Lactic acid fermentation processes employing dried compositions of spores

Abstract

Systems and methods for recycling of organic waste to produce lactic acid by fermentation are provided, which utilize dried compositions of spores of a spore-forming lactic acid-producing bacterium (e.g., Bacillus coagulans). The dried spores are first germinated in a small-scale seed fermenter and grown in the seed fermenter to reach defined criteria, after which they are inoculated into a large-scale lactic acid production fermenter. The provided systems and methods avoid the need of lengthy and laborious seed trains yet provide high titers of live bacteria within a short time, that produce lactic acid at high yields with a shortened lag phase and a shortened fermentation time.

Description

[0001] LACTIC ACID FERMENTATION PROCESSES EMPLOYING DRIED COMPOSITIONS OF SPORES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to industrial lactic acid production by fermentation of organic waste.

[0004] BACKGROUND OF THE INVENTION

[0005] 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. PLA is the most widely used plastic filament material in 3D printing.

[0006] Production of 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 of PLA. 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. For production of PLA, the lactic acid generated during the fermentation is separated from the fermentation broth and purified by various downstream processes, and the purified lactic acid is then subjected to polymerization.

[0007] Lactic acid has a chiral carbon atom and therefore exists in two enantiomeric forms, D- and L-lactic acid. In order to generate PLA that is suitable for industrial applications, the polymerization process should utilize only one enantiomer. Presence of impurities or a racemic mixture of D- and L-lactic acid results in a polymer having undesired characteristics such as low crystallinity and low melting temperature. Thus, lactic acid bacteria that produce only L-lactate enantiomer or only D-lactate enantiomer are typically used. In currently available commercial processes, 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.

[0008] An additional source of carbohydrates for lactic acid fermentation that has been proposed is complex organic waste, such as mixed food waste from municipal, industrial and commercial origin. Such organic waste is advantageous as it is readily available and less expensive compared to other carbohydrate sources for lactic acid fermentation. However, the conversion of complex organic wastes to useful fermentation products such as lactic acid on an industrial scale faces numerous technical challenges and requires precise control over operational conditions, including pretreatment, pH, temperature, microbes and more. Improvements are needed in order to make the process economically feasible on an industrial scale.

[0009] Rosenberg et al. (2005 Biotechnology Leters, 2 . 1943-1947 report the immobilization of Bacillus coagulans spores in polyvinylalcohol (PVA) hydrogel, lensshaped capsules known as LentiKats®, and use of the immobilized spores in a lactic acid production from glucose.

[0010] WO 2008 / 043368 discloses a method of producing endospores of thermophilic sporogenic microbial strains, for example, Bacillus coagulans SIM7 DSM 14043, and the use thereof for inoculation of fermentation processes.

[0011] WO 2017 / 068012 discloses a method for fermenting a microorganism for producing a protein product where seed tanks are omitted with the result that the process is much shorter providing additional flexibility in operating the fermentation plant.

[0012] WO 2019 / 222168 discloses methods of producing and preserving stable, standardized reference cultures for use in inoculating fermentation reactors used in research labs and other commercial applications.

[0013] WO 2021 / 191901, assigned to the Applicant of the present invention, discloses systems and methods for recycling of organic waste to produce lactic acid by fermentation, which utilize dried or partially-dried compositions of spores of the lactic acid-producing bacterium Bacillus coagulans.

[0014] WO 2023 / 053121, assigned to the Applicant of the present invention, discloses methods and systems for pretreatment of organic waste prior to large-scale production of lactic acid from the organic waste, which employ dried or partially-dried compositions of Bacillus coagulans spores.

[0015] There remains a need to improve the production of lactic acid from organic waste on an industrial scale, in order to make the process more economically feasible. It would be highly advantageous to have systems and methods that simplify the process, reduce costs and improve the overall yield.

[0016] SUMMARY OF THE INVENTION

[0017] The present invention provides systems and methods for lactic acid production on an industrial scale using fermentation of organic waste. In particular, the present invention is directed to an improved lactic acid production line employing dried compositions of spores of a spore-forming lactic-acid producing bacterium (e.g. Bacillus coagulans), which avoids the need of lengthy and laborious seed trains yet provides high titers of vegetative bacteria within a short time, that produce lactic acid at high yields with a shortened lag phase and a shortened fermentation time (namely, shortened time for reaching a plateau such that substantially no further lactic acid is produced).

