Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M45/00—Means for pre-treatment of biological substances
  • C12M45/06—Means for pre-treatment of biological substances by chemical means or hydrolysis
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
  • B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
  • B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
  • B09B3/60—Biochemical treatment, e.g. by using enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M45/00—Means for pre-treatment of biological substances
  • C12M45/09—Means for pre-treatment of biological substances by enzymatic treatment
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/56—Lactic acid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/07—Bacillus

Definitions

• the present invention relates to recycling of organic waste.
• methods and systems for treating of organic waste prior to large-scale production of lactic acid by fermentation are provided.
• Lactic acid fermentation namely, production of lactic acid from carbohydrate sources via microbial fermentation, has been gaining interest in recent years due to the ability to use lactic acid as a building block in the manufacture of bioplastics.
• Lactic acid can be polymerized to form the biodegradable and recyclable polyester, polylactic acid (PLA), which is considered a potential substitute for plastics manufactured from petroleum.
• PLA is used in the manufacture of various products including food packaging, disposables, fibers in the textile and hygiene products industries, and more, and is the most common plastic filament utilized in 3D printing.
• lactic acid by fermentation bioprocesses is preferred over chemical synthesis methods for various considerations, including environmental concerns, costs and the difficulty to generate enantiomerically pure lactic acid by chemical synthesis, which is desired for most industrial applications.
• the conventional fermentation process is typically based on anaerobic fermentation by lactic acid-producing microorganisms, which produce lactic acid as the major metabolic end product of carbohydrate fermentation.
• the lactic acid generated during the fermentation is separated from the fermentation broth and purified by various processes, and the purified lactic acid is then subjected to polymerization.
• Lactic acid has a chiral carbon atom and therefore exists in two enantiomeric forms, D- and L-lactic acid.
• the D- or L- lactic acid entering the production process must be highly purified to meet the specification required for polymerization. Therefore, lactic acid bacteria that produce only L-lactate enantiomer or only D-lactate enantiomer are typically used in order to produce one discreet enantiomer (L or D).
• the carbohydrate source for lactic acid fermentation is typically a starch-containing renewable source such as corn and cassava root. Additional sources, such as the cellulose-rich sugarcane bagasse, have also been proposed.
• lactic acid bacteria can utilize reducing sugars like glucose and fructose, but do not have the ability to degrade polysaccharides like starch and cellulose.
• the process requires adding glycolytic enzymes, optionally in combination with chemical treatment, to degrade the polysaccharides and release reducing sugars, a process defined as saccharification.
• Saccharification may precede the fermentation process or may be done simultaneously therewith. Saccharification before fermentation is known as separate hydrolysis and fermentation (SHF), and a process which combines saccharification and fermentation is known as simultaneous saccharification and fermentation (SSF).
• SHF separate hydrolysis and fermentation
• SSF simultaneous saccharification and fermentation
• the SSF has the advantage of requiring only one step, saving time and expenses. It has the further advantage of using-up the reducing sugars as they are released from the polysaccharides, thereby maintaining a relatively low concentration of reducing sugars and high productivity of the hydrolyzing enzymes.
• An additional source of carbohydrates for lactic acid fermentation is complex organic waste, such as mixed food waste from municipal, industrial and commercial origin, which typically includes varied ratios of reducing sugars (glucose, fructose, lactose, etc.), starch and lignocellulosic material.
• organic waste is advantageous as it is readily available and less expensive compared to other carbohydrate sources for lactic acid fermentation.
• organic waste typically contains high levels of endogenous microorganisms, naturally occurring microbial processes take place within the waste material before initiation of the intended industrial fermentation process. These microbial processes often involve utilizing the reducing sugars which are the key substrate of the controlled fermentation, thus compromising the efficiency of the controlled fermentation process and lowering the yield of lactic acid.
• the microbial processes may further produce products and side products which inhibit various stages of the fermentation process, and may also produce a different enantiomer of lactic acid than the desired enantiomer, which elevates the impurity of the lactic acid final product and may necessitate applying time-consuming and costly purification processes.
• Complex organic waste such as mixed food waste, at its raw condition, typically includes various solids, which make it difficult to effectively sterilize the waste, and even if sterilized, it is difficult to maintain the waste under sterile conditions throughout its processing until the lactic acid production stage. Repeated or extensive sterilization may result in the formation of unwanted glucose-degradation products, which also reduce the substrate available for fermentation and may interfere with fermentation.
• EP 1320388 discloses a method for reducing the number of viable microbial organisms and/or prions present in an organic material, said method comprising the steps of: i) providing an organic material comprising solid and/or liquid parts, ii) subjecting said organic material to the processing steps of: a) lime pressure cooking at a temperature of between 100°C and 220°C resulting in hydrolysis of the organic material, wherein lime is Ca(OH)2 and/or CaO, and b) stripping ammonia from said lime pressure cooked organic material, wherein lime added in connection with stripping of ammonia and sanitation of the organic material precipitates dissolved orthophosphate, and iii) obtaining a processed organic material comprising a reduced number of viable microbial organisms and/or prions.
• WO 00/02457 discloses a fermented, pasteurized preferment comprising: the fermentation product of a mixture of gluten and/or bran resulting from the hydrolysis thereof with a protease and/or lipase and/or glycosidase and/or glycanase, preferably a glucanase, followed by a fermentation with an acid forming bacterium, preferably a lactic acid forming bacterium, and a yeast, preferably in the presence of enzymes capable of liberating flavor ingredients or flavor precursors from proteins and/or carbohydrates, preferably selected from proteases and another glycosidase and/or glycanase, in particular amylases.
• WO 2016/016235 discloses a process for preparing lactic acid and/or a lactate salt via fermentation of carbohydrates obtained from lignocellulosic material.
• the process comprises: treating a lignocellulosic material with an alkaline agent comprising 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 comprising fermentable carbohydrates and a solid lignocellulosic fraction; fermenting the fermentable carbohydrates in the saccharified aqueous lignocellulosic material by means of lactic acid producing microorganism in the presence of an alkaline agent comprising a caustic magnesium salt, to provide an aqueous fermentation broth comprising a magnesium lactate; and isolating lactic acid and/or lactate salt from the fermentation broth
• the present invention provides improved methods for pretreating organic waste, particularly starch-containing organic waste, prior to industrial fermentation processes that utilize the organic waste as a substrate for fermentation, particularly industrial production of lactic acid or a salt thereof.
• the improved methods reduce the loss of substrate for fermentation and reduce the formation of unwanted inhibitory compounds during the pretreatment.
• the pretreatment methods according to the present invention reduce or even completely inhibit endogenous microbial activity within the waste during the pretreatment stage, and reduce or even completely prevent formation of glucose-degradation products during the pretreatment.
