of low temperature, e.g., temperatures below those used in conventional processes. In some cases, the use of one or more enzymes or an enzyme composition and / or low temperatures are specific to certain steps (e.g., unit operations) of the crude palm oil production process. Thus, in an aspect is provided a method for producing a final crude palm oil product, including contacting a crude palm oil (CO) with an enzyme composition, where the enzyme composition includes a cellulase. In some embodiments, the enzyme composition includescellulolytic and / or hemicellulolytic activity. In some embodiments, the enzyme composition is a combination of one or more cellulases.A. Methods Steps and Enzyme Compositions

[0075] As described above, processes for extracting crude palm oil are generally well known. The conventional process includes a series of unit operations so connected (e.g., in operable contact), to allow extraction and purification of crude palm oil from palm-oil containing raw materials, e.g., palm fresh fruit bunches (FFB). See, e.g., Section II and FIG. 4. An exemplary conventional process for crude palm oil extraction includes a sterilizing unit, a stripping unit, a digester unit, a pressing unit, a screening unit, a clarifying unit, a holding tank unit (DCO / UDCO tank), a pure oil tank unit, a purifying unit, and a drying unit. The methods provided herein include additional steps (e.g., unit operations) for increasing crude palm oil recovery.

[0076] Sterilization is the first step in the process and plays a critical role in the final oil quality as well as the separation of FFB to derive mass passing to digester (MPD) in the stripping step. Steam sterilization of FFB facilitates fruits being cooked and sterilized via steam injection and later stripped from bunches at a stripping unit to yield the palm fruitlet or MPD. The sterilization step has several advantages, one being that it softens the fruit mesocarp for subsequent digestion. Typically, the palm fruitlets coming out of the sterilization step have a temperature of up to or above 95 °C. Thus, in some embodiments, the methods include sterilizing FFB to produce sterilized FFB (SFB). In some embodiments, the sterilizing step further produces a sterilizer condensate. In some embodiments the sterilizer condensate is one of the components contributing to palm oil milling effluent (POME).

[0077] After sterilization, stripping, digestion, and pressing may be performed using unit operations as shown in FIG. 4. Stripping, also referred to as threshing, may be carried out in a mechanized system having a rotating drum that facilitates detachment of the fruitlets from the bunch, leaving the spikelets on the stem and empty fruit bunch (EFB). After stripping, the palm fruitlets (also referred to as mass passing to digester or MPD) are moved to a digester unit by a transportation means either by screw or scrapper conveyor or a combination of both methods. During the stripping step the palm fruitlets start to cool, as the stripped mass of fruitlets or MPD passes towards the digester by means of one or more conveyors. Thus, in some embodiments, the methods include separating the SFB to produce an MPD. In some embodiments, the method further includes producing EFB. In some embodiments, the method includes pressing the EFB to produce an EFB liquor.

[0078] In some embodiments, the SFB and / or MPD may be contacted with an enzyme composition to increase crude palm oil extraction. Thus, in some embodiments, the SFB arecontacted with an enzyme composition during the stripping step. For example, the SFB may be contacted with an enzyme composition while in the stripping unit. In some embodiments, the SFB may be contacted with an enzyme composition during transport to the stripping unit, e.g., contacted while present on a conveyor belt or other means of transport to the stripping unit. In some embodiments, the MPD is contacted with an enzyme composition in the digester unit. In some embodiments, the MPD is contacted with an enzyme composition during transport to the digester unit, e.g., contacted while present on a belt, screw, or scrapper conveyor or other means of transport to the digester unit. In some embodiments, an enzyme composition is applied at any one of these steps to the SFB and / or the MPD. In some embodiments, an enzyme composition is applied at any one or more of these steps to the SFB and / or the MPD. The contacting may occur by any suitable means including spraying or an intermediate step allowing the SFB and / or the MPD to incubate with an enzyme composition in an enzyme reaction tank unit.

[0079] In the digester, the MPD is reheated above 90°C to loosen the pericarp. The digester is typically a steam-heated vessel with rotating shafts to which are attached a combination of digester short and long sharp-edged arms with an expeller arm at the bottom. The steam-heated digester reheats the MPD to temperatures above 90°C. The MPD are rotated and stirred, causing pericarps to loosen from the nuts and the mesocarp to degrade. The digester is continuously refilled with MPD and kept at a high level as the digested fruit is removed and passed to the next unit operation. Thus, in some embodiments, the method includes digesting MPD to produce a digested MPD, also alternatively referred to as a fruit mash.

[0080] As with the SFB and / or MPD, the digested MPD may be contacted with an enzyme composition to increase crude palm oil extraction. Thus, in some embodiments, the digested MPD is contacted with an enzyme composition during the digesting step. For example, the MPD may be contacted with an enzyme composition while in the digester unit. In some embodiments, the digested MPD may be contacted with an enzyme composition during transport of the digested MPD to the next unit operation, e.g., the pressing unit.

[0081] It should be appreciated that the enzyme composition for contacting the SFB, the MPD, and / or the digested MPD (fruit mash) is a second enzyme composition, while the enzyme composition contacting the crude palm oil (CO) is a first enzyme composition. In some embodiments, the first and second enzyme compositions are identical. In some embodiments, the first and second enzyme compositions are different. In some embodiments, the second enzyme composition contains one or more of a cellulase, a hemicellulase, a beta-glucanase, a xylanase, a mannanase, a pectinase, a protease, or a phospholipase.

[0082] The digested MPD (fruit mash) is next fed into a pressing unit, such as a screw press, from which a mixture of oil, water, and solids and fiber, free shells, whole nuts / broken nuts, and whole / broken kernels are discharged. The liquid discharge, containing oil, water, fiber and nonoil solids, is passed through a screening unit, for example a vibrating screen, to separate the insoluble fibers from the liquid phase (oil and water) and extract the crude palm oil (CO). Thus, in some embodiments, the method includes pressing the digested MPD to produce a CO. In some embodiments, the CO is not diluted. Thus, in some embodiments, the CO is an undiluted crude palm oil (UDCO). In some embodiments, the CO may be diluted to produce a diluted crude palm oil (DCO). In some cases, the CO is diluted, e.g., with water, to reduce viscosity and prevent chokage at unit operation e.g., pumps, vibrating screen.

[0083] The methods provided herein include contacting the CO with an enzyme composition (e.g., first enzyme composition). In some embodiments, the CO contacted with the enzyme composition is UDCO. For example, in some cases, the UDCO extracted from the MPD is contacted with the enzyme composition. In some embodiments, the UDCO contacted with the enzyme composition is subsequently diluted to produce a DCO contacted with the enzyme composition. In some embodiments, the subsequent dilution of the UDCO may occur at any step or at an intermediate step among the remainder of unit operations for producing a final crude palm oil product. In some embodiments, the CO contacted with the enzyme composition is DCO. For example, in some cases, the UDCO may be diluted to produce a DCO prior to contacting with the enzyme composition. In some embodiments, a UDCO and DCO may be contacted with the enzyme composition. For example, in some cases, the UDCO is contacted with the enzyme composition, subsequently diluted to produced DCO, and the produced DCO is contacted with the enzyme composition.

