Method and system for pre-fermentation treatment of organic waste

Abstract

Methods and systems are provided for pretreating organic waste prior to the large-scale production of lactic acid from the organic waste, resulting in increased production yields. The pretreatment methods and systems of the present invention use specific temperature and pH conditions that inhibit or completely prevent endogenous microbial activity within the waste during the pretreatment stage. The pretreatment methods and systems of the present invention thereby reduce or even prevent unwanted utilization of reducing sugars naturally present in the waste, preserving the glucose potential of the organic waste.

Description

[Technical Field]

[0001] The present invention relates to the recycling of organic waste. In particular, methods and systems are provided for treating organic waste prior to the large-scale production of lactic acid by fermentation. [Background technology]

[0002] Lactic acid fermentation Lactic acid fermentation, i.e., the production of lactic acid from carbohydrate sources via microbial fermentation, has attracted interest in recent years due to the ability to use lactic acid as a building block in the production of bioplastics. Lactic acid can be polymerized to form polylactic acid (PLA), a biodegradable and recyclable polyester, which is a promising alternative to petroleum-derived plastics. PLA is used to produce a variety of products, including food packaging, disposables, and fibers in the textile and hygiene industries, and is the most commonly used plastic filament in 3D printing.

[0003] Fermentative bioprocessing for the production of lactic acid is preferred over chemical synthesis for a variety of reasons, including environmental concerns, cost, and the need to produce enantiomerically pure lactic acid, which is difficult to achieve through chemical synthesis for most industrial applications. Traditional fermentation processes are typically based on anaerobic fermentation by lactic acid-producing microorganisms, which produce lactic acid as the primary metabolic end product of carbohydrate fermentation. To produce PLA, the lactic acid produced during fermentation is separated from the fermentation broth and purified through various processes, after which the purified lactic acid is polymerized.

[0004] Lactic acid has a chiral carbon atom and therefore exists in two enantiomeric forms, D- and L-lactic acid. To produce PLA suitable for industrial use, the D- and L-lactic acid entering the production process must be highly purified to meet the specifications required for polymerization. Therefore, to produce one distinct enantiomer (L or D), lactic acid bacteria that produce only the L- or D-lactic acid enantiomer are typically used.

[0005] In currently available commercial processes, carbohydrate sources for lactic acid fermentation are typically renewable starch-containing sources, such as corn and cassava root. Additional sources, such as cellulose-rich sugarcane bagasse, have also been proposed. Typically, lactic acid bacteria can utilize reducing sugars, such as glucose and fructose, but do not have the ability to degrade polysaccharides, such as starch and cellulose. Therefore, to utilize such polysaccharides, the process requires the addition of glycolytic enzymes, typically in combination with chemical treatment, to degrade the polysaccharides and release reducing sugars, a process known as saccharification.

[0006] Saccharification may precede or occur simultaneously with the fermentation process. Saccharification before fermentation is known as separate hydrolysis and fermentation (SHF), while a process combining saccharification and fermentation is known as simultaneous saccharification and fermentation (SSF). Each of these techniques has various advantages and disadvantages. SHF allows each of the saccharification and fermentation steps to occur at their own optimum conditions (temperature, pH, etc.), while SSF requires the application of conditions that allow both the hydrolytic enzymes and the fermenting organisms to operate, albeit suboptimally. SSF, on the other hand, has the advantage of requiring only one step, saving time and money. It has the additional advantage of using up reducing sugars as they are released from the polysaccharides, thereby maintaining a relatively low concentration of reducing sugars and a high productivity of the hydrolytic enzymes.

[0007] Another proposed carbohydrate source for lactic acid fermentation is complex organic waste, such as mixed food waste from municipal, industrial, and commercial sources, which typically contains varying proportions of reducing sugars (glucose, fructose, lactose, etc.), starch, and lignocellulosic materials. Such organic waste is advantageous because it is readily available and inexpensive compared to other carbohydrate sources for lactic acid fermentation. However, because such organic waste typically accumulates for several days before being transferred to a waste treatment facility and / or subjected to an industrial fermentation process, spontaneous microbial processes occur within the waste material before the intended industrial fermentation process begins. These microbial processes often utilize reducing sugars, which are the primary substrate for controlled fermentation, thus impairing the efficiency of the controlled fermentation process and reducing the yield of lactic acid. Microbial processes can also produce products and by-products that inhibit various stages of the fermentation process and can also produce enantiomers of lactic acid different from the desired enantiomer, which can increase impurities in the lactic acid end product and require the application of time-consuming and expensive purification processes.

[0008] EP 1 320 388 discloses a method for reducing the number of viable microorganisms and / or prions present in organic material, comprising the steps of: i) providing an organic material comprising a solid and / or liquid portion; ii) lime-pressuring the organic material at a temperature of between 100°C and 220°C to result in hydrolysis of the organic material, the lime being Ca(OH)2 and / or CaO; b) removing ammonia from the lime-pressurized organic material, the lime being added in connection with ammonia removal and sanitation of the organic material to precipitate dissolved orthophosphates; and iii) obtaining treated organic material containing reduced numbers of viable microorganisms and / or prions.

[0009] WO 00 / 02457 discloses a fermented and pasteurized pre-ferment, comprising the fermentation product of a mixture of gluten and / or bran, which fermentation product is obtained by hydrolysis with a protease and / or lipase and / or glycosidase and / or glycanase, preferably a glucanase, followed by fermentation with acid-forming bacteria, preferably lactic acid-forming bacteria and yeast, in the presence of an enzyme capable of liberating flavor components or flavor precursors from proteins and / or carbohydrates, preferably selected from a protease and another glycosidase and / or glycanase, in particular an amylase.

[0010] WO 2016 / 016235 discloses a process for preparing lactic acid and / or lactate salts via fermentation of carbohydrates obtained from lignocellulosic material. Specifically, the process includes treating lignocellulosic material with an alkaline agent containing a caustic magnesium salt in the presence of water to provide a treated aqueous lignocellulosic material; saccharifying the treated aqueous lignocellulosic material in the presence of a hydrolytic enzyme to provide a saccharified aqueous lignocellulosic material containing fermentable carbohydrates and a solid lignocellulosic fraction; fermenting the fermentable carbohydrates in the saccharified aqueous lignocellulosic material with a lactic acid-producing microorganism in the presence of an alkaline agent containing a caustic magnesium salt to provide an aqueous fermentation broth containing magnesium lactate; and isolating the lactic acid and / or lactate salts from the fermentation broth. The saccharification and fermentation processes are carried out as two separate steps.

[0011] There remains a need to improve the yield of lactic acid production from organic waste, particularly to prevent unnecessary loss of substrate. Summary of the Invention

[0012] The present invention provides a method and system for pretreating organic waste prior to large-scale production of enantiomerically pure lactic acid or a salt thereof, particularly L-lactic acid or a salt thereof, from the organic waste, which results in increased production yields.

[0013] Organic wastes, such as mixed food waste, contain a combination of free reducing sugars, such as glucose, and a variety of different polysaccharides, such as starch, glycogen, and cellulose, collectively referred to as the glucose potential of the organic waste. The reducing sugars present in the organic waste are actively consumed by bacteria and / or other microorganisms naturally found in the organic waste, thereby reducing the supply of reducing sugars available for industrial fermentation processes and can produce inhibitors and other contaminants in the organic waste material. In their untreated state, complex organic wastes, such as mixed food waste, can contain a variety of solids, which makes it difficult to effectively sterilize the waste and, even when sterilized, to maintain the waste in a sterile state throughout its processing up to the lactic acid production stage.