• the pretreatment methods according to the present invention inhibit or even completely prevent the loss of reducing and/or non-reducing sugars during the pretreatment stage. The pretreatment methods according to the present invention thus facilitate increased lactic acid production yield from the organic waste.
• the methods of the present invention are preferably employed on a non-sterile slurry of organic waste comprising starch and C5 and/or C6 reducing sugars as soon as possible following collection of the organic waste and initial processing such as grinding and removal of plastics and inorganic solid components such as glass and sand (if present).
• the non-sterile slurry of organic waste is subjected to a heat treatment combined with an alpha-amylase treatment, comprising heating the slurry to a temperature in the range of 65-95°C (typically between 70-85°C) and incubating the slurry at the elevated temperature with an alpha-amylase that is active at the elevated temperature, to obtain a liquified waste comprising alpha-amylase hydrolysis products of starch (particularly, maltodextrins) and C5 and/or C6 reducing sugars in which endogenous microbial activity is inhibited.
• a heat treatment combined with an alpha-amylase treatment comprising heating the slurry to a temperature in the range of 65-95°C (typically between 70-85°C) and incubating the slurry at the elevated temperature with an alpha-amylase that is active at the elevated temperature, to obtain a liquified waste comprising alpha-amylase hydrolysis products of starch (particularly, maltod
• the liquified waste is subjected to solid-liquid separation to separate a liquid phase comprising the alpha-amylase hydrolysis products of starch and reducing sugars, and subsequently the liquid phase is subjected to sterilization.
• the sterilized liquid phase is then subjected to saccharification by a glucoamylase and fermentation.
• a heat treatment combined with an alpha amylase treatment according to the present invention results in an efficient activity of the alpha amylase on a non- sterile organic waste slurry.
• the alpha amylase works efficiently to break down polysaccharides in the organic waste into shorter chains that are preserved in the liquid phase and not lost upon solid-liquid separation, and are also more stable compared to glucose upon sterilization.
• Pretreatment according to the present invention inhibits endogenous microorganisms, liquifies the slurry to assist in effective mixing in the reactor, reduces formation of glucose-degradation products, and allows separating solids (if desired) without losing glucose potential, thus continuing to sterilization, further saccharification and fermentation with a liquid phase that is more simple to handle.
• the present invention is particularly useful for food waste, which is heterogenous and typically characterized by a high level of microbial contaminants and thus susceptible to decay by natural fermentation processes during collection, transport and/or pretreatment.
• an organic waste slurry for use with the present invention is characterized by microbial content of at least 10 5 CFU/ml.
• pretreatment according the present invention is effective in inhibiting microbial activity, achieving efficient saccharification and preserving the glucose potential of the waste for the controlled, large- scale, production stage.
• the present invention provides a method for pretreating organic waste prior to large-scale production of lactic acid or a salt thereof from the organic waste, the method comprising:
• step (c) incubating the non-sterile slurry of organic waste of step (b) with the added alpha-amylase to obtain alpha-amylase hydrolysis products of starch, wherein the incubating is carried out at the first temperature of step (b);
• step (e) subjecting the non-sterile slurry of organic waste of step (c) or step (d) to solidliquid separation to separate a liquid phase comprising the alpha-amylase hydrolysis products of starch and the reducing sugars selected from C5 sugars, C6 sugars and a combination thereof, and subjecting the liquid phase to sterilization to obtain a sterilized liquid phase;
• the present invention provides a method for producing lactic acid or a salt thereof from organic waste, the method comprising the steps of: subjecting the organic waste to pretreatment comprising a pretreatment method as disclosed herein; and adding a lactic acid producing microorganism to the pretreated organic waste and incubating in a fermentation reactor under controlled conditions for lactic acid production by the lactic acid producing microorganism, to thereby produce lactic acid or a salt thereof.
• the present invention provides a for producing lactic acid or a salt thereof from organic waste, the method comprising the steps of:
• step (c) incubating the non-sterile slurry of organic waste of step (b) with the added alpha-amylase to obtain alpha-amylase hydrolysis products of starch, wherein the incubating is carried out at the first temperature of step (b);
• step (e) subjecting the non-sterile slurry of organic waste of step (c) or step (d) to solidliquid separation to separate a liquid phase comprising the alpha-amylase hydrolysis products of starch and the reducing sugars selected from C5 sugars, C6 sugars and a combination thereof, and subjecting the liquid phase to sterilization to obtain a sterilized liquid phase;
• step (g) subjecting the sterilized liquid phase to lactic acid fermentation by adding a lactic acid-producing microorganism, wherein step (f) and step (g) are performed simultaneously, separately or partially- separately, to obtain simultaneous, separate or partially-separate saccharification and lactic acid fermentation.
• the organic waste comprises plastics and/or inorganic solid components and the method comprises subjecting the organic waste to separation of said plastics and/or inorganic solid components prior to step (a).
• the organic waste is food waste.
• the first temperature in step (b) is between 70°C to 85°C. In additional embodiments, the first temperature in step (b) is between 75°C to 85°C.
• the incubating in step (c) is carried out for a time duration in the range of 0.25-5 hours. In additional embodiments, the incubating in step (c) is carried out for a time duration in the range of 1-3 hours.
• the incubating in step (c) is carried out at the natural pH of the non-sterile slurry of organic waste. In additional embodiments, the incubating in step (c) is carried out at a pH in the range of 3.5 to 5.5. In yet additional embodiments, the incubating in step (c) is carried out at a pH in the range of 4 to 5.
• step (d) is performed and the second temperature is between 50°C to 75°C. In additional embodiments, step (d) is performed and the second temperature is between 50°C to 65°C. In yet additional embodiments, step (d) is performed and further comprises maintaining the non- sterile slurry of organic waste of step (c) at the second temperature for a time duration of at least 1 hour. In some embodiments, step (d) comprises maintaining the non- sterile slurry of organic waste of step (c) at the second temperature for a time duration in the range of 1-24 hours. In additional embodiments, step (d) comprises maintaining the non-sterile slurry of organic waste of step (c) at the second temperature for a time duration in the range of 1-18 hours.
• the non-sterile slurry of organic waste contains microorganisms at a concentration of at least 10 5 CFU/ml. In additional embodiments, the non-sterile slurry of organic waste contains microorganisms at a concentration of at least 10 7 CFU/ml.
• the glucoamylase is a thermophilic glucoamylase and the lactic acid-producing microorganism is a thermophilic lactic acid-producing microorganism. In some embodiments, the glucoamylase is a thermophilic glucoamylase and the lactic acid-producing microorganism is Bacillus coagulans.
• Figure 1 shows a block diagram of a method for waste material pretreatment and lactic acid production enhancement, according to certain embodiments of the present invention.