[0084] In some embodiments, the methods include incubating the CO contacted with the enzyme composition for a duration of or of at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 18 hours, at least 20 hours, or at least 24 hours. In some embodiments, the incubation period is at least or about 30 minutes. In some embodiments, the incubation period is at least or about 1 hour. In some embodiments, the incubation period is at least or about 2 hours. In some embodiments, the incubation period is at least or about 3 hours. In some embodiments, the incubation period is at least or about 4 hours. In some embodiments, the incubation period is at least or about 5 hours. In some embodiments, the incubation period is at least or about 6 hour. In some embodiments, the incubation period is in the range of 30 minutes to 6 hours. In someembodiments, the incubation period is in the range of 1 to 6 hours. In some embodiments, the incubation period is in the range of 1 to 5 hours. In some embodiments, the incubation period is in the range of 1 to 4 hours. In some embodiments, the incubation period is in the range of 1 to 3 hours. In some embodiments, the incubation period is in the range of 3 to 6 hours. In some embodiments, the incubation period is in the range of 1 to 2 hours. In some embodiments, the incubating includes mixing or stirring the contacted CO. For example, the CO contacted with the enzyme composition may be mixed, stirred, recirculated or otherwise agitated during the incubation period. In some embodiments, the mixing occurs intermittently or variably, or at regular intervals. In some embodiments, the mixing is continuous. In some embodiments, the contacting of the CO occurs in the UDCO / DCO tank unit. In some embodiments, the contacting of the CO occurs in an enzyme reaction tank unit. In some embodiments, the enzyme reaction tank unit is an enzyme reaction tank unit as described in Section II.

[0085] In some embodiments, the enzyme composition (e.g., first enzyme composition) includes a cellulase. In some embodiments, the cellulase includes a cellobiohydrolase (EC 3.2.1.91 and / or EC 3.2.1.74), an endoglucanase (EC 3.2. 1.4), beta-glucosidase (EC 3.2.1.21), or any combination thereof. In some embodiments, the enzyme composition (e.g., first enzyme composition) includes a cellulase. In some embodiments, the cellulase includes a cellobiohydrolase (EC 3.2.1.91 and / or EC 3.2.1.74), an endoglucanase (EC 3.2.1.4 and / or EC 3.2.1.6), beta-glucosidase (EC 3.2.1.21), or any combination thereof. In some embodiments, the cellulase includes a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta-glucosidase (BGL), or any combination thereof.

[0086] In some embodiments, the cellulase is derived from a fungus. In some embodiments, the cellulase is derived from a strain of Aspergillus. In some embodiments, the strain of Aspergillus is a strain of Aspergillus aurantiacus, Aspergillus niger, or Aspergillus oryzae. In some embodiments, the cellulase is derived from a strain of Chrysosporium. In some embodiments, the strain of Chrysosporium is a strain of Chrysosporium iucknowense. In some embodiments, the cellulase is derived from a strain of Humicola. In some embodiments, the strain of Humicola is a strain of Humicola insolens. In some embodiments, the cellulase is derived from a strain of Penicillium. In some embodiments, the strain of Penicillium is a strain of Penicillium emersonii or Penicillium oxalicum. In some embodiments, the cellulase is derived from a strain of Talaromyces. In some embodiments, the strain of Talaromyces is a strain of Talaromyces aurantiacus or Talaromyces emersonii. In some embodiments, the cellulase is derived from a strain of Trichoderma. In some embodiments, the strain of Trichoderma is a strain of Trichoderma reesei.

[0087] In some embodiments, the fungus from which the cellulase is derived is a host cell. In some embodiments, the fungus is a recombinant fungus (e.g., host cell) that expresses, overexpresses, or does not express one or more cellulases. A recombinant fungus be alternatively referred to herein as an engineered fungus. In some embodiments, the expressed or over-expressed cellulase is a recombinant cellulase. In some embodiments, the expressed or over-expressed cellulase is a heterologous cellulase. In some embodiments, the over-expressed cellulase is a native cellulase. In some embodiments, native, heterologous, and / or recombinant cellulase are derived from the fungus.

[0088] In some embodiments, the cellulase is contained in a cultured cell material, e.g., a cultured cell material from a fungus. In some embodiments, the cellulase is contained in a cultured cell material, e.g., a cultured cell material from a fungus where the fungus is a host cell (e.g., a recombinant fungus). In some embodiments, the cultured cell material is a whole broth. In some embodiments, the cultured cell material is clarified.

[0089] In some embodiments, the cellulase is derived from a strain of Trichoderma reesei. In some embodiments, the cellulase is a cultured cell material, e.g., a cultured cell material from Trichoderma reesei. In some embodiments, the Trichoderma reesei is a recombinant Trichoderma reesei (e.g., engineered Trichoderma reesei) designed to express or over-express one or more cellulases, optionally including a recombinant cellulase. In some embodiments, the cultured cell material is a whole broth. In some embodiments, the cultured cell material is clarified.

[0090] In some embodiments, the enzyme composition further includes hemicellulolytic activity. In some embodiments, the enzyme composition further comprises a hemicellulase. In some embodiments, the hemicellulase includes a xylanase, 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 pectate lyase, a xylanase, a xylosidase, or any combination thereof. In some embodiments, the hemicellulase includes a xylanase.

[0091] In some embodiments, the fungus from which the cellulase is derived also expresses a hemicellulase. In some embodiments, the hemicellulase is a xylanase, 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 pectate lyase, a xylosidase, or any combination thereof. In some embodiments, the hemicellulase includes a xylanase. In some embodiments, the hemicellulase includes an esterase. In some embodiments, the esterase is a feruloyl esterase (EC 3.1.1.73). In some embodiments, the esterase is an acetylxylan esterase (EC 3.1.1.72). In some embodiments, the hemicellulaseincludes an arabinofuranosidase (EC 3.2.1.55). In some embodiments, the hemicellulase is a native hemicellulase, e.g., a native xylanase, esterase, arabinofuranosidase. In some embodiments, the hemicellulase is a recombinant hemicellulase, e.g., a recombinant xylanase, esterase, arabinofuranosidase. In some embodiments, the fungus from which the cellulase is derived is a host cell that expresses, over-expresses, or does not express a hemicellulase. In some embodiments, the expressed or over-expressed hemicellulase is a recombinant hemicellulase. In some embodiments, the over-expressed hemicellulase is a native hemicellulase.

[0092] In some cases, for example if the cellulase is a cultured cell material, the cellulase includes one or more hemicellulases. In some embodiments, the one or more hemicellulases include native and / or recombinant hemicellulases.