[0014] The methods and systems of the present invention advantageously address this limitation, inhibiting or even completely preventing endogenous microbial activity within the waste during the pretreatment stage. In some embodiments, the methods and systems of the present invention inhibit or even completely prevent the loss of reducing and / or non-reducing sugars during the pretreatment stage. In particular, the methods and systems of the present invention are preferably used on a non-sterile slurry of organic waste as soon as possible after initial processing, such as collection of the organic waste and crushing and removal of plastic and inorganic solid components, e.g., glass and sand (if present), and include maintaining the non-sterile organic waste slurry under specified conditions of temperature and pH until further processing. As exemplified herein below, it has surprisingly been found that maintaining the non-sterile organic waste slurry at a temperature of 55-65°C and a pH of 3.5-5.5 effectively inhibits microbial activity in the waste, as evidenced by a substantially constant concentration of available glucose throughout maintenance at the aforementioned conditions and the absence of gas production, such as CO2 and methane. Maintenance at the aforementioned conditions resulted in essentially complete preservation of the glucose potential of the organic waste. Thus, the methods and systems of the present invention are particularly useful for increasing the potential amount of lactic acid that can be produced from a given batch of organic waste, and thus the overall yield of the production process.

[0015] The pretreatment methods and systems of the present invention further include, according to some embodiments, an improved saccharification process that can be advantageously performed on organic waste without first sterilizing it. The lactic acid fermentation process can utilize only reducing sugars as a substrate, and therefore requires the release of reducing sugars through saccharification of polysaccharides present in the waste prior to fermentation. In some embodiments, the saccharification process disclosed herein employs heating a non-sterile organic waste slurry to a temperature of 65-85°C (typically 70-75°C) for at least 0.25 hours (typically about 2 hours), cooling to a temperature of 45-65°C, and adding one or more saccharification enzymes after cooling. The heating step is referred to herein as "thermal treatment." As exemplified below, thermal treatment has surprisingly been found to result in the effective saccharification and conversion of substantially all of the glucose potential of the organic waste to usable glucose, whereas when saccharification enzymes are added to the non-sterile organic waste slurry prior to thermal treatment, no significant change in usable glucose or only limited, partial conversion to glucose is measured.

[0016] The present invention is particularly useful for food waste, which is heterogeneous and typically characterized by high levels of microbial contaminants, and therefore susceptible to spoilage by natural fermentation processes during collection, transportation, and / or pretreatment. In some embodiments, organic waste slurries for use in the present invention comprise at least 10 5 The microbial content is characterized by CFU / mL. Advantageously, maintaining the organic waste slurry at the temperature and pH conditions disclosed herein throughout the various stages of pretreatment, even when high levels of microbial contaminants are present, is effective in inhibiting microbial activity, achieving efficient saccharification, and preserving the glucose potential of the waste until the controlled large-scale production stage. Preventing naturally occurring microbial processes that wastefully consume free reducing sugars substantially increases the yield of lactic acid that can be obtained from a batch of organic waste by fermentation, along with facilitating the saccharification of the organic waste to recover the maximum amount of free reducing sugars.

[0017] According to a first aspect, the present invention provides a method for pre-treating organic waste prior to the mass production of lactic acid or a salt thereof from the organic waste, comprising: (a) providing a non-sterile slurry of organic waste containing non-reducing polysaccharides and reducing sugars; (b) maintaining the non-sterile slurry of organic waste from step (a) at a temperature in the range of 55°C to 65°C and a pH in the range of 3.5 to 5.5 for a first duration of at least 1 hour, thereby inhibiting endogenous microbial activity in the organic waste; (c) increasing the temperature of the non-sterile slurry of organic waste of step (b) above the temperature of step (b) to a temperature of between 65°C and 85°C for a second duration of at least 0.5 hours; (d) optionally adjusting the temperature to a temperature between 45°C and 65°C; (e) adding one or more saccharification enzymes to the non-sterile slurry of organic waste from step (c) or step (d) to hydrolyze polysaccharides in the organic waste to release reducing sugars, thereby obtaining saccharified organic waste.

[0018] In some embodiments, steps (c) and (e) are performed simultaneously.

[0019] In some embodiments, the organic waste comprises plastic and / or inorganic solid components and the method comprises subjecting the organic waste to separation of said plastic and / or inorganic solid components prior to step (a).

[0020] In some embodiments, the method further comprises separating the saccharified organic waste into a liquid phase containing reducing sugars and a solid phase, and sterilizing the liquid phase.

[0021] According to another aspect, the present invention provides a method for pre-treating organic waste prior to large-scale production of lactic acid or a salt thereof from the organic waste, comprising the steps of: (a) providing a non-sterile slurry of organic waste containing non-reducing polysaccharides and reducing sugars; (b) maintaining the non-sterile slurry of organic waste from step (a) at a temperature in the range of 55°C to 65°C and a pH in the range of 3.5 to 5.5 for a first duration of at least 1 hour, thereby inhibiting endogenous microbial activity in the organic waste; (c) increasing the temperature of the non-sterile slurry of organic waste of step (b) above the temperature of step (b) to a temperature of between 65°C and 85°C for a second duration of at least 0.5 hours; (d) adjusting the temperature to 45°C to 65°C and adding one or more saccharification enzymes to hydrolyze the polysaccharides in the organic waste, releasing reducing sugars and obtaining saccharified organic waste; (e) separating the saccharified organic waste into a liquid phase containing reducing sugars and a solid phase, thereby pretreating the organic waste and obtaining raw material for the large-scale production of lactic acid or a salt thereof.

[0022] In some embodiments, the one or more saccharification enzymes include at least one of glucoamylase, α-amylase, and pullulanase.

[0023] In some embodiments, the second duration of step (c) ranges from 0.5 hours to 5 hours.

[0024] According to a further aspect, the present invention provides a method for pre-treating organic waste prior to large scale production of lactic acid or a salt thereof from said organic waste, comprising the steps of: (A) providing a non-sterile slurry of organic waste containing non-reducing polysaccharides and reducing sugars; (B) heating the non-sterile slurry of organic waste to a temperature of 65°C to 85°C for at least 1 hour; (C) optionally adjusting the temperature to a temperature between 45°C and 65°C; (D) adding one or more saccharification enzymes to the non-sterile slurry of organic waste from step (B) or step (C) to hydrolyze polysaccharides in the organic waste, thereby releasing reducing sugars and obtaining saccharified organic waste.

[0025] In some embodiments, steps (B) and (D) are performed simultaneously.

[0026] According to a further aspect, the present invention provides a method for inhibiting endogenous microbial activity in organic waste material prior to subjecting the waste material to the controlled large-scale production of lactic acid or a salt thereof, comprising: (a) providing a non-sterile slurry of organic waste material comprising at least one of non-reducing polysaccharides and reducing sugars; (b) maintaining the waste material of step (a) at a temperature in the range of 55°C to 65°C and a pH in the range of 3.5 to 5.5 for a first duration of at least 1 hour, thereby inhibiting endogenous microbial activity in the waste material prior to subjecting the waste material to the controlled large-scale production of lactic acid or a salt thereof.

[0027] In some embodiments, the organic waste comprises plastic and / or inorganic solid components and the method comprises subjecting the organic waste to separation of said plastic and / or inorganic solid components prior to step (a).

[0028] In some embodiments, step (b) is carried out at a pH in the range of 4-5.

[0029] In some embodiments, the first duration of step (b) ranges from 1 to 96 hours, hi additional embodiments, the first duration of step (b) ranges from 12 to 48 hours.

[0030] In some embodiments, the method further comprises applying a mechanical treatment to the waste material prior to, concurrently with, and / or after step (b), hi some embodiments, the mechanical treatment is selected from the group consisting of crushing, chipping, shredding, milling, agitating, and combinations thereof.

[0031] In some embodiments, the waste material is selected from the group consisting of food waste, municipal waste, agricultural waste, plant material, and combinations thereof.

[0032] In some embodiments, the non-sterile slurry of organic waste is at least 10 5 In a further embodiment, the non-sterile slurry of organic waste contains microorganisms at a concentration of at least 10 CFU / mL. 7 Contains microorganisms at a concentration of CFU / mL.