• Figure 2 shows changes in glucose (g/L), lactate (g/L), fructose, and hydroxymethylfurfural (HMF, mg/L) that was measured in a fermentation broth of mixed food waste containing solids following a pretreatment that included extensive sterilization.
• the present invention provides methods and systems for preserving glucose potential of a starch-containing organic waste material prior to industrial fermentation processes that utilize the organic waste as a substrate for fermentation, particularly lactic acid fermentation.
• the methods and systems of the present invention are useful for increasing the yield of lactic acid in the controlled fermentation process, e.g., by suppressing unwanted substrate consumption by endogenous microorganisms present in the organic waste and by further preventing formation of inhibitory side products and other impurities within the waste material.
• the methods and systems of the present invention involve subjecting a provided waste material to a heat treatment combined with an alpha amylase treatment, followed by solid-liquid separation, sterilization and saccharification with a glucoamylase.
• the methods and systems of the present invention are employed on a non-sterile slurry of organic waste, preferably as soon as possible following collection of the organic waste and initial processing such as grinding and removal of plastics and inorganic solid components (if present).
• a "slurry" of organic waste refers to a mixture of the organic waste and water, typically containing solid particles of the organic waste.
• a slurry of organic waste as used herein is typically formed by collecting waste material from various sources; subjecting the waste material to separation of plastics and inorganic solid components such as glass, metal and sand, to remove most and preferably all of the plastics and inorganic solid components; reducing the particle size of the waste material, e.g., by shredding or grinding; adding water if necessary; and creating a suspension of organic waste material in the water.
• forming a slurry of organic waste according to the present invention comprises subjecting the waste to depackaging, namely, removal of packaging material, including plastic, metal and glass packaging material.
• the organic waste may naturally be in the form of a slurry.
• An organic waste slurry according to the present invention (e.g. a food waste slurry) is characterized by a solid content (dry matter content) in the range of 5-50% (namely, characterized by a liquid or moisture content in the range of 50-95%), including each value within the range.
• an organic waste slurry according to the present invention is characterized by a solid content in the range of 10-30% (namely, a liquid or moisture content in the range of 70%-90%), including each value within the range.
• an organic waste slurry according to the present invention is characterized by a solid content in the range of 15-35% (namely, a liquid or moisture content in the range of 65%-85%) including each value within the range.
• an organic waste slurry according to the present invention is characterized by a water content in the range of 50-95%, including each value within the range. In some embodiments, an organic waste slurry according to the present invention is characterized by a water content in the range of 70%-90%, including each value within the range. In some embodiments, an organic waste slurry according to the present invention is characterized by a water content in the range of 65%-85%, including each value within the range.
• the organic waste for use with the present invention naturally contains the aforementioned solid/liquid/water content.
• water is added to the organic waste (e.g., food waste) to obtain a slurry characterized by the aforementioned solid/liquid/water content.
• An organic waste slurry according to the present invention (e.g. a food waste slurry) is pumpable and mixable, and thus suitable for further handling and processing according to the present invention.
• lactic acid refers to the hydroxycarboxylic acid with the chemical formula CH3CH(OH)CO2H.
• lactate unprotonated lactic acid
• L-lactic acid/L-lactate D-lactic acid/D- lactate, or to a combination thereof.
• reducing sugar(s) As used herein, the terms “reducing sugar(s)”, “free sugar(s)” and “available sugar(s)” are used interchangeably and refer to soluble sugar molecules which can be utilized in their current state as a substrate for lactic acid fermentation.
• the reducing sugars typically comprise C5 sugars (pentoses), C6 sugars (hexoses) or a combination thereof.
• the reducing sugars comprise glucose.
• the reducing sugars comprise xylose.
• non-reducing carbohydrate(s) As used herein, the terms “non-reducing carbohydrate(s)”, “polysaccharide(s)” and “non-reducing sugar(s)” are used interchangeably, and refer to polymeric sugar molecules which cannot be utilized in their current state for lactic acid fermentation and require saccharification/hydrolysis in order to become available for fermentation. Examples include starch, cellulose, hemicellulose, and combinations thereof.
• maltodextrins refers to the products of starch hydrolysis by alpha amylase and encompasses maltose, maltooligosaccharides, linear and branched dextrins and combinations thereof. Maltodextrins can be further hydrolyzed, e.g., by a glucoamylase, to release free glucose.
• the term “maintaining at a temperature/pH in a range of X for a time 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 time duration, allowing for possible deviations of the temperature/pH from the defined range.
• L-lactic acid monomers with high purity are required in order to produce PLA with suitable properties.
• the methods and systems of the present invention are directed, in particular, to processes for the production of L- lactate salts at high yields, which can then be converted to L-lactic acid suitable for industrial use.
• Figure 1 is a block diagram of a process, referenced 100, for waste material pretreatment followed by industrial lactic acid production by fermentation, according to some embodiments of the present invention.
• Step 102 includes providing an organic waste material, typically an organic waste material in the form of a slurry.
• An organic waste for use with the methods and systems disclosed herein was not subjected to sterilization.
• Organic waste suitable for use according to the present invention is typically a complex, heterogenous, organic waste comprising solid and non-solid components.
• a complex, heterogenous, organic waste includes carbohydrates for fermentation (soluble carbohydrates available for fermentation and/or polysaccharides that need to be decomposed via enzymes to release soluble carbohydrates for fermentation) and further contains impurities such as salts, fats, oils, lipids, proteins, color components, and/or inert materials and more.
• organic waste for use with the present invention comprises starch and reducing sugars comprising C5 sugars, C6 sugars or a combination thereof.
• organic waste for use with the present invention comprises at least 2% starch (w/v).
• organic waste for use with the present invention comprises between 20-150 gr/1 starch, for example between 30-140 gr/1 starch, or between 30-130 gr/1 starch, including each value within the specified ranges. Each possibility represents a separate embodiment of the present invention.
• Organic waste for use with the present invention may also comprise inorganic solid components such as plastics, glass and the like.
• Organic waste for use with the present invention comprises endogenous microorganisms.
• the organic waste material may be a product of municipal waste, food waste, agricultural waste and/or plant matter.
• the provided waste material includes water and/or other aqueous and/or liquid solutions.
• water or other aqueous and/or liquid solutions may be added to the waste material throughout process 100, according to need, once or several times, intermittently and/or continuously. Each possibility represents a separate embodiment.
• the organic waste for use with the present invention is food waste.
• Food waste in accordance with the present invention encompasses food waste and beverages of plant origin and/or animal origin.
• Food waste in accordance with the present invention encompasses household food waste, commercial food waste, and/or industrial food waste.
• the organic food waste may originate from vegetable and fruit residues, plants, cooked food, protein residues, slaughter waste, and/or combinations thereof.