[0093] In some embodiments, the enzyme composition is dosed at a concentration of at least about 0.01% weight (wt) enzyme / wt per FFB input into sterilization. In some embodiments, the enzyme composition is dosed at a concentration of at least about 0.1% wt enzyme / wt per FFB input into sterilization. In some embodiments, the enzyme composition is dosed at a concentration of at least about 0.5% wt enzyme / wt per FFB input into sterilization. In some embodiments, the enzyme composition is dosed at a concentration of at least about 1% wt enzyme / wt per FFB input into sterilization. In some embodiments, the enzyme composition is dosed at a concentration of at least about 2% wt enzyme / wt per FFB input into sterilization. In some embodiments, the enzyme composition is dosed at a concentration of at least about 5% wt enzyme / wt per FFB input into sterilization. In some embodiments, the enzyme composition is dosed at a concentration in a range of about 0.05% to 5% wt enzyme / wt per FFB input into sterilization. In some embodiments, the enzyme composition is dosed at a concentration in a range of about 0.1% to 5% wt enzyme / wt per FFB input into sterilization. In some embodiments, the enzyme composition is dosed at a concentration in a range of about 0.5% to 5% wt enzyme / wt per FFB input into sterilization. In some embodiments, the enzyme composition is dosed at a concentration in a range of about 1% to 5% wt enzyme / wt per FFB input into sterilization. In some embodiments, the enzyme is dosed into the CO based on the enzyme wt / wt per FFB input into sterilization. In some embodiments, the enzyme is dosed into the UDCO based on the enzyme wt / wt per FFB input into sterilization. In some embodiments, the enzyme is dosed into the DCO based on the enzyme wt / wt per FFB input into sterilization.

[0094] The pH of the incubation may range from about 3.0 to about 8.0. In some embodiments, the pH is in a range of about 4.0 to 8.0. In some embodiments, the pH is in a range of about 4.0 to 6.0. In some embodiments, the pH is in a range of about 4.0 to 5.0.

[0095] In a conventional process, from the digester unit through to the pressing unit, the temperatures are typically between about 90 and 95 °C. However, in aspects of the methods provided herein, the temperature of the CO may be reduced below conventional temperatures. For example, in some embodiments, contacting the CO with the enzyme composition occurs at a temperature of or of less than about 90°C, 80°C, or 70°C. In some embodiments, contacting the CO with the enzyme occurs at a temperature of or of less than about 75°C, 70°C, 65°C, 60°C, 55 °C, or 50°C. In some embodiments, contacting the CO with the enzyme occurs at a temperature of or of less than about 70°C, 65°C, 60°C, 55°C, or 50°C. In some embodiments, contacting the CO with the enzyme occurs at a temperature of or of less than about 65°C, 60°C, 55°C, or 50°C. In some embodiments, the contacting occurs at a temperature of less than 75°C. In some embodiments, the contacting occurs at a temperature of less than 70°C. In some embodiments, the contacting occurs at a temperature of less than 65°C. In some embodiments, the contacting occurs at a temperature of less than 60 °C. In some embodiments, the contacting occurs at a temperature of less than 55 °C. In some embodiments, the contacting occurs at a temperature of less than 50°C. In some embodiments, the contacting occurs at a temperature of less than 45°C. In some embodiments, the contacting occurs at a temperature between 50 and 75°C. In some embodiments, the contacting occurs at a temperature between 50 and 70°C. In some embodiments, the contacting occurs at a temperature between 50 and 65 °C. In some embodiments, the contacting occurs at a temperature between 50 and 60°C. In some embodiments, the contacting occurs at a temperature of about 75°C. In some embodiments, the contacting occurs at a temperature of about 70°C. In some embodiments, the contacting occurs at a temperature of about 65 °C. In some embodiments, the contacting occurs at a temperature of about 60°C. In some embodiments, the contacting occurs at a temperature of about 55 °C. In some embodiments, the contacting occurs at a temperature of about 50°C.

[0096] In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature of or of less than about 90°C, 80°C, or 70°C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature of or of less than about 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature of or of less than about 65°C, 60°C, 55°C, or 50°C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature of less than 75°C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature of less than 70°C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature of less than 65 °C. In some embodiments, prior to contacting theCO with the enzyme composition, the CO is cooled to a temperature of less than 60°C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature of less than 55 °C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature of less than 50°C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature of less than 45°C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature between 50 and 75°C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature between 50 and 70°C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature between 50 and 65 °C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature between 50 and 60°C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature of about 75 °C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature of about 70°C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature of about 65 °C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature of about 60 °C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature of about 55°C. In some embodiments, prior to contacting the CO with the enzyme composition, the CO is cooled to a temperature of about 50°C.

[0097] In some embodiments, following the incubating, the CO is heated to a temperature in the range of about 65 to about 100°C. In some embodiments, following the incubating, the CO is heated to a temperature in the range of between about 70 and about 95°C. In some embodiments, following the incubating, the CO is heated to a temperature in the range of between about 80 and about 95°C. In some embodiments, following the incubating, the CO is heated to a temperature in the range of between about 65 and about 75°C. In some embodiments, following the incubating, the CO is heated to a temperature in the range of between about 70 and about 75°C. In some embodiments, following the incubating, the CO is heated to a temperature in the range of between about 65 and about 70°C. In some embodiments, following the incubating, the CO is heated to a temperature of about 70°C. In some embodiments, following the incubating, the CO is heated to a temperature of about 75°C. In some embodiments, following the incubating, the CO is heated to a temperature of about 80°C. In some embodiments, following the incubating, the CO is heated to a temperature of about 85 °C. In some embodiments, following the incubating, the CO is heated to a temperature of about 95 °C. In some embodiments, after the incubating, the temperature of the CO is maintained at a temperature described in this paragraph for one or more of the remainingunit operations in the crude palm oil process. In some embodiments, after the incubating, the temperature of the CO is maintained at a temperature described in this paragraph for the remainder of the crude palm oil process.

[0098] In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature of or of less than about 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature of or of less than about 70°C, 65°C, 60°C, 55°C, or 50°C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature of or of less than about 65°C, 60°C, 55°C, or 50°C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature of less than 75 °C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature of less than 70°C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature of less than 65°C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature of less than 60°C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature of less than 55°C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature of less than 50°C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature of less than 45 °C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature between 50 and 75°C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature between 50 and 70°C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature between 50 and 65 °C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature between 50 and 60°C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature between 65 and 75°C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature between 65 and 70°C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature between 70 and 75 °C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature of about 75 °C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature of about 70°C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature of about 65°C. In some embodiments, after the incubatingthe CO with the enzyme composition, the CO is maintained at a temperature of about 60°C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature of about 55 °C. In some embodiments, after the incubating the CO with the enzyme composition, the CO is maintained at a temperature of about 50°C. In some embodiments, after the incubating, the temperature of the CO is maintained at a temperature described in this paragraph for one or more of the remaining unit operations in the crude palm oil process. In some embodiments, after the incubating, the temperature of the CO is maintained at a temperature described in this paragraph for the remainder of the crude palm oil process.

[0099] In some cases, palm oil extraction may be further increased by recycling oil-containing waste streams back into unit operations of the extraction process. Exemplary methods for performing such recycling are described in published international application WO2017 / 182667, which is incorporated herein by reference. In some embodiments, the sterilized condensate and / or the fruit press liquor is added to the MPD, fruit mash, or CO.