[0033] According to another aspect, the present invention provides a method for producing lactic acid from organic waste, comprising the steps of: (i) subjecting organic waste to pretreatment, including pretreatment according to the methods disclosed herein; (ii) adding lactic acid producing microorganisms to the pretreated organic waste and incubating in a fermentation reactor under controlled conditions for lactic acid production by the lactic acid producing microorganisms, thereby producing lactic acid.

[0034] Other objects, features, and advantages of the present invention will become apparent from the following description, examples, and drawings. [Brief explanation of the drawings]

[0035] [Figure 1] FIG. 1 shows a block diagram of a method for pre-treating waste materials and enhancing lactic acid production, according to certain embodiments of the present invention. [Figure 2] 1 shows a graph illustrating maintaining waste material at temperature and pH conditions according to the present invention for 16 hours. [Figure 3] 1 shows a graph depicting maintaining waste material at 60°C and pH 4.5 with a first dose of saccharification enzymes for 18 hours, followed by heat treatment at 70°C for 2 hours, then reducing the temperature to 60°C and adding a second dose of saccharification enzymes. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention provides methods and systems for preserving the glucose potential of organic waste material before subjecting the waste material to controlled lactic acid fermentation. The methods and systems are useful for increasing the yield of lactic acid in controlled fermentation processes, for example, by suppressing unwanted substrate consumption by endogenous microorganisms present in the organic waste and by further preventing the formation of inhibitory by-products and other impurities within the waste material. The methods and systems include subjecting the provided waste material to specific temperature and pH conditions that inhibit microbial activity and maintaining the waste material under these conditions until further processing.

[0037] The methods and systems of the present invention are used on non-sterile slurries of organic waste, preferably as soon as possible after collection of the organic waste and initial processing such as grinding and removal of plastic and inorganic solid components (if present).

[0038] As used herein, an organic waste "slurry" typically refers to a mixture of organic waste and water containing solid particles of organic waste. Organic waste slurries, as used herein, are typically formed by collecting waste materials from various sources, separating the waste materials into plastics and inorganic solid components such as glass, metal, and sand to remove most, preferably all, of the plastics and inorganic solid components, reducing the particle size of the waste material, for example, by shredding or grinding, adding water as needed, and producing a suspension of the organic waste material in water. In some embodiments, forming an organic waste slurry, particularly a food waste slurry, according to the present invention includes depackaging the waste, i.e., subjecting the waste to removal of packaging materials, including plastic, metal, and glass packaging materials. Naturally, in some embodiments, the organic waste may be in the form of a slurry.

[0039] Organic waste slurries (e.g., food waste slurries) according to the present invention are characterized by a solids content (dry matter content) in the range of 5-50%, inclusive (i.e., a liquid or moisture content in the range of 50-95%). In some embodiments, organic waste slurries according to the present invention are characterized by a solids content in the range of 10-30%, inclusive (i.e., a liquid or moisture content in the range of 70-90%). In some embodiments, organic waste slurries according to the present invention are characterized by a solids content in the range of 15-35%, inclusive (i.e., a liquid or moisture content in the range of 65-85%).

[0040] In some embodiments, organic waste slurries according to the present invention are characterized by a moisture content in the range of 50-95%, inclusive. In some embodiments, organic waste slurries according to the present invention are characterized by a moisture content in the range of 70-90%, inclusive. In some embodiments, organic waste slurries according to the present invention are characterized by a moisture content in the range of 65-85%, inclusive.

[0041] In some embodiments, organic waste (e.g., food waste) for use in the present invention naturally contains the solids / liquids / water content described above. In other embodiments, water is added to the organic waste (e.g., food waste) to obtain a slurry characterized by the aforementioned solids / liquids / water content.

[0042] The organic waste slurry (eg, food waste slurry) according to the present invention is pumpable and mixable, and therefore suitable for further handling and processing according to the present invention.

[0043] In some embodiments, the waste material is maintained under temperature and pH conditions that inhibit microbial activity until the waste material is saccharified (preferably, post-thermal saccharification as disclosed herein), hi further embodiments, the waste material is maintained under these conditions between saccharification and controlled lactic acid fermentation.

[0044] Maintenance under temperature and pH conditions that inhibit microbial activity may optionally begin at any point in the waste management process after extraction of the waste material from the source. While maintained under the specific conditions disclosed herein, several processes may be applied to the waste material, including mechanical treatment, solid-liquid separation, saccharification, or any other related waste treatment process. Alternatively, if a particular waste treatment process requires a temperature and / or pH that differs from the specific conditions disclosed herein (e.g., the temperature and / or pH required for saccharification enzyme activity), the temperature and / or pH may be adjusted to that required for the treatment process and then adjusted back to that disclosed herein after the treatment process is complete.

[0045] As used herein, the term "lactic acid" refers to a hydroxycarboxylic acid having the chemical formula CHCH(OH)COH. The term lactic acid or lactate (unprotonated lactic acid) can refer to the stereoisomers of lactic acid: L-lactic acid / L-lactate, D-lactic acid / D-lactate, or combinations thereof.

[0046] As used herein, the terms "reducing sugars," "free sugars," and "available sugars" are used interchangeably and refer to soluble sugar molecules currently available as substrates for lactic acid fermentation. Reducing sugars typically include C5 sugars (pentoses), C6 sugars (hexoses), or combinations thereof. In some embodiments, reducing sugars include glucose. In some embodiments, reducing sugars include xylose.

[0047] As used herein, the terms "non-reducing carbohydrate," "polysaccharide," and "non-reducing sugar" are used interchangeably and refer to polymeric sugar molecules that are not currently available for lactic acid fermentation and require saccharification / hydrolysis to become available for fermentation. Examples include starch, cellulose, hemicellulose, and combinations thereof.

[0048] As used herein, the term "maintaining a temperature / pH within X range for a duration" refers to maintaining the temperature / pH anywhere within the defined range for at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% of the duration, taking into account possible deviations of the temperature / pH from the defined range.

[0049] In most industrial applications, high purity L-lactic acid monomer is required to produce PLA with suitable properties. Therefore, the method and system of the present invention particularly relates to a process for producing L-lactate salt in high yield, which can then be converted into L-lactic acid suitable for industrial use.

[0050] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or illustrated by the examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology or terminology used herein is for the purpose of description and should not be regarded as limiting.

[0051] Referring now to the drawings, Figure 1 is a block diagram of a process, generally designated 100, for pretreatment of waste material followed by industrial lactic acid production by fermentation according to some embodiments of the present invention. According to the illustrated embodiment, the pretreatment process involves maintaining a non-sterile organic waste slurry at a temperature ranging from 55 to 65°C (e.g., a temperature of about 60°C) and a pH of 3.5 to 5.5 (e.g., a pH of about 4.5) for a first period of time (e.g., 1 to 96 hours), followed by a thermal treatment involving heating the non-sterile organic waste slurry to a temperature of 65 to 85°C (e.g., to 70°C) for a second period of time (e.g., 15 minutes to 3 hours), and saccharification, with or without cooling prior to saccharification, as described in further detail below. The process also includes solid-liquid separation and mechanical treatment, which may be applied at various points during the process, as described in further detail below.

[0052] Step 102 involves providing an organic waste material, typically in the form of a slurry. The organic waste for use in the methods and systems disclosed herein has not been sterilized.

[0053] Organic waste suitable for use in accordance with the present invention is typically complex, heterogeneous organic waste, containing solid and non-solid materials. Complex, heterogeneous organic waste contains carbohydrates for fermentation (soluble carbohydrates available for fermentation and / or polysaccharides that must be broken down by enzymes to release soluble carbohydrates for fermentation), and also contains impurities such as salts, fats, oils, lipids, proteins, color components, and / or inert materials. Organic waste for use in the present invention may also contain inorganic solid components such as plastics, glass, etc. Organic waste for use in the present invention contains endogenous microorganisms. Organic waste materials may be municipal waste, food waste, agricultural waste, and / or plant matter products.