• Industrial organic food waste may include factory waste such as by products, factory rejects, market returns or trimmings of inedible food portions (such as peels).
• Commercial organic food waste may include waste from shopping malls, restaurants, supermarkets, etc.
• Food waste according to the present invention is typically mixed food waste, comprising one or more of: bakery waste, dairy waste, animal-origin food waste including meat, poultry and fish waste, fruit and vegetable waste, and grain-based food waste (e.g., rice, couscous, pasta, noodles).
• mixed food waste according to the present invention comprises a combination of food wastes selected from bakery waste, dairy waste, animal-origin food waste including meat, poultry and fish waste, fruit and vegetable waste, and grain-based food waste (e.g., rice, couscous, pasta, noodles).
• Food waste typically comprises organic solid components originating from food products or residues, e.g., food particles and debris, bones and bone fragments, shells and shell fragments, seeds and seed fragments, peels and the like, and also solids that do not originate from food products or residues, e.g., plastics, glass and metals, originating, for example, from packaging material.
• pretreatment according to the present invention is carried out on a slurry of food waste after it was subjected to depackaging, to remove most or even all of the packaging material.
• Plant material in accordance with the present invention encompasses agricultural waste and manmade products such as paper waste.
• the waste material may be provided to a designated waste treatment facility or may be provided to a temporary collection point.
• the temporary collection point may be stationary, e.g., an underground sump or a storage house, or may include a waste transporting means, such as a truck, a boat, and the like.
• the waste does not undergo any treatment prior to being subjected to pretreatment process 100, particularly does not undergo sterilization or pasteurization.
• the waste undergoes separation of plastics and inorganic solid components and grinding to form a slurry.
• the slurry of waste material includes at least 10 5 CFU/ml (Colony Forming Units) of endogenous microbes (i.e., bacteria, yeast, fungi, and the like).
• the slurry of waste material includes at least 10 7 CFU/ml. It is noted that the present invention is particularly beneficial for waste streams which contain viable microbes, which, when not treated, can contaminate the waste and exploit the beneficial compounds therein in their metabolic processes.
• Step 104 includes heating the non-sterile slurry of organic waste to a first temperature between 65°C to 95°C (e.g., about 80°C as in the illustrated embodiment), including each value within the range, adding an alpha-amylase that is active at the first temperature, and incubating the non-sterile slurry of organic waste with the added alphaamylase to obtain alpha-amylase hydrolysis products of starch and liquify the organic waste, wherein the incubating is carried out at the first temperature.
• the alpha-amylase may be added to the slurry before the slurry is heated to the first temperature (e.g., at room temperature), during the heating or after reaching the first temperature.
• the first temperature is between 65°C to 85°C, including each value within the range. In some embodiments, the first temperature is between 70°C to 85°C, including each value within the range. In additional embodiments, the first temperature is between 75°C to 85°C, including each value within the range. In some particular embodiments, the first temperature is 80°C.
• the incubation with the alpha-amylase may be carried out for 0.25-5 hours, including each value within the range, for example 1-3 hours or for 2 hours. Each possibility represents a separate embodiment.
• sufficient liquification of the waste may be determined by sampling the waste during the alpha-amylase treatment and measuring glucose potential of each sample before and after solid-liquid separation. Sufficient liquification is determined when a sample shows substantially no glucose loss after the solids are separated.
• sufficient liquification of the waste may be determined by measuring viscosity of the waste and determining a reduction of the viscosity by a predefined percentage (e.g., reduction of a least 5% of the viscosity or at least 10% reduction of the viscosity) and/or determining that viscosity reached a desired predefined value, typically measured in cps.
• a predefined percentage e.g., reduction of a least 5% of the viscosity or at least 10% reduction of the viscosity
• determining that viscosity reached a desired predefined value typically measured in cps.
• the initial viscosity of organic waste depends on its source and composition, and may vary greatly from batch to batch. Viscosity can be measured using methods known in the art using a suitable viscometer in a setup that is compatible for viscous materials.
• known methods for determining liquification of starch for example based on determining dextrose equivalents (DE) value, may be used for
• the incubation with the alpha-amylase is preferably carried out at the natural pH of the non-sterile slurry of organic waste. In some embodiments, the incubation with the alpha amylase is carried out at a pH in the range of 3.5 to 5.5, including each value within the range. In additional embodiments, the incubation with the alpha amylase is carried out at a pH in the range of 4 to 5, including each value within the range.
• Step 106 is optional and includes adjusting the temperature to a second temperature below the first temperature and between room temperature to 75°C, for example between 25°C to 65°C, typically between 50°C to 65°C (e.g., about 60°C as in the illustrated embodiment).
• the non-sterile slurry of organic waste is maintained at the second temperature for a time duration of at least 1 hour.
• the non-sterile organic waste slurry is maintained at the second temperature for a time duration in the range of 1-24 hours, including each value within the range, or for a time duration in the range of 1-18 hours or 10-18 hours, including each value within the specified ranges.
• the second temperature that is selected is preferably in the range of 50°C to 75°C, including each value within the range, for example in the range of 50°C to 65°, including each value within the range, or 60°C as in the illustrated embodiment.
• the second temperature that is selected is preferably in the range of 50°C to 75°C, including each value within the range, for example in the range of 50°C to 65°, including each value within the range, or 60°C as in the illustrated embodiment.
• step 106 is not performed and the organic waste is subjected to solid-liquid separation (see next step 108) directly after the treatment with the alphaamylase, without adjusting the temperature between the two steps.
• Step 108 includes subjecting the non-sterile slurry of organic waste to solid-liquid separation.
• Solid-liquid separation may include, for example, filtration, decantation, and the like.
• the waste material or portions thereof may be processed by a screw press, a filter press, may be centrifuged, and/or may be processed by any other liquid-solid separating method known in the art or a combination of methods.
• the liquid phase usually includes maltodextrins that were generated by the alpha-amylase as well as reducing sugars that were already present at the waste material, typically C5 and/or C6 reducing sugars.
• the liquid phase is collected for further processing.
• the solid phase may also be independently collected to be selectively treated.
• Methods for breaking-up or molding materials such as mechanical grinding, chipping, laser cutting, etc., are usually more effective when the processed material is dry and hard, as compared with a moist mixture. Therefore, the separated solid phase material may be subjected to at least one such trimming or breaking-up method after being at least partially dewatered.
• the trimming or breaking-up of the solid material may produce smaller solid particles which may be reintroduced into the liquid phase of the waste material, for further processing.
• Step 110 includes subjecting the liquid phase to sterilization to obtain a sterilized liquid phase.
• Sterilization may be performed, for example, by subjecting the liquid phase to a short incubation (typically up to 5 minutes, e.g., 1-5 minutes, and preferably up to 2- 3 minutes) at ⁇ 140°C.