[0100] Conventional processes further include clarifying the crude palm oil, purifying the oil, and drying the oil before storing the oil. Thus, in some embodiments the methods include clarifying the CO contacted and incubated with the enzyme composition in a clarifying unit. In some embodiments, the clarifying unit produces a pure oil. As used herein, and in the field, pure oil refers to the oil layer that is extracted from the clarifying unit, however the oil is not yet purified. In some embodiments, the pure oil is processed by a purifying unit and drying unit to produce a final crude palm oil (CPO) product.

[0101] The methods described herein may be carried out using an apparatus described herein. It is contemplated herein that mass balance may be used to determine the composition of outputs, e.g., CO, final crude palm oil product, sludge, POME, to assess the quality and optimize the methods and systems described herein. In some embodiments, the mass balance analysis is used to optimize the method. In some embodiments, the mass balance analysis is used to optimize the enzyme composition dose, temperature, and / or pH of the CO. In some embodiments, the mass balance analysis is used to optimize the enzyme composition dose and / or temperature of the CO.IL SYSTEM FOR PRODUCING A FINAL CRUDE PALM OIL PRODUCT

[0102] In an aspect is provided a system for producing a final crude palm oil product. The system described herein is suitable for use according to the methods provided herein. See, Section I. Thus, provided herein is a system for producing a final crude palm oil product including a sterilizing unit, a stripping unit, a digester unit, a pressing unit, a screening unit, a buffer tank unit, a cooling unit, an enzyme reaction tank unit, a holding tank unit, a clarifying unit, a pure oil tank unit, a purifying unit, and a drying unit, where the units are in operable contact. In someembodiments, the system is a conventional system for palm oil extraction to which additional units are added. In some embodiments, the additional units are in operable contact with units of an existing conventional system. In some embodiments, the additional units are a buffer tank unit, a cooling unit, and an enzyme reaction tank unit. Thus, in an aspect is provided a system for producing a final crude palm oil product, including a buffer tank unit, a cooling unit, and an enzyme reaction tank unit, wherein the units are in operable contact, and wherein the buffer tank unit is in operable contact with a screening unit of an existing palm oil extraction system and receives crude palm oil (CO) from the screening unit, and the enzyme reaction tank unit is in operable contact with a holding tank unit of the existing palm oil extraction system, and the holding tank unit receives a CO contacted with one or more enzymes or an enzyme composition, e.g., as described herein, from the enzyme reaction tank unit. In some embodiments, the system is a continuous system. FIG. 4 shows a conventional system for palm oil extraction. FIG. 5 shows an exemplary system of the present invention for producing a final crude palm oil product.

[0103] With reference to FIG. 5, the system 100 includes a sterilizing unit 10, for receiving palm-oil containing raw material 110 such as fresh fruit bunches (FFB). In some embodiments, the FFB is sterilized using steam as the heating medium. The sterilizing unit 10 may be any suitable type of sterilizer, including but not limited to, a horizontally or vertically positioned cylindrical pressure vessel. Such pressure vessels are filled with steam, for example by injection, under pressure and the sterilizing may be performed as a batch process. In some embodiments, the temperature in the sterilizing unit, e.g., horizontal or vertical sterilizer, may vary' from about 120-130°C and the pressure may be about 3.0 bar. The sterilizing duration may vary and can be, e.g., up to 90 minutes or less. In some embodiments, for example when using a horizontal sterilizer, the FFB may be stacked for example in cages across the length of the vessel and the steam can be injected to the interior of the vessel. In some embodiments, for example when vertical sterilizers are used, the FFB may be stacked into the vessel without cages. In some embodiments, a continuous sterilizer is used. For continuous sterilizers, the sterilizing is carried out in a heating cabin injected with steam at atmospheric pressure as a continuous process. In some embodiments, the FFB is crushed before it is transported through the heating cabin to facilitate steam penetration. In the heating cabin, the FFB is exposed to steam at atmospheric pressure. In some embodiments, palm oil mill effluent (POME) 129 generated in the sterilizing unit is referred to as a sterilizer condensate from the steam and is sent to an effluent pond 25 for treatment. In some embodiments, the POME, e.g., sterilizer condensate, may be recycled back into a unit operation, for example as described in Section I.

[0104] In some embodiments, the process steam consumption of the sterilizing unit is up to 550 Kg per ton FFB. In some embodiments, the amount of steam consumption depends on the type of sterilization method used. In some embodiments, the steam is generated in a mill or biomass boiler 27, which typically requires use of a large amount of fresh water. By fresh water is meant water that is not salty, and / or water, which is considered suitable for consumption as for example watering of plants and / or as drinking water.

[0105] Downstream of the system’s sterilizing unit, the sterilized raw material 111 (SFB) is supplied to a stripping unit 11 for the removal of palm fruitlets from the raw material (FFB). The FFB may be conveyed to the stripping unit by any suitable system, including, but not limited to, a screw conveyor. In some embodiments, the stripping unit is a mechanized system having a rotating drum that facilitates detachment of the fruit from the bunch. Empty fresh fruit bunches (EFB) are separated from the palm fruitlets and recycle to plantation estates to be used as natural fertilizer, or optionally incinerated to generate an extra source of energy.

[0106] Palm fruitlet 112 or MPD from the stripping unit 11 is subsequently transferred to the system’s digester unit 12. The MPD may be conveyed using any suitable means for conveyance, including, but not limited to, a scrapper conveyor. In some embodiments, palm oil is released from the palm fruitlet in the digester unit by rupturing or breaking down oil-bearing cells. In some embodiments, the rupturing or breaking action can be obtained by mechanical beaters or stirrers, which are arranged to pound the stripped palm fruitlets. In some embodiments, the digester unit 12 is a cylindrical vessel. In some embodiments, the digester unit 12 can be heated e.g., using steam.

[0107] From the digester unit 12, the digested palm fruitlets 113 are transported to a pressing unit 13 located downstream of the digester unit 12. The digested palm fruitlets are pushed through the expeller arm in the digester and directed into the pressing unit that is attached below the digester unit. Thus, in some embodiments, the system provided herein includes a digester unit 12 and a stripping unit 11 located upstream of the pressing unit 13. In the pressing unit 13, crude palm oil (CO) 114 is pressed from the digested palm fruitlets 113. In some embodiments, the pressing unit 13 includes mechanical presses. In some embodiments, the pressing unit is a screw press. In some embodiments, a press cake 127 containing solids from the pressing unit is further supplied to a depericarping process 26, in which fibers and shells 128 and nuts are obtained. The press cake may be conveyed using any suitable means for conveyance, including, but not limited to, a screw conveyor. The nuts may be further treated using a nutcracker and further processed in a hydrocyclone to separate kernels and shells and hydrocyclone wastewater. In someT1embodiments, the hydrocyclone wastewater is contained in the POME of the palm oil mill and is provided to the effluent pond 25 for treatment.

[0108] The system further includes a screening unit 14. In some embodiments, the screening unit separates or extracts the CO from insoluble fibers. In some embodiments the screening unit is a vibrating screen.