[0054] Reducing sugars are the substrate that needs to be preserved for the controlled fermentation process. In some embodiments of the invention, the provided waste material comprises water and / or other aqueous and / or liquid solutions. In additional embodiments of the invention, water or other aqueous and / or liquid solutions may be added to the waste material one or more times, intermittently and / or continuously, as needed throughout process 100. Each option represents a separate embodiment.

[0055] In some particular embodiments, the organic waste used in the present invention is food waste.

[0056] Food waste according to the present invention includes food waste and beverages of plant and / or animal origin. Food waste according to the present invention includes household food waste, commercial food waste, and / or industrial food waste. Each option represents a separate embodiment. Organic food waste can come from vegetable and fruit residues, plants, cooked food, protein residues, slaughter waste, and / or combinations thereof. Each option represents a separate embodiment. Industrial organic food waste can include factory waste such as by-products, factory rejects, market returns, or off-cuts of inedible food parts (such as peels). Commercial organic food waste can include waste from shopping malls, restaurants, supermarkets, etc.

[0057] Food waste according to the present invention is typically mixed food waste comprising one or more of bakery waste; dairy waste; animal food waste including meat, poultry and fish waste; fruit and vegetable waste; and grain-based food waste (e.g., rice, couscous, pasta, noodles). In some embodiments, mixed food waste according to the present invention comprises a combination of food waste selected from bakery waste; dairy waste; animal food waste including meat, poultry and fish waste; fruit and vegetable waste; and grain-based food waste (e.g., rice, couscous, pasta, noodles).

[0058] Food waste typically includes solid components derived from food or residues, such as food particles and food scraps, bones and bone fragments, shells and shell fragments, seeds and seed fragments, peels, etc., as well as solids not derived from food or residues, such as from packaging materials, e.g., plastic, glass, and metal. In some embodiments, pretreatment according to the present invention is performed on a slurry of food waste after it has been depackaged to remove most or all of the packaging material.

[0059] Plant material according to the present invention includes man-made products such as agricultural waste and paper waste.

[0060] The waste material may be provided to a designated waste treatment facility or may be provided to a temporary collection point, which may be fixed, for example, an underground collection basin or reservoir, or may include a waste transport vehicle, such as a truck, boat, or the like.

[0061] According to some embodiments of the present invention, the waste material is not subjected to any treatment, in particular is not subjected to sterilization or pasteurization, before being subjected to the pretreatment process 100. In other embodiments, the waste material is subjected to separation and grinding of plastic and inorganic solid components to form a slurry. According to some embodiments, the slurry of waste material is subjected to a grinding process of at least 10 5 CFU / mL (colony forming units) of endogenous microorganisms (i.e., bacteria, yeast, fungi, etc.). According to some further embodiments, the waste material slurry contains at least 10 7 CFU / mL. It should be noted that the present invention is particularly beneficial for waste streams that contain viable microorganisms that would contaminate the waste if untreated and that are capable of utilizing the beneficial compounds therein in metabolic processes.

[0062] Step 104 includes maintaining the waste material at a temperature in the range of 55-65°C, inclusive, and a pH in the range of 3.5-5.5, inclusive, for a first duration. In some embodiments, the waste material is maintained at a temperature in the range of about 50°C to about 85°C, inclusive, within the specified range. In additional embodiments, the waste material is maintained at a temperature in the range of about 55°C to about 85°C, inclusive, within the specified range. In yet additional embodiments, the waste material is maintained at a temperature in the range of about 55°C to about 75°C, inclusive, within the specified range. In yet additional embodiments, the waste material is maintained at a temperature in the range of about 55°C to about 70°C, inclusive, within the specified range. In one embodiment, the temperature is in the range of 57-62°C, inclusive, within the specified range. In another embodiment, the temperature is about 60°C. In some particular embodiments, the temperature is 60°C.

[0063] In some embodiments, the pH ranges from about 4 to about 5.5, inclusive. In one embodiment, the pH ranges from about 4 to about 5, inclusive. In another embodiment, the pH ranges from about 4.2 to about 4.6, inclusive.

[0064] The waste material is typically maintained under these conditions for at least 0.5 hours, preferably at least 1 hour. In some embodiments, the waste material is maintained under these conditions for 1 to 96 hours. Thus, in one embodiment, the first duration is at least 0.5 hours, preferably at least 1 hour. In additional embodiments, the first duration is in the range of 1 to 96 hours, inclusive, within the specified range. In another embodiment, the first duration is in the range of 1 to 10 hours, inclusive, within the specified range. In additional embodiments, the first duration is in the range of 1 to 5 hours, inclusive, within the specified range. In another embodiment, the first duration is in the range of 12 to 48 hours, inclusive, within the specified range. In additional embodiments, the first duration is in the range of 10 to 20 hours, inclusive, within the specified range. In some embodiments, the waste material is maintained under the specified temperature and pH conditions for up to 4 days before undergoing additional processing and / or controlled fermentation. In other embodiments, the waste material undergoes additional processing, such as mechanical processing, solid-liquid separation, or introduction of saccharification enzymes, while being maintained at specific temperature and pH conditions, as further described with reference to steps 108, 112, and 114.

[0065] Maintaining waste materials under the conditions disclosed herein is believed to lead to lactic acid production via controlled fermentation from several perspectives. First, the temperature and pH conditions disclosed herein suppress the activity of endogenous microorganisms, i.e., those naturally found within the waste material or those found in the surrounding environment that contaminate the waste material when it is transported from one location to another. In waste materials not maintained under conditions that inhibit microbial activity, the endogenous microorganisms feed on the free reducing sugars in the waste material to produce various products, such as D- and L-lactic acid, pyruvate, succinate, acetic acid, formic acid, and ethyl alcohol. Many of the products of naturally occurring microbial reactions are harmful to controlled lactic acid production. In particular, some of these compounds act as inhibitors and toxins for the enzymes and bacteria introduced into the waste material at different stages of the controlled fermentation process. In addition, reducing sugars, the substrate for lactic acid production, are consumed. Finally, uncontrolled microbial reactions can induce racemization of lactic acid, necessitating time-consuming and costly purification of the final fermentation product. Maintaining waste materials under the conditions disclosed herein has been found to have a highly effective inhibitory effect on microbial activity.

[0066] Second, the relatively high temperatures at which waste materials are maintained according to the present invention, compared to naturally occurring ambient or chilled conditions, promote the liquefaction of the waste material. Liquefaction is the process by which a solid substance becomes liquid by melting or dissolving in water or any other solvent. As some of the solids in the waste material mix with the liquid phase, components beneficial to controlled fermentation become more readily accessible to the enzymes and / or microorganisms utilized in the different stages of lactic acid production, improving the efficiency and yield of the process. Even without actually liquefying the solid waste material, the pH and temperature conditions disclosed herein contribute to the softening and increased flowability of strong, stiff fibers that could otherwise damage or clog waste handling equipment.