• sterilization may be performed, for example, by subjecting the liquid phase to incubation of approximately 20 minutes at ⁇ 121°C.
• Steps 112 and 114 include subjecting the sterilized liquid phase to saccharification with a glucoamylase and fermentation with a lactic acid-producing microorganism.
• glucoamylase saccharification and lactic acid fermentation i.e., steps 112 and 114, may be carried out simultaneously (simultaneous saccharification and fermentation, SSF).
• glucoamylase saccharification may be carried out before lactic acid fermentation, in the same reactor in which fermentation is carried out or in a different reactor (separate hydrolysis and fermentation, SHF).
• a semiSHF process may be applied, wherein the glucoamylase is added at its optimal temperature and pH conditions and incubated for a time duration that provides partial saccharification, for example, between 0.25-5 hours, typically for 1-5 hours (including each value within the specified range), and after that the temperature and pH are adjusted to best fit the lactic acid-producing microorganism and inoculation is done.
• the glucoamylase is typically still active at the temperature that is optimal for the lactic acid-producing microorganism, and thus saccharification can continue also during fermentation.
• SSF, SHF, semi-SHF represents a separate embodiment of the present invention.
• a method for pretreating organic waste and producing lactic acid comprises adding a glucoamylase and a lactic acid-producing microorganism to the sterilized liquid phase and subjecting the sterilized liquid phase to saccharification and lactic acid fermentation, wherein the glucoamylase is added prior to or simultaneously with the lactic acid-producing microorganism, to obtain simultaneous, separate or partially-separate saccharification and lactic acid fermentation
• the glucoamylase is a thermophilic glucoamylase (e.g., a glucoamylase of A. niger) and the lactic acid-producing microorganism is a thermophilic lactic acid-producing microorganism, for example Bacillus coagulans.
• a semi-SHF process using a thermophilic glucoamylase (e.g., a glucoamylase of A. niger) and Bacillus coagulans may be carried out by adding the glucoamylase to the sterilized liquid phase and incubating for 1-5 hours at a temperature in the range of 55-65°C (e.g.
• Saccharification according to step 112 includes adding a glucoamylase to further hydrolyze maltodextrins generated by the alpha amylase to glucose.
• Saccharification generally includes adding one or more saccharide-degrading enzymes (also termed saccharifying enzymes or saccharification enzymes), typically glycolytic enzymes which hydrolyze the glycosidic bonds of the saccharides in the waste material, to release reducing sugars for fermentation.
• saccharide-degrading enzymes also termed saccharifying enzymes or saccharification enzymes
• glycolytic enzymes typically glycolytic enzymes which hydrolyze the glycosidic bonds of the saccharides in the waste material, to release reducing sugars for fermentation.
• the saccharides include bi- saccharides (di-saccharides), oligosaccharides, polysaccharides and glycoconjugates.
• Saccharide-degrading enzymes may be selected from the group consisting of glycoside hydrolases, polysaccharide lyases and carbohydrate esterases. Each possibility represents a separate embodiment.
• the saccharide-degrading enzymes may be modified enzymes (i.e., enzymes that have been modified and are different from their corresponding wild-type enzymes).
• the modification may include one or more mutations that result in improved activity of the enzyme.
• the saccharidedegrading enzymes are wild type (WT) enzymes.
• the broad group of saccharide-degrading enzymes is divided into enzyme classes and further into enzyme families according to a standard classification system (Cantarel et al. 2009 Nucleic Acids Res 37: D233-238). An informative and updated classification of such enzymes is available on the Carbohydrate- Active Enzymes (CAZy) server (www.cazy.org).
• CAZy Carbohydrate- Active Enzymes
• Alpha-amylase (may be denoted also as a -amylase), also identified as 1,4-alpha-D-glucan glucanohydrolase, glycogenase and by EC number 3.2.1.1, acts on starch, glycogen and related polysaccharides and oligosaccharides in a random manner, yielding shorter chains thereof, dextrins, and maltose, through the following biochemical process:
• the present invention also employs glucoamylases.
• Glucoamylase also identified as glucan 1,4-alpha-glucosidase and by the EC number 3.2.1.3, catalyzes hydrolysis of terminal (1 — >4)-linked a-D-glucose residues successively from non-reducing ends of the chains with release of P-D-glucose.
• saccharide-degrading enzymes are selected from amylases, cellulases and hemicellulases. Each possibility represents a separate embodiment of the present invention.
• a cellulase may be selected from, but not limited to: endo-(l ,4)- -D-glucanase, s%o- (1 ,4)-P-u-glucanase, P-glucosidases, Carboxymethylcellulase (CMCase); endoglucanase; cellobiohydrolase; avicelase, celludextrinase, cellulase A, cellulosin AP, alkali cellulase, and pancellase SS. Each possibility is a separate embodiment.
• a hemicellulase may be a xylanase.
• additional hemicellulases include arabinofuranosidases, acetyl esterases, mannanases, a-D- glucuronidases, P-xylosidases, P-mannosidases, P-glucosidases, acetyl-mannanesterases, a-galactosidases, -a-Larabinanases, and P-galactosidases.
• arabinofuranosidases include arabinofuranosidases, acetyl esterases, mannanases, a-D- glucuronidases, P-xylosidases, P-mannosidases, P-glucosidases, acetyl-mannanesterases, a-galactosidases, -a-La
• An amylase may be selected from, but not limited to: glucoamylase, a -amylase; (1,4-a-D-glucan glucan ohydrolase; glycogenase) P- Amylase; (1 ,4-a-D-glucan maltohydrolase; glycogenase; saccharogen amylase) y- Amylase; (Glucan 1 ,4-a- glucosidase; amyloglucosidase; Exo-1 ,4-a-glucosidase; lysosomal a-glucosidase; 1 ,4-a- D-glucan glucohydrolase) and pullulanase (limit dextrinase; amylopectin 6- glucanohydrolase; bacterial debranching enzyme; debranching enzyme; alpha-dextrin endo-l,6-alpha-glucosidase; R-enzyme; pullulan alpha
• saccharide-degrading enzymes are disaccharide-degrading enzymes.
• disaccharide-degrading enzymes are selected from lactases and invertases. Each possibility represents a separate embodiment of the present invention.
• Saccharide-degrading enzymes may be from a bacterial source.
• the bacterial source is a thermophilic bacterium.
• the term "thermophilic bacterium” as used herein indicates a bacterium that thrives at temperatures higher than about 45°C, preferably above 50°C.
• thermophilic bacteria according to the present invention have optimum growth temperature of between about 45 °C to about 75°C, preferably about 50-70°C.
• Non-limiting examples of thermophilic bacterial sources for saccharide-degrading enzymes include: Cellulases and hemicellulases - Clostridium sp. (e.g.