[0018] As disclosed herein, dried spores of a spore-forming lactic acid-producing bacterium are first germinated in a small-scale seed fermenter and grown in the seed fermenter to reach defined criteria, after which they are inoculated into a large-scale lactic acid production fermenter. In particular, the spores are germinated and grown in the seed fermenter to provide at least l*10A5 CFU / ml of vegetative cells in the production fermenter upon inoculation of the production fermenter, wherein the volume of the inoculum corresponds to 0.5-30% of the volume of the medium in the production fermenter. Typically, the incubation time in the seed fermenter is in the range of 2-12 hours, after which the production fermenter is inoculated and lactic acid fermentation is carried out. Such an inoculum preparation process was surprisingly found to provide improved results in terms of lag phase and total fermentation time compared to seed trains and compared to direct seeding of spores into the production fermenter. In addition, the use of just a single seed fermenter, that is operated for only a few hours prior to the main production fermenter, is highly advantageous and cost effective over complicated seed trains.

[0019] The present invention advantageously allows simple integration of lactic acid production into organic waste management facilities, for on-site production of lactic acid from the organic waste. Conventionally, industrial fermentation processes involve seed lines, also termed seed trains, where banked cell samples are expanded to finally provide sufficient biomass to inoculate the main fermenter. A conventional seed train process begins with thawing of a cryopreserved cell bank vial, followed by multiple successive propagations into progressively larger culture vessels. When culture volume and cell density meet predetermined criteria, the culture is transferred to a production bioreactor in which cells continue to grow and divide and produce the desired product. Conventional seed train processes are time-consuming due to the number of culturing steps, and due to the low cell numbers in the cryopreserved cell-bank vial. In addition, sterility is required for inoculating each culture vessel, including the main production fermenter.

[0020] The present invention avoids the need for seed lines at the production site and provides simple means for sterile inoculation, thus saving both capital expenditure (CAPEX) and operational expenditure (OPEX). Compositions of dried spores can be easily transported to organic waste management sites, stored and removed from storage upon need. Advantageously, the dried compositions of spores do not require cooling and sustain various storage conditions for prolonged periods of time.

[0021] The utilization of organic waste as a substrate for fermentation as described herein is highly advantageous compared to previously described lactic acid production processes which utilize source materials that are of high value as human food.

[0022] According to one aspect, the present invention provides a method for producing lactic acid or a salt thereof from organic waste, the method comprising:

[0023] (i) providing a dried composition of spores of a spore-forming lactic acid-producing bacterium, optionally suspended in an aqueous medium to obtain a suspension of spores;

[0024] (ii) providing a first fermenter containing therein a first fermentation medium suitable for growth of the spore-forming lactic acid-producing bacterium, and a second fermenter containing therein a second fermentation medium comprising pretreated organic waste;

[0025] (iii) inoculating the dried composition or suspension of spores into the first fermentation medium to obtain at least IxlO5CFU / ml upon inoculation, and incubating to induce spore germination and obtain vegetative cells of the spore-forming lactic acidproducing bacterium; (iv) inoculating vegetative cells obtained in step (iii) into the second fermentation medium, wherein the volume of the inoculum corresponds to 0.5-30% of the volume of the second fermentation medium, and wherein the incubating in step (iii) is carried out to obtain an amount of vegetative cells that provides at least IxlO5CFU / ml in the second fermentation medium after inoculation;

[0026] (v) fermenting the pretreated organic waste to produce lactic acid; and

[0027] (vi) recovering the produced lactic acid or a salt thereof.

[0028] In some embodiments, the volume of the first fermentation medium corresponds to 0.5-30% of the volume of the second fermentation medium, and step (iv) comprises inoculating the entire first fermentation medium with vegetative cells obtained in step (iii) into the second fermentation medium.

[0029] In some embodiments, the volume of the inoculum corresponds to 1-10% of the volume of the second fermentation medium. In additional embodiments, the volume of the inoculum corresponds to 1-5% of the volume of the second fermentation medium.

[0030] In some embodiments, the dried composition of spores is characterized by a spore concentration in the range of l*10A8-l*10 12 CFU / gr.

[0031] In some embodiments, the first fermentation medium comprises pretreated organic waste that is the same as the pretreated organic waste in the second fermentation medium.

[0032] In some embodiments, the organic waste is selected from the group consisting of food waste, municipal waste, agricultural waste, plant material and a mixture or combination thereof. In some particular embodiments, the organic waste is food waste.

[0033] In some embodiments, the incubating in step (iii) is carried out for a period of time in the range of 2-12 hours.

[0034] In some embodiments, the spore-forming lactic acid-producing bacterium is Bacillus coagulans.

[0035] Other objects, features and advantages of the present invention will become clear from the following description and examples.

[0036] BRIEF DESCRIPTION OF THE FIGURES

[0037] Figure 1. Schematic illustration of industrial lactic acid production using seed train, spore inoculum or an inoculum of germinated spores according to the present invention.