[0067] Step 106 involves raising the temperature of the waste material to a temperature above 65°C for a second duration (this step is referred to herein as "thermal treating"). The temperature may range from 65 to 85°C, e.g., 65 to 80°C, 70 to 85°C, 70 to 80°C, or 70 to 75°C, inclusive of any value within the specified range. Each option represents a separate embodiment. In one embodiment, the temperature ranges from 65 to 85°C, inclusive of each value within the specified range. In a further embodiment, the temperature ranges from 68 to 73°C, inclusive of each value within the specified range. In some embodiments, the second duration ranges from 15 minutes to 5 hours, inclusive of each value within the specified range. In additional embodiments, the second duration ranges from 15 minutes to 3 hours, inclusive of each value within the specified range. In one embodiment, the second duration ranges from 15 minutes to 2.5 hours, inclusive of each value within the specified range. In some embodiments, the second duration ranges from 0.5 to 5 hours, inclusive of each value within the specified range. In additional embodiments, the second duration is 0.5 to 3 hours, inclusive. In some embodiments, the second duration is 0.5 to 2.5 hours, inclusive. The pH of the waste material during heat treatment is typically maintained within the range of 3.5 to 5.5, inclusive. Generally, the required duration is inversely related to temperature; i.e., the higher the temperature, the shorter the required duration. The duration of step 106 may exceed 5 hours. Step 106 is generally performed as a preparatory step for the saccharification procedure, as described in step 108 herein. While saccharification is generally performed at temperatures in the range of 50 to 60°C, it has surprisingly been discovered that heat-treating the waste material prior to saccharification substantially improves the efficiency of saccharification and significantly increases the amount of reducing sugars released from non-reducing polysaccharides. Surprisingly, a relatively modest increase in the temperature of the waste material by only 10°C, to a temperature of approximately 70°C, has been shown to significantly improve the yield of reducing sugars.

[0068] Step 108 involves saccharifying non-reducing polysaccharides in the waste material to release reducing sugars. Saccharification according to the present invention generally involves adding one or more glycolytic enzymes (also referred to as saccharifying enzymes or saccharification enzymes), typically glycolytic enzymes, that hydrolyze glycosidic bonds in sugars in the waste material to release reducing sugars for fermentation. Sugars include disaccharides, oligosaccharides, polysaccharides, and complex carbohydrates. The glycolytic enzymes may be selected from the group consisting of glycoside hydrolases, polysaccharide lyases, and carbohydrate esterases. Each option represents a separate embodiment. In some embodiments, the glycolytic enzymes may be modified enzymes (i.e., enzymes that have been modified and are different from their corresponding wild-type enzymes). In some embodiments, the modifications may include one or more mutations that result in improved activity of the enzyme. In some embodiments, the glycolytic enzymes are wild-type (WT) enzymes.

[0069] The broad group of glycolytic enzymes is divided into enzyme classes and then into enzyme families according to standard classification systems (Cantarel et al. 2009 Nucleic Acids Res 37:D233-238). An informative and up-to-date classification of such enzymes is available on the Carbohydrate-Active Enzymes (CAZy) server (www.CAZy.org).

[0070] In some embodiments, the glycolytic enzyme is selected from amylases, cellulases, and hemicellulases, with each option representing a separate embodiment of the present invention.

[0071] The cellulase may be selected from, but is not limited to, endo-(1,4)-D-glucanase, exo-(1,4)-β-υ-glucanase, β-glucosidase, carboxymethylcellulase (CMCase); endoglucanase; cellobiohydrolase; avicelase, celludextrinase, cellulase A, cellulosin AP, alkaline cellulase, and pancelase SS, each of which is a separate embodiment.

[0072] The hemicellulase may be a xylanase. Non-limiting examples of additional hemicellulases include arabinofuranosidase, acetyl esterase, mannanase, α-D-glucuronidase, β-xylosidase, β-mannosidase, β-glucosidase, acetyl-mannan esterase, α-galactosidase, α-L-arabinanase, and β-galactosidase. Each option represents a separate embodiment of the present invention.

[0073] The amylase may be selected from, but is not limited to, glucoamylase, α-amylase; (1,4-α-D-glucan glucanohydrolase; glycogenase), β-amylase; (1,4-α-D-glucan maltohydrolase; glycogenase; (saccharogen amylase), γ-amylase; (glucan 1,4-α-glucosidase; amyloglucosidase; exo-1,4-α-glucosidase; lysosomal α-glucosidase; 1,4-α-D-glucan glucohydrolase), and pullulanase (limit dextrinase; amylopectin 6-glucanohydrolase; bacterial debranching enzyme; debranching enzyme; α-dextrin endo-1,6-α-glucosidase; R-enzyme; pullulan α-1,6-glucanohydrolase). Each option is a separate embodiment.

[0074] In some embodiments, the glycolytic enzyme used in the present invention is a disaccharidase. In some embodiments, the disaccharidase is selected from lactase and invertase. Each option represents a separate embodiment of the present invention.

[0075] The glycolytic enzymes of the present invention may be derived from a bacterial source. In some embodiments, the bacterial source is a thermophilic bacterium. As used herein, the term "thermophilic bacterium" refers to a bacterium that grows at temperatures greater than about 45°C, preferably greater than 50°C. Typically, thermophilic bacteria of the present invention have an optimal growth temperature of about 45°C to about 75°C, preferably about 50°C to 70°C. Non-limiting examples of thermophilic bacterial sources of glycolytic enzymes include, for cellulases and hemicellulases, Clostridium species (e.g., Clostridium thermocellum), Paenibacillus species, and Thermobifida fusca; and for amylases, Bacillus species (e.g., Bacillus stearothermophilus), Geobacillus species (e.g., Geobacillus thermoleovorans), Chromohalobacter species, and Rhodothermus marinus. Each alternative is a separate embodiment.

[0076] In further embodiments, the bacterial source of glycolytic enzymes is a mesophilic bacterium. As used herein, the term "mesophilic bacterium" refers to bacteria that grow at temperatures between about 20°C and 45°C. Non-limiting examples of mesophilic bacterial sources for glycolytic enzymes include Klebsiella species (e.g., Klebsiella pneumoniae), Cornell species, Streptomyces species, Acetivibrio cellulolyticus, and Ruminococcus albus for cellulases and hemicellulases; and Bacillus species (e.g., Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus licheniformis) and Lactobacillus yeast for amylases. Those skilled in the art will appreciate that some mesophilic bacteria (e.g., some Bacillus species) produce thermostable enzymes.

[0077] The glycolytic enzymes of the present invention may also be derived from fungal sources, including, but not limited to, Trichoderma reesei, Humicola insolens, and Fusarium oxysporum for cellulases and hemicellulases, and Aspergillus niger, Aspergillus oryzae, Penicillium fertanum, and Thermomyces lanuginosus for amylases (e.g., glucoamylases).

[0078] Further sources of glycolytic enzymes for use in accordance with the present invention can be found, for example, on the CAZy server mentioned above.

[0079] According to some embodiments of the invention, saccharification enzymes may be added at a concentration sufficient to saccharify at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.9% of the polysaccharides in the waste material.

[0080] In one embodiment of the present invention, step 108 (i.e., saccharification) is performed at the same temperature as, and optionally simultaneously with, the heat treatment. When saccharification is performed simultaneously with the heat treatment, the second duration is typically extended to the duration required for sufficient or complete saccharification of the polysaccharides in the waste material. In another embodiment, the temperature of the waste material may be adjusted to a temperature typically in the range of 45-65°C, inclusive, prior to the addition of saccharification enzymes. In some embodiments, saccharification is performed at a temperature range of 55-65°C, a temperature range disclosed herein as effective for inhibiting microbial activity in the waste material. In one embodiment, the temperature of the waste material during saccharification is in the range of 50-62°C, inclusive, inclusive. In another preferred embodiment, saccharification is performed at a temperature in the range of 58-62°C, inclusive, inclusive. The pH of the waste material during saccharification is typically maintained within the range of 3.5-5.5, inclusive, inclusive. The use of the temperature and pH conditions disclosed herein during the saccharification process provides benefits such as suppressing endogenous bacterial activity and liquefying waste materials during saccharification.

[0081] Generally, the temperature and pH of the waste material during saccharification must be controlled so that the enzymes utilized can effectively hydrolyze non-reducing carbohydrates. It should be understood that if saccharification is carried out under temperature and pH conditions that inhibit microbial activity, the glycolytic enzymes are enzymes that are active under those conditions.

[0082] The saccharification step may be carried out for a period of 0.25 to 24 hours, typically 1 to 24 hours, 1.5 to 24 hours, 5 to 24 hours, 8 to 24 hours, 1 to 15 hours, or 1 to 10 hours, inclusive of each value within the specified ranges, with each option representing a separate embodiment of the present invention.