• Clostridium thermocellum Paenibacillus sp., Thermobifida fusca; Amylases - Bacillus sp. (e.g. Bacillus stearothermophilus), Geobacillus sp. (e.g. Geobacillus thermoleovorans), Chromohalobacter sp., Rhodothermus marinus.
• Amylases - Bacillus sp. e.g. Bacillus stearothermophilus
• Geobacillus sp. e.g. Geobacillus thermoleovorans
• Chromohalobacter sp. Rhodothermus marinus.
• the bacterial source of the saccharide-degrading enzymes is a mesophilic bacterium.
• mesophilic bacterium indicates a bacterium that thrives at temperatures between about 20°C and 45°C.
• Nonlimiting examples of mesophilic bacterial sources for saccharide-degrading enzymes include: Cellulases and hemicellulases - Klebsiella sp. (e.g. Klebsiella pneumonia), Cohnel sp., Streptomyces sp, Acetivibrio cellulolyticus , Ruminococcus albus', Amylases- Bacillus sp. (e.g.
• Bacillus amyloliquefaciens Bacillus subtilis, Bacillus licheniformis). Lactobacillus fermentum.
• mesophilic bacteria e.g., several Bacillus sp.
• the saccharide-degrading enzymes according to the present invention may also be from a fungal source.
• fungal sources for saccharide-degrading enzymes include: Cellulases and hemicellulases - Trichoderma reesei, Humicola insolens, Fusarium oxysporum', Amylases (e.g., glucoamylases) - Aspergillus niger Aspergillus oryzae, Penicillium fellutanum, Thermomyces lanuginosu.
• saccharide-degrading enzymes for use in accordance with the present invention can be found, for example, at the CAZy server mentioned above.
• Saccharide-degrading enzymes which are active at a temperature in the range of 45°C to 75°C (e.g., between 50-70°C), preferably with an optimal activity in the aforementioned temperature ranges including each value within the ranges, are referred to herein as thermophilic enzymes.
• Saccharide-degrading enzymes which are active at a temperature in the range of 20°C and 45°C, preferably with an optimal activity in the aforementioned temperature range including each value within the ranges, are referred to herein as mesophilic enzymes.
• the saccharifying enzymes may be added at a concentration sufficient for saccharification of 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.
• the saccharification step may be carried out for a period of time between 0.25-24 hours, typically between 1-24 hours, 1.5-24 hours, 5-24 hours, 8-24 hours, 1-15 hours or 1-10 hours, including each value within the specified ranges.
• 0.25-24 hours typically between 1-24 hours, 1.5-24 hours, 5-24 hours, 8-24 hours, 1-15 hours or 1-10 hours, including each value within the specified ranges.
• Each possibility represents a separate embodiment of the present invention.
• a combination of a variety of saccharifying enzymes may be applied, suited for saccharifying a variety of types of saccharides, e.g., cellulose, glucan, starch, and the like.
• one of the factors which controls the types and relative concentrations of enzymes used in the saccharification process may be the relative prevalence of the different types of polysaccharides in the waste material. For example, when treating a batch of waste material known to originate from crop residue, enzymes from the cellulase group may be used predominantly, to saccharify the high concentrations of cellulose.
• the saccharifying enzymes may include a substantial amount of amylase.
• other considerations may affect the types of utilized saccharifying enzymes, such as cost-effectiveness, yield, manufacturer- recommended conditions of the various enzymes, and the like.
• the pretreatment may further include maintaining the waste material at a temperature in the range of 55-65°C, and at a pH in the range of 3.5-5.5, including each value within the specified ranges.
• the waste material is maintained at a temperature in the range of 50-85°C, including each value within the specified range.
• the waste material is maintained at a temperature in the range of 55-85°C, including each value within the specified range.
• the waste material is maintained at a temperature in the range of 55-75°C, including each value within the specified range.
• the waste material is maintained at a temperature in the range of 55-70°C, including each value within the specified range.
• the temperature is in the range of 57-62°C, including each value within the specified range.
• the temperature is about 60°C. In some particular embodiments, the temperature is 60°C.
• the pH is in the range of 4-5.5, including each value within the specified range. In one embodiment, the pH is in the range of 4-5, including each value within the specified range. In another embodiment, the pH is in the range of 4.2- 4.6, including each value within the specified range.
• the waste material is typically maintained under these conditions for at least 0.5 hour, preferably for at least 1 hour. In some embodiments, the waste material is maintained under these conditions for 1-96 hours. In another embodiment, the waste material is maintained under these conditions for 1 to 10 hours, including each value within the specified range. In additional embodiments, the waste material is maintained under these conditions for 1 to 5 hours, including each value within the specified range. In another embodiment, the waste material is maintained under these conditions for 12 to 48 hours, including each value within the specified range. In additional embodiments, the waste material is maintained under these conditions forl0-20 hours, including each value within the specified range. In some embodiments, the waste material is kept under the particular temperature and pH conditions up to 4 days prior to undergoing additional treatments and/or controlled fermentation. In other embodiments, the waste material undergoes additional treatments while being maintained in the particular temperature and pH conditions, e.g., mechanical treatment and solid-liquid separation.
• Maintaining the waste material under the conditions disclosed herein is considered conducive to lactic acid production via controlled fermentation from several perspectives.
• the temperature and pH conditions disclosed herein suppress activity of endogenous microorganisms, i.e., microorganisms which are naturally found within the waste material, or which are found in the ambient surroundings and contaminates the waste material as it is transferred from one location to another.
• the endogenous microorganisms feed on the free reducing sugars in the waste material to produce a variety of products, such as D- and L-lactic acid, pyruvate, succinic acid, acetic acid, formic acid, and ethyl alcohol.
• the relatively elevated temperature in which the waste material is maintained according to the present invention promotes softening and liquification of the waste material.
• Step 114 includes controlled production of lactic acid via fermentation of reducing sugars, naturally found in the waste material or generated via enzymatic hydrolysis as described herein.
• Controlled production or "controlled fermentation” as used herein refers to lactic acid fermentation that is carried out in a fermenter under controlled conditions, for example: lactic acid fermentation under the control of one or more of the following parameters: temperature, pH, levels of nutrients, agitation rate and aeration (aerobic/anaerobic/microaerophilic conditions).
• the fermentation broth is processed to recover the fermentation product, namely, lactic acid or a salt thereof.
• Lactic acid fermentation is performed using a lactic acid-producing microorganism.
• “LA-producing microorganisms” as used herein refers to microorganisms that produce lactic acid as the major metabolic end product of carbohydrate fermentation. Currently preferred is the use of microorganisms which produce only L-lactic acid.