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention is directed to industrial fermentation processes for production of lactic acid from organic waste, in which dried compositions of spores of a sporeforming lactic acid-producing bacterium are used. As disclosed herein, the spores are first germinated in a small-scale seed fermenter and grown in the seed fermenter to provide at least l*10A5 CFU / ml of vegetative cells (in some preferred embodiments, at logarithmic growth) in a large-scale production fermenter upon inoculation of the production fermenter, wherein the volume of the inoculum corresponds to 0.5-30% of the volume of the medium in the production fermenter. It was surprisingly found that the initial germination and growth in a seed fermenter provides a high titer of live bacteria within a short time (several hours), which produce lactic acid at high yields with a shortened lag phase and a shortened fermentation time (namely, shortened time for reaching a plateau such that substantially no further lactic acid is produced). The use of just a single seed fermenter, that is operated for only a few hours prior to the main production fermenter, is highly advantageous and cost effective over complicated seed trains, and surprisingly provides improved results in terms of lag phase and total fermentation time.

[0040] Referring now to the drawings, Figure 1 shows a comparison of industrial fermentation processes employing a seed train, a spore inoculum or an inoculum of germinated spores according to the present invention. The processes will be described below with respect to exemplary embodiments in which a production fermenter containing 8000 -10,000 L fermentation medium is inoculated.

[0041] Seed train:

[0042] A seed train generally involves thawing of a cryopreserved vial ("Cryovial" in Figure 1) containing vegetative cells of the lactic-acid producing microorganism followed by a plurality of successive propagations into progressively larger culture vessels (for example, two successive propagations, identified as "Seed 1", "Seed 2" in Figure 1). When the culture volume and cell density meet predetermined criteria, the culture is transferred to the production fermenter ("Fermenter" in Figure 1) in which cells continue to grow and divide and produce the desired product, namely, lactic acid.

[0043] Typically, a plurality of cryovials are thawed and seeded into lab scale shake flasks. The number of cryovials to be thawed and seeded into lab scale shake flasks is determined, inter alia, according to the concentration of cells in each cryovial, the volume of the subsequent culture vessel and the desired concentration of cells in the subsequent culture vessel following inoculation. For example, five frozen cryovials, each containing 0.5ml of frozen cells of a lactic acid-producing bacterium at a concentration of ~ l*10 8 CFU / ml may be thawed. The thawed bacteria from each cryovial may be added to a IL shake flask containing 0.5L of a standard growth medium (e.g., LB medium) - total of five shake flasks, providing 2.5L of medium with bacteria, identified as "Seed 1 " in Figure 1. Seed 1 is incubated at a growth temperature of the lactic acid-producing bacterium until a desired concentration of cells is obtained, for example at least l*10A6 CFU / ml, or even at least 5*10A6 CFU / ml (typically ~ l*10A8 CFU / ml) at the end of Seed 1. Typically, the incubation time for Seed 1 is in the range of 15-24 hours.

[0044] Next, the medium with bacteria obtained at the end of Seed 1 (e.g., 2.5L medium with bacteria) is transferred into a larger fermenter - "Seed 2". Seed 2 fermenter typically contains a standard growth medium (e.g., LB medium). For a Seed 1 volume of 2.5L, the volume of the medium in the Seed 2 fermenter may range from 500-1000L, for example, 750L.

[0045] Seed 2 is incubated at a growth temperature of the lactic acid-producing bacterium until a desired concentration of cells is obtained, for example at least l*10A6 CFU / ml, or even at least 5*10A6 CFU / ml, typically at least l*10A8 CFU / ml, or even at least 2*10A8 CFU / ml at the end of Seed 2. Typically, the incubation time for Seed 2 is in the range of 15-24 hours.

[0046] In the illustrated example, the medium with bacteria obtained at the end of Seed 2 (e.g. 750L medium with bacteria) is transferred into the production fermenter ("Fermenter" in Figure 1). For a Seed 2 volume in the range of 500-1000L, the volume of the medium in the production fermenter may range from 8000-10,000L, for example, 8000L. The production fermenter contains the substrate for fermentation, namely pretreated organic waste. The concentration of cells after inoculation is typically at least 10A6 CFU / ml. Lactic acid fermentation is then carried out at a growth temperature of the lactic acid-producing bacterium with pH adjustment until reaching a plateau such that substantially no further lactic acid is produced. The inoculum preparation using a seed train takes about 1-2 days. Spore inoculum:

[0047] A spore inoculum can start with direct inoculation of a spore powder, or as in the illustrated example with a spore powder that is suspended in a suspension medium (e.g., water) at room temperature. The suspension ("Seed 1") is used to directly inoculate a production fermenter containing pretreated organic waste. The suspension may be inoculated into the production fermenter immediately or after a short mixing at room temperature, for example, for up to 30 minutes. The amount of spore powder depends, inter alia, on the concentration of spores in the powder, the volume of the medium in the production fermenter and the desired concentration of spores in the production fermenter upon inoculation. Exemplary concentrations of spores in the powder are typically in the range of l*10A8-l*10A12 CFU / gr, for example about 5*10A10 CFU / gr.