[0083] Combinations of various saccharification enzymes suitable for saccharifying various types of sugars, such as cellulose, glucans, and starches, can be applied. In one embodiment of the present invention, one of the factors controlling the type and relative concentrations of enzymes used in the saccharification process may be the relative abundance of different types of polysaccharides in the waste material. For example, when processing a batch of waste material known to be derived from crop residues, enzymes from the cellulase group may be primarily used to saccharify the high concentration of cellulose. If the waste material contains high levels of starch, such as in waste from a bakery or cafeteria, the saccharification enzymes may include significant amounts of amylase. Alternatively, other considerations, such as the cost-effectiveness of various enzymes, yield, and manufacturer-recommended conditions, may influence the type of saccharification enzymes utilized.

[0084] In some embodiments of the present invention, the order of steps 104, 106, and 108 may be changed. For example, step 106 may be performed before step 104, such that the provided waste material of step 102 is directly subjected to heat treatment in step 106. Following heat treatment, the waste material may be saccharified (i.e., step 108) or maintained at temperature and pH conditions that inhibit microbial activity before undergoing saccharification (i.e., step 104). Indeed, the waste material may be maintained under conditions that inhibit microbial activity prior to heat treatment, between heat treatment and saccharification, and / or after saccharification prior to fermentation in step 110. Each option represents a separate embodiment.

[0085] Step 110 involves the controlled production of lactic acid by fermentation of reducing sugars of the waste material. As used herein, "controlled production" or "controlled fermentation" refers to lactic acid fermentation carried out in a fermentor under controlled conditions, such as controlling one or more of the following parameters: temperature, pH, nutrient levels, agitation rate, and aeration (aerobic / anaerobic / microaerobic conditions). Typically, separation of solid components (step 114, as described in more detail below) and / or sterilization occurs prior to step 110. Following the controlled production stage, the fermentation broth is processed to recover the fermentation product, i.e., lactic acid or a salt thereof.

[0086] Lactic acid fermentation is carried out using lactic acid-producing microorganisms. As used herein, "LA-producing microorganisms" refers to microorganisms that produce lactic acid as the main metabolic end product of carbohydrate fermentation. Preferred herein is the use of microorganisms that produce only L-lactic acid. LA-producing microorganisms can naturally produce only L-lactic acid, or can be genetically modified to produce only L-lactic acid, for example, by knocking out one or more enzymes involved in the synthesis of the unwanted D-enantiomer. LA-producing microorganisms include various bacteria, fungi, and yeasts, including, for example, Lactobacillus species and Bacillus species. Each option represents a separate embodiment.

[0087] Saccharification and lactic acid fermentation, i.e., steps 108 and 110, can be carried out simultaneously (simultaneous saccharification and fermentation, SSF). Alternatively, enzymatic digestion of polysaccharides can be carried out prior to lactic acid fermentation, either in the same reactor as fermentation or in a different reactor (separate hydrolysis and fermentation, SHF). Each option (SSF, SHF) represents a separate embodiment of the present invention.

[0088] Fermentation is typically carried out in the presence of an alkaline compound, such as a metal oxide, carbonate, or hydroxide, as detailed above. Suitable alkaline compounds include, but are not limited to, MgO, CaO, CaCO3, MgCO3, NaOH, KOH, NH4OH, Ca(OH)2, Mg(OH)2, and mixtures or combinations thereof. Each option represents a separate embodiment. The alkaline compound is added to adjust the pH of the fermentation broth to a desired value, typically in the range of 5 to 7, including each value within the specified range. The alkaline compound also neutralizes L-lactic acid to lactate. During fermentation, the pH in the fermenter decreases due to the production of lactic acid, which adversely affects the productivity of the lactic acid-producing microorganism. The pH is adjusted by adding a base, such as magnesium hydroxide, sodium hydroxide, potassium hydroxide, or calcium hydroxide, to neutralize the lactic acid, thereby resulting in the formation of lactate.

[0089] Typically, fermentation is carried out under anaerobic or microaerobic conditions using batch, fed-batch, continuous, or semi-continuous fermentation, with each option representing a separate embodiment of the present invention.

[0090] In batch fermentation, the carbon substrate and other components are added to a reactor, and the product is collected when the fermentation is complete. Except for the alkaline compounds mentioned above for pH control, no other components are added to the reaction before completion. The size of the inoculum is typically about 5-10% of the liquid volume in the reactor. The fermentation is maintained at a substantially constant temperature and pH, and the pH is maintained by adding alkaline compounds.

[0091] In fed-batch fermentation, substrate is fed continuously or sequentially to the reactor without removing the fermentation broth (i.e., product remains in the reactor until the end of the run). Common feeding methods include intermittent, constant, pulsed, and exponential feeding. Each option represents a separate embodiment.

[0092] In continuous fermentation, substrate is added continuously to a reactor at a fixed rate and fermentation product is removed continuously.

[0093] In a semi-continuous process, portions of the culture are removed at intervals and fresh medium is added to the system. Repeated fed-batch cultures that can be maintained indefinitely are also considered semi-continuous processes.

[0094] Lactic acid fermentation is typically carried out for about 1-4 days, or any period therebetween, for example, 1-2 days, or 2-4 days, or 3-4 days, inclusive of each value within the range specified.

[0095] The waste material subjected to the lactic acid production process 100 is often large in volume and contains a heterogeneous mixture of various liquids and solids. Therefore, potentially beneficial compounds in the waste material are not always accessible to treatment reagents, such as reactive compounds, enzymes, and the like, and therefore do not contribute to lactic acid production. Furthermore, some of the macromolecules in the waste material are degraded through surface erosion, progressing gradually through one or more erosion fronts to the bulk, which can be slow and not always effective. Now, referring to step 112, the waste material is subjected to mechanical treatment to increase the surface area and / or improve the turnover of organic and active components within the waste material. Mechanical treatment can include, for example, grinding, chipping, shredding, chopping, and / or milling.

[0096] Mechanical treatment can also include agitating the waste material or portions thereof. Agitation is intended to thoroughly mix the waste material and allow all portions of the waste material to interact with the treatment reagents. Mixing can also enhance the effectiveness of processes such as heating, aeration, and / or vacuum applied to the waste material. Agitation may be at speeds ranging from 10 to 1000 RPM, e.g., 30 to 100 RPM, 50 to 200 RPM, 150 to 500 RPM, 300 to 700 RPM, or 500 to 1000 RPM, including any value within the specified range. The agitation speed, or any other parameters such as power, force, directionality, agitation means (e.g., internal or external), may be substantially fixed throughout process 100 or may be adjusted and / or turned on and off at different steps. For example, the agitation speed and / or force may be highest during step 104, when the waste material is in a relatively untreated state, and may decrease as the process progresses, correlating with the liquefaction of the waste material. Alternatively, agitation may begin in a gentle manner, e.g., at a relatively low agitation speed, and may be intensified as treatment agents are added to the waste material, e.g., in steps 108 and 110, to accelerate the treatment procedure. Agitation speed and power may be increased in unison, or optionally, may be inversely related, such that as speed increases, power decreases. Each option represents a separate embodiment. Any other combination of agitation parameters during the various steps may be utilized. Some variables that may determine the selected agitation parameters include, but are not limited to, gas-liquid mass transfer, feed distribution, local oxygen concentration, shear rate distribution, and local mixing intensity.