[0109] It should be appreciated that the system may further include one or more apparatuses for delivering an enzyme composition (e.g., a second enzyme composition, see, Section I) to contact one or more of the SFB, the palm fruitlets (MPD), and / or the digested palm fruitlets (fruit mash, digested MPD). One or more of the units or conveyance systems between units may be fitted with an apparatus for delivering the second enzyme composition. Non-limiting examples of apparatuses for enzyme delivery may be found, for example, in international applications WO2017202983 and WO2018135937, which are incorporated herein by reference.

[0110] In some embodiments, the CO 115 output from the screening unit is subsequently provided to a buffer tank unit 15 of the system. In some embodiments, the CO 115 may be a UDCO or a DCO. In some embodiments, the CO 115 is UDCO. In some embodiments, the CO 115 is DCO. Thus, in some cases, the CO may be diluted or not diluted. From the buffer tank unit 15, the CO is transported to an enzyme reaction unit 17 by first passing through a cooling unit 16. In some embodiments, the cooling unit 16 decreases the temperature of the CO. In some embodiments, the cooling unit 16 is a heat exchanger. In some embodiments, the cooling system includes an automated chiller. In some embodiments, the automated chiller ensures consistency in cooling the CO 116. In some embodiments, the automated chiller decreases the temperature of the CO to a temperature as described in Section I, e.g., about or at least 75, 70, 65, 60, 55, or 50°C. In some embodiments, the automated chiller decreases the temperature of the CO to a temperature as described in Section I, e.g., about or at least 70, 65, 60, 55, or 50°C. In some embodiments, the automated chiller decreases the temperature of the CO to a temperature as described in Section I, e.g., about or at least 65, 60, 55, or 50°C. In some embodiments, the automated chiller decreases the temperature of the CO to a temperature of about or below 75 °C. In some embodiments, the automated chiller decreases the temperature of the CO to a temperature of about or below 70°C. In some embodiments, the automated chiller decreases the temperature of the CO to a temperature of about or below 65 °C. In some embodiments, the automated chiller decreases the temperature of the CO to a temperature of about or below 60°C. In some embodiments, the automated chiller decreases the temperature of the CO to a temperature of about or at least 60°C. In some embodiments, the automated chiller decreases the temperature of the CO to a temperature of aboutor at least 55 °C. In some embodiments, the automated chiller decreases the temperature of the CO to a temperature of about or at least 50°C.

[0111] In some embodiments, the buffer tank unit 15 and the cooling unit 16 are operably connected by a pump. For example, in some embodiments, the pump provides consistent flow of the CO 116 from the buffer tank unit 15 to the cooling unit 16. In some embodiments, the pump controls the flow of the CO from the buffer tank unit 15 to the cooling unit 16 and to the enzyme reaction tank unit 17, e.g., a single pump controls the flow of the CO from the buffer tank unit 15 to the enzyme reaction tank unit 17. In some embodiments, one pump controls the flow of the CO 116 from the buffer tank unit 15 to the cooling unit 16 and another pump controls the flow of the CO 117 from the cooling unit 16 to the enzyme reaction tank unit 17. In some embodiments, the pump is a continuous pump. In some embodiments, the pump is an inverter control pump. In some embodiments, the pump is a positive displacement pump, e.g., a peristaltic pump, centrifugal pump. In some embodiments, the pump is a peristaltic pump. In some embodiments, the pump is a centrifugal pump. In some embodiments, the pump is a gear pump. In some embodiments, the pump is an air operated diaphragm pump. In some embodiments, the pump is a mechanical diaphragm pump. In some embodiments, the pump is a fixed speed pump. In some embodiments, the pump is a variable speed pump. Pumps may be selected based upon the viscosities and suction requirements of the pump relative to the buffer tank unit and the CO.

[0112] In some embodiments, a flow meter or similar sensor is used to detect the flow of CO 116 from the buffer tank unit 15 to the cooling unit 16. In some embodiments, a flow meter or similar sensor is used to detect the flow of CO 117 from the cooling unit 16 to the enzyme reaction tank unit 17. Any flow meter or sensor suitable for detecting the rate, e.g., flow rate, of CO (e.g., CO 116 and / or CO 117) is contemplated for use in the system. In some embodiments, the pump includes a flow meter or sensor. For example, in some cases, the pump has a flow meter or sensor integrated into the pump apparatus. In some embodiments, the flow meter or sensor is separate from the pump. For example, the pump and the flow meter or sensor are two separate apparatuses. In some embodiments, the flow meter is a magnetic flow meter. In some embodiments, the flow meter or sensor measures the flow rate with about or at least 5% accuracy. In some embodiments, the flow meter or sensor is in operable contact with the pump. In some embodiments, the flow meter or sensor measures a flow rate and controls the pump. In this way, the flow meter or sensor can regulate, e.g., automatically, the flow rate of the CO. In some embodiments, information from the pump and / or flow meter is communicated to an operator or a programmable logic controller (PLC) capable of receiving and processing information from the pump and / or flow meter. In some embodiments, the flow rate is controlled manually, e.g., by an operator monitoring the flow meterand / or PLC. In some embodiments, the flow rate is controlled automatically, e.g., by a feedback command from, e.g., a PLC.

[0113] In some embodiments, the cooled CO 117 is provided to the enzyme reaction tank unit 17 from the cooling unit 16. In some embodiments, the CO is incubated with the enzyme composition (first enzyme composition) 118 in the enzyme reaction tank unit. In some embodiments, the enzyme reaction tank unit 17 is temperature controlled. For example, in some cases the enzyme reaction tank unit 17 further includes a water jacket to regulate the temperature inside the reaction tank. In some embodiments, the reaction tank unit maintains the CO at a decreased temperature described in Section I, e.g., about or at least 75, 70, 65, 60, 55, or 50°C. In some embodiments, the reaction tank unit maintains the CO at a decreased temperature described in Section I, e.g., about or at least 70, 65, 60, 55, or 50°C. In some embodiments, the reaction tank unit maintains the CO at a decreased temperature described in Section I, e.g., about or at least 65, 60, 55, or 50°C. In some embodiments, the water jacket includes a temperature sensor for regulating the temperature of the enzyme reaction tank unit 17. In some embodiments, the enzyme reaction tank unit 17 maintains the temperature of the CO at a temperature of about or below 75 °C. In some embodiments, the enzyme reaction tank unit 17 maintains the temperature of the CO at a temperature of about or below 70°C. In some embodiments, the enzyme reaction tank unit 17 maintains the temperature of the CO at a temperature of about or below 65°C. In some embodiments, the enzyme reaction tank unit maintains the temperature of the CO at a temperature of about or below 60°C. In some embodiments, the enzyme reaction tank unit maintains the temperature of the CO at a temperature of about or at least 75 °C. In some embodiments, the enzyme reaction tank unit maintains the temperature of the CO at a temperature of about or at least 70°C. In some embodiments, the enzyme reaction tank unit maintains the temperature of the CO at a temperature of about or at least 65 °C. In some embodiments, the enzyme reaction tank unit maintains the temperature of the CO at a temperature of about or at least 60°C. In some embodiments, the enzyme reaction tank unit maintains the temperature of the CO at a temperature of about or at least 55°C. In some embodiments, the enzyme reaction tank unit maintains the temperature of the CO at a temperature of about or at least 50°C. In some embodiments, the enzyme reaction tank unit includes one or more sensors. In some embodiments, the sensors include a temperature sensor and / or a pH sensor. In some embodiments, the sensors are positioned at different depths within the enzyme reaction tank unit to monitor the temperature and / or pH. In some embodiments, the sensors are positioned in the middle portion of the enzyme reaction tank unit, which includes predominantly oil. In some embodiments, the sensors are positioned in the bottom portion of the enzyme reaction tank unit, which includes predominantly sludge. In someembodiments, the sensors are positioned in the middle and bottom portions of the enzyme tank. In some embodiments, the sensors are positioned in the upper, middle, and bottom portions of the enzyme tank. In some embodiments the enzyme reaction tank unit includes an agitator mechanism to enable homogenous mixing of enzyme and all phases in the CO. In some embodiments, the agitator mechanism may be a single or multiple shaft stirrer with rotating blades or arms. In some embodiments, the agitator mechanism may be baffle and recirculation. In some embodiments, the enzyme reaction tank unit is a continuous stirred tank reactor.