• the LA- producing microorganisms may naturally produce only L-lactic acid, or may be genetically modified to produce only L-lactic acid, for example by knocking out one or more enzymes involved in the synthesis of the undesired D- enantiomer.
• LA-producing microorganisms include various bacteria, including for example Lactobacillus species and Bacillus species, fungi, and yeast. Each possibility represents a separate embodiment.
• Fermentation is typically performed in the presence of an alkaline compound, such as a metal oxide, a carbonate or a hydroxide as detailed above.
• alkaline compounds include, but are not limited to, MgO, CaO, CaCCL, MgCCL, NaOH, KOH, NH4OH, Ca(0H)2, Mg(0H)2, and a mixture or combination thereof. Each possibility 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 further results in the neutralization of the L-lactic acid to a lactate salt.
• the pH in the fermenter decreases due to the production of the lactic acid, which adversely affects the productivity of the lactic acid-producing microorganism.
• bases such as magnesium -, sodium -, potassium -, or calcium-hydroxide adjusts the pH by neutralizing the lactic acid thereby resulting in the formation of a lactate salt.
• the fermenting is carried out under anaerobic or microaerophilic conditions, using batch, fed-batch, continuous or semi-continuous fermentation.
• anaerobic or microaerophilic conditions using batch, fed-batch, continuous or semi-continuous fermentation.
• the carbon substrates and other components are loaded into the reactor, and when the fermentation is completed, the product is collected. Except for the alkaline compound discussed above for pH control, other ingredients are not added to the reaction before it is completed.
• the inoculum size is typically about 5-10% of the liquid volume in the reactor.
• the fermentation is kept at substantially constant temperature and pH, where the pH is maintained by adding the alkaline compound.
• the substrate is fed continuously or sequentially to the reactor without the removal of fermentation broth (i.e., the product(s) remain in the reactor until the end of the run).
• Common feeding methods include intermittent, constant, pulse-feeding, and exponential feeding. Each possibility represents a separate embodiment.
• the substrate is added to the reactor continuously at a fixed rate, and the fermentation products are taken out continuously.
• Lactic acid fermentation is typically carried out for about 1-4 days or any amount therebetween, for example, 1-2 days, or 2-4 days, or 3-4 days, including each value within the specified ranges.
• the waste material subjected to lactic acid production process 100 is often of a large volume and includes a non-uniform mixture of a variety of liquids and solids. Therefore, potentially beneficial compounds in the waste material are not always accessible to treatment reagents, e.g., reactive compounds, enzymes, and the like, and so do not contribute to lactic acid production.
• the waste material may be subjected to mechanical treatment for increasing the surface area and/or for improving the turnover of the organic components and the active ingredients within the waste material.
• the mechanical treatment may include, for example, grinding, chipping, shredding, mincing and/or milling.
• the mechanical treatment may also include stirring the waste material or a portion thereof.
• the stirring is intended to thoroughly mix the waste material, allowing interaction of all parts of the waste material with the treatment reagents.
• the mixing may also enhance the effectivity of processes such as heating, aerating and/or depressurizing applied to the waste material.
• Stirring may be at a rate in the range of 10 to 1000 RPM, e.g., in the range of 30-100 RPM, 50-200 RPM, 150-500 RPM, 300-700 RPM, or 500- 1000 RPM, including any value within the specified ranges.
• the rate of stirring may be substantially fixed throughout process 100, or may be adjusted, and/or activated and deactivated, at different steps.
• the rate and/or force of the stirring may be highest when the waste material is at a relatively raw state, and may be reduced as the process advances, in correlation with liquification of the waste material.
• the stirring may begin in a mild manner, e.g., at a relatively low stirring rate, and may intensify as treatment agents are added into the waste material, so as to accelerate the treatment procedures.
• the rate and power of the stirring may be increased in accord, or may optionally be reverse correlated, such that when the rate is increased the power is reduced.
• Each possibility represents a separate embodiment. Any other combination of stirring parameters during the various steps may be utilized.
• Some variables which may determine the selected stirring parameters include, but are not limited to, gas-liquid mass transfer, feed distribution, local oxygen concentration, shear rate distribution, and local mixing intensity.
• any embodiment of mechanical treatment may be performed at any one or more of the various stages of process 100, or throughout the entire process.
• the waste material may undergo grinding, chipping, etc., proximately to when the waste material is provided.
• the waste material may be mechanically treated to enlarge the surface area of the polysaccharide compounds prior to commencement of saccharification, and/or simultaneously therewith.
• the waste material may be crudely ground, chipped, shredded, milled, or otherwise broken up, at an early stage of the pre-treatment, and more finely ground, chipped, etc., at a later stage of the process.
• Mechanical treatment in the form of stirring may also be applied continuously, or at selected stages of process 100.
• the term “about” refers to ⁇ 10%, for example +/-5%, +/-1%, and +/-0.1% from the specified value.
• a non- sterile slurry of mixed food waste collected from supermarket logistic 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 of inorganic solid components (plastics, etc.) and griding to form the slurry.
• the glucose potential of the slurry namely, the concentration of both free glucose and glucose that is in a non-reducing state (polysaccharides), was 82 g/1.
• the available glucose concentration namely, the concentration of free glucose available for fermentation, was 2 g/1.
• the slurry was heated to 80°C and an alpha amylase that is active at 80°C was added (Bacillus licheniformis, 1 gr per 1 Kg of waste).
• the slurry with the added alpha amylase was then incubated for 2 hours with stirring at the waste's native pH of 4.2. During this incubation time the liquification of the waste material and reduction of viscosity was visually observed.
• the temperature was decreased to 60°C and the slurry was incubated overnight (12-16 hours). Following the overnight incubation, the slurry was subjected to solid-liquid separation (centrifugation at 4,000g, 5 min). The supernatant was collected and subjected to sterilization (120°C for 20min).
• a glucoamylase (GA) was added to the sterilized supernatant (Aspergillus niger. 0.5 gr per 1 Kg of waste), and the sterilized supernatant with the added GA was incubated at 60°C pH 4.2 (native pH of the waste) for 2 h with stirring. Following this incubation, the temperature was adjusted to 52°C, the pH was adjusted to 6.2, and B. coagulans was inoculated to a final concentration of lxlO A 6 cells/ml. Lactic acid fermentation was carried out for 16 hours. All the glucose in the waste was depleted and the fermentation ended with 82 gr/L lactate.
• GA glucoamylase
• the alpha amylase works efficiently to break down polysaccharides in the organic waste into shorter chains that are preserved in the liquid phase and not lost upon solid-liquid separation.
• the above exemplified pretreatment inhibits endogenous microorganisms, liquifies the slurry to assist in effective mixing in the reactor, reduces formation of glucose degradation products, and allows separating solids (if desired) without losing glucose, thus continuing to sterilization, further saccharification and fermentation with a liquid phase that is more simple to handle.