[0097] Any embodiment of mechanical treatment, such as that described in step 112, may be performed at any one or more of the various stages of process 100 or throughout the process. As a non-limiting example, the waste material may be subjected to grinding, chipping, etc., immediately prior to, contemporaneously with, and / or after being subjected to conditions that inhibit microbial activity (i.e., step 104) when the waste material is provided (i.e., step 102). Alternatively, or in addition, the waste material may be mechanically treated to increase the surface area of ​​polysaccharide compounds prior to and / or contemporaneously with the initiation of saccharification (i.e., step 108). In another embodiment, the waste material may be coarsely ground, chipped, shredded, milled, or otherwise disrupted at an early stage of pretreatment (e.g., before applying conditions that inhibit microbial activity) and more finely ground, chipped, etc. at a later stage of the process (e.g., before saccharification and / or fermentation). Mechanical treatment in the form of agitation may be applied continuously or at selected stages of process 100.

[0098] Organic waste materials according to the present invention typically contain solid components, including organic and inorganic solid components. While pretreatment induces liquefaction of the waste material, solid particles of various shapes, sizes, and textures may remain within the waste material. These solid particles often cannot be effectively processed to produce reducing sugars for controlled fermentation. They may interfere with the treatment process the waste material undergoes, clog machinery, and / or prevent treatment reagents from accessing the productive waste material. Now, referring to step 114, solid-liquid separation is applied to the waste material. Examples of solid-liquid separation include filtration, decantation, and the like. The waste material, or a portion thereof, may be processed by a screw press, a filter press, centrifuged, and / or any other liquid-solid separation method or combination of methods known in the art. After separation, the liquid phase, typically containing free reducing sugars and / or saccharifiable carbohydrates, is collected for further processing.

[0099] The solid phase can also be collected separately for selective processing. Methods of crushing or shaping materials, such as mechanical grinding, chipping, laser cutting, etc., are typically more effective when the processed material is dry and hard compared to a wet mixture. Therefore, the separated solid phase material can be subjected to at least one such trimming or crushing method after being at least partially dewatered. Trimming or crushing the solid material can produce smaller solid particles that can be reintroduced into the liquid phase of the waste material for further processing.

[0100] The solid-liquid separation of step 114 can be utilized simultaneously with any one or more of the steps of process 100, and / or before or after any one or more of the steps. As a non-limiting example, the waste material may be subjected to solid-liquid separation prior to saccharification. Alternatively, solid-liquid separation may be performed as a pre-step to maintain the waste material under conditions that inhibit microbial activity (i.e., step 104), and the resulting liquid phase may be subjected to conditions that inhibit microbial activity, while the solid phase may be subjected to a disruption treatment as described above, after which the degraded solid phase is returned to the liquid phase. Additionally, alternatively, or in addition, solid-liquid separation may be performed prior to fermentation (i.e., step 110).

[0101] In some embodiments, after solid-liquid separation, the liquid phase is concentrated to obtain a concentrated liquid or syrup containing reducing sugars. In some embodiments, the concentrated liquid or syrup containing reducing sugars is produced at a first location and then transported to a second location where lactic acid is produced from the concentrated liquid or syrup. In some embodiments, the first location is an organic waste collection facility and the second location is a lactic acid production facility. In some embodiments, sterilization of the concentrated liquid or syrup occurs at the second location prior to lactic acid production from the concentrated liquid or syrup by fermentation. In some embodiments, the concentrated liquid or syrup is maintained under conditions disclosed herein during transport.

[0102] As used herein and in the appended claims, the term "about" refers to ±10% from the specified value, for example, + / -5%, + / -1%, and + / -0.1%.

[0103] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to an "enzyme" includes a plurality of enzymes and types of enzymes unless the context clearly dictates otherwise. It should be noted that the term "and" or "or" is generally employed in its "and / or" sense unless the context clearly dictates otherwise.

[0104] The following examples are presented in order to more fully illustrate certain embodiments of the present invention. However, they should in no way be construed as limiting the broad scope of the invention. Those skilled in the art can readily devise numerous variations and modifications of the principles disclosed herein without departing from the scope of the invention. [Example]

[0105] Example 1 Inhibition of endogenous microbial activity in organic waste materials. To demonstrate the effectiveness of maintaining organic waste at the temperature and pH conditions of the present invention (e.g., at a temperature of 55°C to 65°C and a pH of 3.5 to 5.5) in inhibiting endogenous microbial activity, the following experiment was conducted.

[0106] procedure: A non-sterile slurry of mixed food waste collected from supermarket distribution returns and surplus was provided. The mixed food waste contained expired bakery and dairy products, beverages, and fruits and vegetables, and was subjected to separation and grinding of inorganic solid components (e.g., plastics). The slurried waste contained 60 g / L of latent glucose and 17 g / L of available glucose. Latent glucose, also referred to as "glucose potential," refers to the concentration of both sugars in a non-reduced state prior to saccharification and fermentable sugars in a reduced state, i.e., available for fermentation ("available glucose").

[0107] The waste material was pumped into a bioreactor heated to a temperature of 60 °C (thin black line in Figure 2). The pH of the waste material was 4.3-4.4, and therefore no pH adjustment was performed. The waste material was maintained in the heated bioreactor for 16 h. pH, base weight [kg], pressure (mbar), potential glucose concentration, and available glucose concentration (g / L) were measured intermittently during the 16 h period by calibrated instruments.

[0108] result: The results are shown in Figure 2.

[0109] The thick black line indicates pH. The pH remained stable at 4-5, as confirmed by offline measurements at pH 4.3-4.4. Because glucose consumption and subsequent acid production indicate microbial activity, the essentially constant pH indicates that no microbial activity occurred during the experiment. Therefore, no base was added to the waste material (dashed gray line). The suppression of microbial activity is also evident from the pressure measurements (thick gray line), which remained stable with no production of gases, e.g., CO2, methane.

[0110] The black triangles (▲) indicate glucose potential. The black dots (●) indicate the concentration of available fermentable reducing sugars, e.g., glucose alone. The initial glucose potential and available glucose concentration of 60 g / L and 17 g / L, respectively, were maintained throughout the 16-hour experiment, indicating that no microbial activity accompanied the utilization of free glucose occurred.

[0111] When non-sterile mixed food waste slurries are not maintained under the conditions of the present invention (e.g., maintained at room temperature), significant gas production is observed, indicating undesirable microbial activity within the waste. The above results demonstrate that maintaining non-sterile organic waste slurries under conditions according to the present invention, e.g., at a temperature of 60°C and a pH of 4-5, inhibits microbial activity, including glucose consumption and gas production.

[0112] Example 2 Heat treatment followed by accelerated saccharification To test the effect of heat treatment on subsequent saccharification, the following experiment was performed.

[0113] procedure: A non-sterile slurry of mixed food waste material similar to that of Example 1 was provided, which contained a glucose potential of 60 g / l and an effective glucose concentration of 17 g / l.

[0114] The waste material was pumped into the bioreactor. The first dose of saccharification enzymes (glucoamylase (GA) and α-amylase (AA)) was added, and then the temperature was heated to 60°C. The pH of the waste material was 4.3-4.4, so no pH adjustment was performed. The waste material was maintained at 60°C with stirring for 18 hours. After 18 hours, the temperature was steadily increased to reach 70°C and maintained in the 70-73°C range for 2 hours. After cooling to 60°C, at 22 hours, the second dose of saccharification enzymes was added, and the temperature was maintained at 60°C for an additional 2.5 hours.

[0115] pH, pressure (mbar), potential glucose concentration and available glucose concentration (g / l) were measured intermittently during the process by calibrated instruments.

[0116] result: The results are shown in Figure 3.

[0117] The thick black line indicates pH. The pH was maintained between 4 and 5 and confirmed by offline measurements at pH 4.3 and 4.4. Because glucose consumption and subsequent acid production indicate microbial activity, a substantially constant pH indicates that no microbial activity occurred during the experiment. Therefore, no base was added to the waste material (gray dashed line).

[0118] The vertical black bar at 22 h indicates the addition of enzymes after heat treatment at 70°C (temperature indicated by the thin black line). Two enzymes, glucoamylase (GA) and α-amylase (AA), were added to induce saccharification.