[0114] As described in Section I above, the CO is incubated with the enzyme composition (first enzyme composition) 118 described herein in the enzyme reaction tank unit. In some embodiments, the CO is contacted with the enzyme composition described herein as it is provided to the enzyme reaction tank unit. For example, in some cases, the enzyme composition is contacted with the CO by inline delivery. In some embodiments, the enzyme composition is dosed into the inlet CO pipe of the enzyme reaction tank unit 17. In some embodiments, the enzyme composition is contacted with the CO in the enzyme reaction tank unit 17. For example, in some embodiments, the enzyme composition is directly injected into the reaction tank unit 17. In some embodiments, the enzyme composition is sprayed directly into the enzyme reaction tank unit 17. In some embodiments, the delivery of the enzyme composition, e.g., by inline dosing, direct injection, and / or spraying, is controlled by a pump. In some embodiments, the pump controls the dosing rate of the enzyme. In some embodiments, the pump is an inverter pump. In some embodiments, the pump is a metered dosing pump including a control. In some embodiments, the pump includes a flow meter, for example a flow meter as described above. In some embodiments, a dose rate may be set, e.g., a set value, and the flow may be monitored by the flow meter. In some embodiments, the flow meter controls the flow of the enzyme composition into the enzyme reaction tank unit by the pump. In some embodiments, the flow rate is controlled manually, e.g., by an operator monitoring the flow meter and / or PLC. In some embodiments, the flow rate is controlled automatically, e.g., by a feedback command from, e.g., a PLC.

[0115] In some embodiments, following an incubating period, e.g., as described in Section I, the contacted CO 119 from the enzyme reaction tank 17 passes to a clarifying unit 19 for clarifying the extracted crude palm oil. In some embodiments, the contacted CO 119 is provided to a holding tank unit 18 before passing to the clarifying unit.

[0116] In the clarifying unit 19, the contacted CO 119 is clarified and / or separated. The CO clarification process may include settling of the oil in the clarifying unit 19. The clarifying unit may be of any type. In some embodiments, the clarifying unit is a horizontal type tank. In some embodiments, the clarifying unit is a vertical clarifying unit. The contacted CO 119 includes amixture of palm oil, water, cell debris, fibrous material, and solids. In some embodiments, the CO 119 may have a high viscosity. Therefore, in some embodiments, hot water or steam may be added to the contacted CO. In this way, the solids, which are heavier than the water and oil, fall to the bottom of the clarifying unit 19, while the lighter oil settles above a water layer. From the clarifying unit 19, the clarified CO 120 may be supplied to a pure oil unit 22 before passing (121) to a purifying unit 23 for purification. In some embodiments, the purifying unit removes water and dirt.

[0117] The remaining liquor or sludge 123 containing solids from the clarifying unit 19 may be passed to a sludge tank unit 20 and a separator 21 in which solids are separated into a Heavy Phase 125 and Decanter Cake 124. The Heavy Phase 125, which is a component of POME, is sent to the effluent ponds 25 for treatment. The recovered crude oil 126 from the separator 21 can be returned back to the clarifying unit 19 for further recovery. The decanter cake 124 is waste, and optionally can be used as fertilizer.

[0118] In some embodiments, the purified CO 122 from the purifying unit 23 is supplied to a drying unit 24. In some embodiments, the drying unit further removes any remaining moisture to -0.02% wt / wt of the final crude palm oil product. In some embodiments, the drying unit 24 is a vacuum dryer. In some embodiments, the drying unit 24 is located downstream of the purifying unit 23 in the system provided herein. The water content may be reduced to, for example, from 0.15 to 0.25 percent by weight. Thus, in some embodiments, the oil fraction from the purifying unit 23 is supplied to a vacuum dryer to reduce the water content of the CO. The final crude palm oil product (CPO) 130 from the drying unit 24 is sent to storage. Any wastewater generated in the drying process can be sent to the effluent pond 25 for treatment. The effluent pond 25 is used for POME storage and treatment. It is configured to receive POME generated in the sterilizing unit 10, POME generated in the hydrocyclone process, POME generated in the sludge separation process 21, and POME generated as wastewater in other sections of the palm oil mill. The POME may thus include effluents generated during one or more of the unit operations including sterilizing, digesting, pressing, clarifying, drying, and / or hydrocyclone operation.

[0119] As suggested above, it is contemplated that mass balance may be used to quantify and optimize the methods and systems provided herein. Thus, in some embodiments, to effectively understand performance for optimization of the unit operations and the system as a whole, and to quantify on-going performance, the use of mass balance may be used.

[0120] In some embodiments, one or more sampling points in the system or a unit operation are identified for sampling. In some embodiments, a flow rate is determined at the sampling point. In some embodiments, a sample, e.g., CO, final crude palm oil (CPO) product, sludge, POME, orother output, is taken at the sampling point and the phase composition of the sample is determined. In some embodiments, the flow rate is determined at the sampling point and the phase composition of the sample, e.g., CO, final crude palm oil (CPO) product, sludge, POME, or other output, from the sampling point is determined. In some embodiments, the flow rate and the phase composition are used to determine mass balance. For example, flow rate and phase composition data from the one or more sample points may be used to determine mass balance within a unit operation and / or over the entire system. In some embodiments, flow meters are placed at the one or more sampling points. In some embodiments, the methods for phase quantitation are placed at the one or more sampling points. In some embodiments, the phase quantification methods are performed at a location different from the one or more sampling points. Thus, in some embodiments, the system provided herein further includes one or more sampling points, e.g., for purposes of conducting mass balance analyses. In some embodiments, the one or more sampling points are located after the screening unit 14. In some embodiments, the one or more sampling points are located at any one or more of the connection between the reaction tank unit 17 and the holding tank unit 18, at the holding tank unit 18, at the clarifying unit 19, at the pure oil tank unit 22, at the sludge tank unit 20, at a connection between the sludge tank unit 20 and the separator unit 21, at the separator unit 21, at a connection between the separator unit 21 and the clarifying unit 19, or at the drying unit 24, for example the outlet of the drying unit to capture the final crude palm oil product. In some embodiments, the one or more sampling points are at steam inlets. In some embodiments, the sample points at one or more of the holding tank unit 18, the clarifying unit 19, the pure oil tank unit 22, and the sludge tank unit 20 are at the steam inlets into the unit. In some embodiments, one or more sampling points are located at any one or more of the buffer tank unit 15, the holding tank unit 18, the clarifying unit 19, the pure oil tank unit 22, the sludge tank unit 20, the separator unit 21, including at the outlets of the separator unit 21 for the decanter cake and the outlet for the Heavy Phase, or the drying unit 24, including at the outlet of the drying unit for the final crude palm oil product. In some embodiments, the sampling points in the foregoing sentence are useful for overall performance quantification. In some embodiments, the one or more sampling points are located at any one or more of a connection between the reaction tank unit 17 and the holding tank unit 18, a connection between the sludge tank unit 20 and the separator unit 21, or a connection between the separator unit 21 and clarifying unit 22. In some embodiments, the sampling points in the foregoing sentence are useful for enzyme optimization and / or separation optimization.EXEMPLARY EMBODIMENTS