• the results show that pretreatment according to the present invention preserves the glucose potential of the organic waste and minimizes or even completely prevents glucose loss during the pretreatment.
• Example 1 Samples of mixed food waste as described in Example 1 were subjected to separation of inorganic solid components (glass, plastics, etc.) and griding to form slurries.
• the slurries were subjected to saccharification with a glucoamylase (Aspergillus niger, 0.5 gr per 1 Kg of waste) at 52-60°C, pH ⁇ 4.7 (native pH of the waste).
• a glucoamylase Aspergillus niger, 0.5 gr per 1 Kg of waste
• pH ⁇ 4.7 native pH of the waste.
• the saccharified slurries were subjected to sterilization (120°C for 20min).
• Glucose concentration (g/L) was measured before and after sterilization. The results are summarized in Table 1.
• Complex organic waste such as mixed food waste, typically includes various solids, making it difficult to effectively sterilize the waste on an industrial scale.
• To effectively sterilize mixed food waste containing solids long and extensive sterilization is required.
• an excessively long sterilization process was performed to mimic the procedure required for large scale effective sterilization of mixed food waste containing solids.
• a slurry of mixed food waste as described in Example 1 was saccharified with a glucoamylase at 60°C, pH ⁇ 4.7 (native pH of the waste) and subsequently subjected to sterilization according to the procedure specified in Table 2, which included 180 minutes at 90°C, gradual increase to 121°C (210 minutes until reaching 121°C), 3 minutes at 121°C and subsequently gradual cool down. Overall, the waste was exposed to temperatures above 100°C for almost 5 hours, of which approximately 2 hours at temperatures above 110°C. Following sterilization and cooling the waste was inoculated with a lactic acid producing microorganism to initiate lactic acid production from the waste.
• glucose concentration decreased from over lOOg/L to 77 g/L, and fructose concentration doubled.
• the increase in fructose indicates glucose isomerization to fructose.
• HMF concentration increased significantly, from 20 mg/L to 435 mg/L, which indicates formation of inhibitory early Maillard reaction products (MRP).
• the sterilized food waste was then inoculated with a lactic acid-producing microorganism (“First inoculation”) (B. coagulans, lxlO A 6 CFU/ml) and incubated at 52°C, pH 6.2. Approximately 80% of the live count decreased between inoculation and 15 hours later, indicating massive mortality and/or strong growth inhibition likely due to growth inhibitors formed during the long sterilization.
• Alpha-amylase breaks down polysaccharides in organic waste into shorter chains that are preserved in the liquid phase upon solid-liquid separation
• Example 1 A non- sterile slurry of mixed food waste as described in Example 1 was provided and divided into the following treatments:
• the slurry was heated to 80°C (Treatment I) or 70°C (Treatment II) and a high- temperature alpha- amylase was added (Bacillus licheniformis, 1 gr per 1 Kg of waste).
• the slurry with the added alpha-amylase was incubated for 2 hours with stirring at the waste's native pH ( ⁇ 4.7).
• the slurry was subjected to overnight saccharification with a glucoamylase (Aspergillus niger, 0.5 gr per 1 Kg of waste) at 60°C, and then to solidliquid separation (centrifugation at 4000g, 5 min).
• the slurry was heated to 80°C (Treatment III) or 70°C (Treatment IV) and the high- temperature alpha-amylase was added.
• the slurry with the added alpha-amylase was incubated for 2 hours with stirring at the waste's native pH ( ⁇ 4.7).
• the slurry was first subjected to solid-liquid separation (centrifugation at 4000g, 5 min
• Example 2 A non-sterile slurry of mixed food waste as described in Example 1 was provided and divided into the following treatments:
• the slurry was heated to 80°C and a high-temperature alpha-amylase was added (Bacillus licheniformis, 1 gr per 1 Kg of waste).
• the slurry with the added alpha-amylase was incubated for 2 hours with stirring at the waste's native pH ( ⁇ 4.7).
• the slurry was subjected to overnight saccharification with a glucoamylase (Aspergillus niger, 0.5 gr per 1 Kg of waste) at 60°C and subsequently to solid-liquid separation (4000g, 5 min) (Treatment I), or first subjected to solid-liquid separation and after that the SN and pellet fractions were each subjected to overnight saccharification with a glucoamylase at 60°C (Treatment II).
• glucoamylase Aspergillus niger, 0.5 gr per 1 Kg of waste
• the slurry was heated to 80°C and incubated for 2 hours without alpha- amylase addition. Next, the slurry was subjected to overnight saccharification with a glucoamylase (Aspergillus niger, 0.5 gr per 1 Kg of waste) at 60°C and subsequently to solid-liquid separation (4000g, 5 min) (Treatment III), or first subjected to solid-liquid separation and after that the SN and pellet fraction were each subjected to overnight saccharification with a glucoamylase at 60°C (Treatment IV).
• glucoamylase Aspergillus niger, 0.5 gr per 1 Kg of waste
• a non-sterile slurry of mixed food waste as described in Example 1 was provided.
• the slurry contained 4.92 g/L lactate, 11.42 g/L glucose and 16.07 g/L fructose.
• the slurry was heated to 80°C, and an alpha-amylase that is active at 80°C was added (Bacillus licheniformis, 1 gr per 1 Kg of waste).
• the slurry with the added alpha-amylase was then incubated for 2 hours with stirring at the waste's native pH of 4.5. During this incubation time, the liquification of the waste material and reduction of viscosity was visually observed.
• the slurry was subjected to solid-liquid separation (centrifugation at 4,000g, 5 min). The supernatant was collected, loaded to a 15L fermenter and subjected to sterilization (120°C for 20min).
• a glucoamylase was added to the sterilized supernatant (Aspergillus niger, 0.5 gr per 1 Kg of waste), and the sterilized supernatant with the added glucoamylase was incubated at 60°C pH 4.5 (native pH of the waste) for 12 hours with stirring. At the end of the saccharification step, glucose and fructose concentrations were 91 and 21 g/L, respectively. The temperature was then adjusted to 52°C, the pH was adjusted to 6.2, and B. coagulans was inoculated to a final concentration of 10 A 6 cells/ml.
• Lactate production started 6 hours after inoculation, and glucose was depleted entirely 15 hours after inoculation. Lactate final titer at the end of fermentation was 102.44 g/L and the total fermentation yield was 93.44%.
• a pretreatment method according to the present invention enables efficient extraction of fermentable sugars from complex and viscous waste streams, such as mixed food waste.
• the pretreatment method according to the present invention facilitates a highly productive lactic acid fermentation process, with a high yield and high concentration of lactate at a relatively short fermentation time.

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