[0119] The black triangles (▲) indicate glucose potential. Glucose potential indicates the concentration of both non-reduced (pre-saccharification) sugars and reduced, i.e., fermentable, sugars available for fermentation. The black dots (●) indicate the concentration of available, fermentable, reducing sugars, e.g., glucose, only. The waste contained 60 g / L of latent glucose, of which 17 g / L constituted available glucose. After the addition of the first dose of saccharification enzyme and 18 hours at 60°C and pH 4.4, no significant change in available glucose was measured. After heat treatment and the addition of the second dose of saccharification enzyme, virtually all of the latent glucose was converted to available glucose (59.5 g / L).

[0120] The thick gray line indicates pressure measurements (mbar). It is worth noting that the pressure began to rise from approximately 40 mbar to approximately 65 mbar at 18 hours, but this increase in pressure was the result of the simultaneous increase in temperature from 60°C to over 70°C. The elevated pressure was not the result of gas generation due to microbial utilization of available glucose, as the stable pH and glucose levels clearly indicated the absence of such microbial activity. This is also evident by the decrease in pressure beginning at approximately 21 hours, which corresponds to the simultaneous cooling of the waste material.

[0121] Taken together, these results demonstrate that subjecting non-sterile organic waste material to heat treatment according to the present invention (e.g., at a temperature of 70°C for 2 hours as carried out in this example) prior to saccharification substantially increases the efficiency of saccharification and, therefore, the yield of reducing sugars available for fermentation.

[0122] Saccharification of polysaccharides for fermentation processes is typically performed on polysaccharide-containing substrates after sterilization. Organic waste slurries, particularly food waste slurries, contain various solids in their untreated state, making it difficult to effectively sterilize the waste, and even if sterilized, maintaining the waste in a sterile state throughout its processing up to the lactic acid production stage. The above experiments demonstrated that heat treatment according to the present invention can efficiently saccharify non-sterilized organic waste slurries, thereby improving the organic waste pretreatment process.

[0123] The foregoing description of specific embodiments fully reveals the general nature of the present invention, so that others, by applying the knowledge herein, can easily modify and / or adapt such specific embodiments to various uses without undue experimentation and without departing from the general concept; therefore, such adaptations and modifications should be understood within the meaning and range of equivalents of the embodiments of the present disclosure, and are so intended. It is to be understood that the phraseology or terminology used herein is for purposes of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the spirit and scope of the invention, as set forth by the following claims.

Claims

1. 1. A method for pretreating organic waste prior to large-scale production of lactic acid or a salt thereof from said organic waste, comprising: (a) providing a non-sterile slurry of organic waste containing non-reducing polysaccharides and reducing sugars; (b) maintaining the non-sterile slurry of organic waste of step (a) at a temperature in the range of 55°C to 65°C and a pH in the range of 3.5 to 5.5 for a first duration of at least 1 hour, thereby inhibiting endogenous microbial activity in the organic waste; (c) increasing the temperature of the non-sterile slurry of organic waste of step (b) above the temperature of step (b) to a temperature of between 65°C and 85°C for a second duration of at least 0.5 hours; (d) optionally adjusting the temperature to a temperature between 45°C and 65°C; (e) adding one or more saccharification enzymes to the non-sterile slurry of organic waste of step (c) or step (d) to hydrolyze polysaccharides in the organic waste to release reducing sugars and obtain saccharified organic waste.

2. 10. The method of claim 1, wherein steps (c) and (e) are performed simultaneously.

3. 3. The method according to claim 1 or 2, wherein the organic waste comprises plastic and / or inorganic solid components, and the method comprises subjecting the organic waste to separation of the plastic and / or inorganic solid components prior to step (a).

4. The method according to any one of claims 1 to 3, further comprising the steps of separating the saccharified organic waste into a liquid phase containing reducing sugars and a solid phase, and sterilizing the liquid phase.

5. 1. A method for pretreating organic waste prior to large-scale production of lactic acid or a salt thereof from said organic waste, comprising: (a) providing a non-sterile slurry of organic waste containing non-reducing polysaccharides and reducing sugars; (b) maintaining the non-sterile slurry of organic waste of step (a) at a temperature in the range of 55°C to 65°C and a pH in the range of 3.5 to 5.5 for a first duration of at least 1 hour, thereby inhibiting endogenous microbial activity in the organic waste; (c) increasing the temperature of the non-sterile slurry of organic waste of step (b) above the temperature of step (b) to a temperature of 65°C to 85°C for a second duration of at least 0.5 hours; (d) adjusting the temperature to a temperature of 45°C to 65°C and adding one or more saccharification enzymes to hydrolyze polysaccharides in the organic waste to release reducing sugars and obtain saccharified organic waste; (e) separating the saccharified organic waste into a liquid phase containing reducing sugars and a solid phase, and sterilizing the liquid phase; thereby pretreating said organic waste to obtain raw material for large scale production of lactic acid or a salt thereof.

6. The method according to any one of claims 1 to 5, wherein the one or more saccharifying enzymes include at least one of glucoamylase, α-amylase, and pullulanase.

7. 7. The method of any one of claims 1 to 6, wherein the second duration of step (c) is in the range of 0.5 hours to 5 hours.

8. 1. A method for pretreating organic waste prior to large-scale production of lactic acid or a salt thereof from said organic waste, comprising: (A) providing a non-sterile slurry of organic waste containing non-reducing polysaccharides and reducing sugars; (B) heating the non-sterile slurry of organic waste to a temperature of 65°C to 85°C for at least 1 hour; (C) optionally adjusting the temperature to a temperature of between 45°C and 65°C; (D) adding one or more saccharification enzymes to the non-sterile slurry of organic waste from step (B) or step (C) to hydrolyze polysaccharides in the organic waste to release reducing sugars and obtain saccharified organic waste.

9. 1. A method for inhibiting endogenous microbial activity in organic waste materials prior to subjecting said waste materials to the controlled large-scale production of lactic acid or a salt thereof, comprising: (a) providing a non-sterile slurry of organic waste material comprising at least one of non-reducing polysaccharides and reducing sugars; (b) maintaining the waste material of step (a) at a temperature in the range of 55°C to 65°C and a pH in the range of 3.5 to 5.5 for a first duration of at least 1 hour, thereby inhibiting endogenous microbial activity in the waste material prior to subjecting the waste material to controlled large-scale production of lactic acid or a salt thereof.

10. 10. The method of claim 9, wherein the organic waste comprises plastic and / or inorganic solid components, and the method comprises subjecting the organic waste to separation of the plastic and / or inorganic solid components prior to step (a).

11. 11. The method of claim 9 or 10, wherein step (b) is carried out at a pH in the range of 4 to 5.

12. 12. The method of any one of claims 9 to 11, wherein the first duration of step (b) is in the range of 1 to 96 hours.

13. 13. The method of any one of claims 9 to 12, wherein the first duration of step (b) is in the range of 12 to 48 hours.

14. 14. The method of any one of claims 9 to 13, further comprising the step of applying a mechanical treatment to the waste material prior to, simultaneously with, and / or after step (b).

15. 15. The method of any one of claims 9 to 14, wherein the waste material is selected from the group consisting of food waste, municipal waste, agricultural waste, plant material, and combinations thereof.

16. The non-sterile slurry of organic waste is ５ The method of any one of claims 9 to 15, containing microorganisms at a concentration of CFU / mL.

17. The non-sterile slurry of organic waste is ７ The method of any one of claims 1 to 15, containing microorganisms at a concentration of CFU / mL.

18. 1. A method for producing lactic acid from organic waste, comprising: (i) subjecting the organic waste to a pretreatment process comprising the pretreatment method according to any one of claims 1 to 8; (ii) adding lactic acid producing microorganisms to the pretreated organic waste and incubating in a fermentation reactor under controlled conditions for lactic acid production by the lactic acid producing microorganisms, thereby producing lactic acid.

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