[0121] Among the provided embodiments are:1. A method for producing a final crude palm oil product, comprising contacting a crude palm oil (CO) with an enzyme composition, wherein the enzyme composition comprises a cellulase.2. The method of embodiment 1, wherein the contacting occurs at a temperature of or of less than about 65°C, 60°C, 55°C, or 50°C.3. The method of embodiment 1 or embodiment 2, wherein prior to the contacting, the CO is cooled to a temperature of or of less than about 65 °C, 60°C, 55 °C, or 50°C.4. The method of any one of embodiments 1-3, comprising incubating the CO contacted with the enzyme composition for a duration of at least about 30 minutes.5. The method of embodiment 4, wherein the CO is heated to a temperature in a range of about 70°C to about 100°C after the incubating.6. The method of embodiment 4 or embodiment 5, wherein the CO is maintained at a temperature of or of less than about 65°C, 60°C, 55°C, or 50°C after the incubating.7. The method of any one of embodiments 1-6, comprising prior to the contacting: sterilizing fresh fruit bunches (FFB) to produce sterilized FFB (SFB) and sterilizer condensate; separating the SFB to produce a mass passing to digester (MPD) and empty sterilized FFB (EFB); optionally, pressing the EFB to produce an EFB liquor; digesting the MPD to produce a fruit mash; pressing the fruit mash to produce an undiluted crude palm oil (UDCO); and extracting the UDCO by separation.8. The method of embodiment 7, comprising diluting the UDCO to produce a diluted crude palm oil (DCO).9. The method of any one of embodiments 1-8, wherein the CO is UDCO or DCO.10. The method of any one of embodiments 1-9, wherein the contacting occurs in an enzyme reaction tank unit.11. The method of any one of embodiments 1-10, wherein the cellulase comprises a cellobiohydrolase, an endoglucanase, a beta-glucosidase, or any combination thereof.12. The method of any one of embodiments 1-11, wherein the cellulase comprises a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta-glucosidase (BGL), or any combination thereof.13. The method of any one of embodiments 1-12, wherein the cellulase is derived from a fungus.14. The method of any one of embodiments 1-13, wherein the cellulase is derived from a strain of Trichoderma, optionally Trichoderma reesei.15. The method of any one of embodiments 1-14, further comprising clarifying the CO contacted with the enzyme composition to produce a pure oil and purifying and drying the pure oil to produce a final crude palm oil product.16. A system for producing a final crude palm oil product, comprising a sterilizing unit, a stripping unit, a digester unit, a pressing unit, a screening unit, a buffer tank unit, a cooling unit, an enzyme reaction tank unit, a holding tank unit, a clarifying unit, a pure oil tank unit, a purifying unit, and a drying unit, wherein the units are in operable contact.17. The system of embodiment 16, wherein the cooling unit receives crude palm oil (CO) from the buffer tank unit and decreases a temperature of the CO prior to the CO entering the enzyme reaction tank unit.18. The system of embodiment 16 or embodiment 17, wherein the buffer tank unit and the cooling unit are in operable contact by a pump.19. The system of any one of embodiments 16-18, wherein the pump is located between the buffer tank unit and the cooling unit.20. The system of any one of embodiments 16-19, wherein the enzyme reaction tank unit comprises an inlet for dosing one or more enzymes or an enzyme composition.21. The system of embodiment 20, wherein the one or more enzymes or the enzyme composition is dosed to the enzyme reaction tank unit by inline dosing and / or direct injection.22. The system of embodiment 20 or embodiment 21, wherein the one or more enzymes or the enzyme composition is dosed using a pump.23. The system of any one of embodiments 18-22, wherein the pump is a continuous pump, optionally comprising a flow meter.24. The system of any one of embodiments 16-23, wherein the enzyme reaction tank unit comprises temperature and / or pH sensors.25. The system of any one of embodiments 16-24, wherein the enzyme reaction tank unit comprises a water jacket.26. The system of any one of embodiments 16-25, wherein the enzyme reaction tank unit comprises an agitator mechanism and / or is a continuous stirred tank reactor.27. The system of any one of embodiments 16-26, comprising one or more sampling points for performing mass balance analysis.28. The system of embodiment 27, wherein the one or more sampling points comprise a flow meter.III. EXAMPLES

[0122] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.Example 1: Enzymes and temperature for palm oil extraction

[0123] This Example describes the impact of enzymes and temperatures on the extraction of palm oil.

[0124] Methods: Undiluted crude oil (UDCO) 100 grams was placed in a shake flask and dosed with one of three enzyme compositions, a Trichoderma Whole Cellulase I, a Trichoderma Engineered Whole Cellulase, or a Trichoderma Whole Cellulase II, each of which included cellobiohydrolase (CBHI and CBHII), endoglucanase (EGI and EGII), and beta-glucosidase, at 0.2% wt / wt. A shake flask containing UDCO without enzyme was prepared as a control. The flasks were incubated for 2 hours in a shaker (200 rpm first 10 minutes, 150 for remaining time) at a temperature of 50°C (Trichoderma Engineered Whole Cellulase), 60°C (Trichoderma Whole Cellulase I, Trichoderma Whole Cellulase II), or 95°C (Control). After the 2 hours, the flasks were transferred to a 95 °C water bath (Trichoderma Whole Cellulase I, Trichoderma Whole Cellulase II, and Control) or kept in the shaker at 50°C (Trichoderma Engineered Whole Cellulase) for a further 1 hour.

[0125] Following the 1-hour incubation, the preparations were centrifuged (5000 rpm, 8 minutes) to separate liquids from solids. The liquid portion was passed through a separatory funnel to separate the oil from the water. The solid portion was subjected to an oil extraction process yielding dried solids and oil. Briefly, the oil extraction process included subjecting the solid portion to weighted ANKOM Technology filter bags (ANKOM Technology, Macedon, NY) and sealing. The bags were oven dried for moisture analysis. After drying, the bags were placed in an extraction chamber (ANKOMAT75 Extraction System) and petroleum ether was added to the solvent room. The extraction proceeded for 2 hours at a temperature of 90°C. After extraction, the bags were taken out and dried in an oven to constant weight.

[0126] Analysis: The National Renewable Energy Laboratory method of structure carbohydrates and lignin in biomass was used to determine the composition of non-oil solids. Briefly, cellulose (0.3 grams) was pretreated with H2SO4 (3 mL, 72%) at 30°C for 1 hour. The mixture was subsequently diluted to 4% H2SO4 and hydrolyzed at 121 °C for 1 hour. Liquids were used for detection of carbohydrates and solids were used for non-soluble lignin analysis.

[0127] UDCO was fractionated and the amount of oil, water, and sludge was determined.

[0128] Results: As shown in FIG. 1, treatment with Trichoderma Whole Cellulase I, Trichoderma Whole Cellulase II, or Trichoderma Engineered Whole Cellulase improved separation of the oil phase, resulting in higher oil recovery as compared to Control (no enzyme) on a wt / wt basis of UDCO. Additionally, enzyme treatments yielded lower residual sludge volumes.

[0129] As shown in FIG. 2, Trichoderma Whole Cellulase I and Trichoderma Engineered Whole Cellulase were effective at hydrolysing the cellulosic component in UDCO.

[0130] As shown in FIG. 3A, moisture in dried sludge from the enzyme treated samples was lower than the Control (no enzyme) condition. Trichoderma Whole Cellulase I and Trichoderma Engineered Whole Cellulase treated samples also had a lower oil content in dried sludge (FIG. 3B). Considered in combination with FIG. 1, the total oil loss in sludge is reduced with enzyme treatment because the mass of the solid phase was also reduced.

[0131] These results support the use of cellulases to improve separation of the oil phase to deliver higher oil recovery, even at low temperatures.Example 2: Impact of enzymes, lower temperature, and no dilution on palm oil extraction

[0132] This Example describes the impact of enzymes on palm oil extraction when low temperatures and no dilution are used.

[0133] Methods: Undiluted crude oil (UDCO, 30 grams) was placed in a Teflon tube and dosed with Trichoderma Whole Cellulase I, including cellobiohydrolase (CBHI and CBHII), endoglucanase (EGI and EGII), and beta-glucosidase, at 0.1% or 0.2% wt / wt. The enzyme treated tubes were incubated for 4 hours in a water bath (tubes were mixed occasionally in the 1sthour) at a temperature of 60°C. A control of diluted crude oil (DCO Control), produced by adding water to 30 grams UDCO to obtain 35% water, was incubated for 1 hour at 95°C. Trichoderma Whole Cellulase I was not added to the DCO Control. The DCO Control was used as a control because dilution of UDCO is standard industry practice. However, because dilution requires the use of water, a valuable resource, and has the potential to increase effluent (e.g., POME), it may be considered undesirable. Thus, the use of enzymes at low temperatures in UDCO was compared to DCO Control to identify a potential benefit of not requiring dilution. All preparations underwent post-hydrolysis processing at either 95°C (DCO Control) or 70°C (Trichoderma Whole Cellulase I and UDCO Control) for 4 hours.

[0134] The post-hydrolysis preparations were centrifuged (2119g, 30 seconds) to separate liquids from solids. The solid portion was subjected to an oil extraction process yielding dried solids and oil.

[0135] Results: As shown in FIG. 6, treatment with Trichoderma Whole Cellulase I yielded lower residual sludge volumes compared to DCO Control. The moisture of the sludge was also reduced by treatment with Trichoderma Whole Cellulase I compared to DCO Control (FIG. 7). Trichoderma Whole Cellulase I treatment also resulted in a lower oil content in dried sludge compared to DCO Control (FIG. 8). Considered in combination with FIG. 6, the total oil loss in sludge is reduced with enzyme treatment because the mass of the sludge was also reduced.

[0136] FIG. 9 shows the reduction in oil loss to sludge with the use of the Trichoderma Whole Cellulase I compared to DCO Control, where the DCO Control is considered to not have any reduction in oil loss (i.e., reduction in oil loss to sludge is 0%). As shown in FIG. 9, treatment with Trichoderma Whole Cellulase I reduced the amount of oil lost to sludge compared to the DCO Control condition.

[0137] Conclusion: These results support the use of cellulase to lower palm oil recovery temperatures while reducing oil losses. These results further support the use of enzymes to reduce the need for dilution of UDCO.

[0138] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure. Although the invention may be described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method for producing a final crude palm oil product, comprising contacting a crude palm oil (CO) with an enzyme composition, wherein the enzyme composition comprises a cellulase.

2. The method of claim 1 , wherein the contacting occurs at a temperature of or of less than about 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C.

3. The method of claim 1 or claim 2, comprising incubating the CO contacted with the enzyme composition for a duration of at least about 30 minutes.

4. The method of claim 3, wherein the CO is heated to a temperature in a range of about 70°C to about 100°C after the incubating.

5. The method of claim 3 or claim 4, wherein the CO is maintained at a temperature of or of less than about 75°C, 70°C, 65 °C, 60°C, 55°C, or 50°C after the incubating.

6. The method of any one of claims 1-5, comprising prior to the contacting:- sterilizing fresh fruit bunches (FFB) to produce sterilized FFB (SFB);- separating the SFB to produce a mass passing to digester (MPD) and empty sterilized FFB (EFB);- optionally, pressing the EFB to produce an EFB liquor;- digesting the MPD to produce a fruit mash;- pressing the fruit mash to produce an undiluted crude palm oil (UDCO); and- extracting the UDCO by separation.

7. The method of claim 6, comprising diluting the UDCO to produce a diluted crude palm oil (DCO).

8. The method of any one of claims 1-7, wherein the CO is UDCO or DCO.

9. The method of any one of claims 1-8, wherein the cellulase comprises a cellobiohydrolase, an endoglucanase, a beta-glucosidase, or any combination thereof.

10. The method of any one of claims 1-9, wherein the cellulase is derived from a fungus, optionally a Trichoderma.

11. The method of any one of claims 1-10, wherein the enzyme composition further comprises a hemicellulase, optionally a xylanase.

12. The method of any one of claims 1-11, further comprising clarifying the CO contacted with the enzyme composition to produce a pure oil and purifying and drying the pure oil to produce a final crude palm oil (CPO) product.

13. A system for producing a final crude palm oil product, comprising a buffer tank unit, a cooling unit, and an enzyme reaction tank unit, wherein the units are in operable contact, and wherein the buffer tank unit is in operable contact with a screening unit of an existing palm oil extraction system and receives crude palm oil (CO) from the screening unit, and the enzyme reaction tank unit is in operable contact with a holding tank unit of the existing palm oil extraction system, and the holding tank unit receives a CO contacted with one or more enzymes or an enzyme composition from the enzyme reaction tank unit.

14. The system of claim 13, wherein the cooling unit receives CO from the buffer tank unit and decreases a temperature of the CO prior to the CO entering the enzyme reaction tank unit.

15. The system of claim 13 or claim 14, wherein the enzyme reaction tank unit comprises an inlet for dosing one or more enzymes or an enzyme composition.

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