[0048] About 50-300gr spore powder may be suspended in 0.2- IE suspension medium and used to inoculate a production fermenter containing 8000 -10,000 E pretreated organic waste, to provide at least l*10A5 CFU / ml after inoculation, or even at least l*10A6 CFU / ml. The spores germinate in the production fermenter, and the vegetative cells that germinate from the spores ferment the pretreated organic waste to produce lactic acid.

[0049] As used herein, the term "germination" refers to the growth of vegetative cells from dormant bacterial spores. The terms "vegetative bacterial cells" or "vegetative cells" refers to bacterial cells that are actively growing, exhibiting metabolic activity, and dividing.

[0050] The inoculum preparation is very short, however as demonstrated in the Examples section below, a spore inoculum is characterized by longer lag phase and longer total fermentation time compared to an inoculum of germinated spores according to the present invention, which will now be described in more detail.

[0051] Inoculum of germinated spores:

[0052] An inoculum of germinated spores according to the present invention starts with a spore powder that is inoculated into a seed fermenter ("Seed 1" in Figure 1) in which the spores germinate and grow to reach defined criteria as described herein, after which they are inoculated into the production fermenter. The powder may be inoculated directly into the seed fermenter or may be suspended first in a suspension medium (e.g., water) to form a spore suspension that is used to inoculate the seed fermenter. Each possibility represents a separate embodiment of the present invention. The seed fermenter may contain a standard / synthetic growth medium of the spore-forming lactic acid-producing bacterium to be used (e.g., LB or other media with a known / defined composition that are suitable for growth of the spore-forming lactic acid-producing bacterium to be used) or may contain pretreated organic waste - preferably same pretreated organic waste as in the production fermenter. Each possibility represents a separate embodiment of the present invention.

[0053] As used herein, a "growth medium of a spore-forming lactic acid-producing bacterium", or a "growth medium suitable for growth of a spore-forming lactic acidproducing bacterium" refers to a liquid composition that provides the necessary nutrients and conditions to support the growth and / or fermentation of such bacteria for the purpose of lactic acid production. This includes, but is not limited to, commercially available culture media commonly used for bacterial cultivation, such as Luria-Bertani (LB), nutrient broth (NB), tryptic soy broth (TSB), yeast extract-peptone-dextrose (YPD), or other suitable media providing essential nutrients for bacterial growth. It also encompasses custom-prepared media formulated to contain appropriate carbohydrate sources and essential nutrients required for bacterial growth. The formulation and selection of such growth media are within the skills of a person skilled in the art, familiar with the physiological requirements of the relevant bacteria.

[0054] A spore-forming lactic acid-producing bacterium may be selected from the group consisting of: Bacillus coagulans, Bacillus stearothermophilus, Bacillus licheniformis, Bacillus subtilis, Bacillus laevolacticus, Bacillus racemilacticus, Bacillus thermoamylovorans, Sporolactobacillus, Sporolactobacillus shoreicorticis, Sporolactobacillus vineae, Sporolactobacillus nakayamae, Terrilactibacillus laevilacticus, and Terrilactibacillus tamarindi. Each possibility represents a separate embodiment of the present invention.

[0055] In some particular embodiments, the spore-forming lactic acid-producing bacterium is Bacillus coagulans.

[0056] The seed fermenter is typically of smaller scale (smaller volume) compared to the production fermenter. In some embodiments, the medium in the seed fermenter is used in its entirety to inoculate the production fermenter. In other embodiments, the medium in a single seed fermenter is used to inoculate a plurality of production fermenters. The size (volume) of the seed fermenter is selected, inter alia, according to the intended use (namely, inoculation of a single or a plurality of production fermenter(s)), the size (volume) of the production fermenter and the inoculum's volume as described herein. As described herein, the inoculum's volume corresponds to 0.5-30% of the volume of the medium in the production fermenter, for example to 1-25%, 1-20%, 1-15%, 1-10%, 1- 5%, 1-4%, 2-4%, or 1.5-3% of the volume of the medium in the production fermenter, with each possibility representing a separate embodiment, and including each value within the specified ranges.

[0057] The seed fermenter is incubated at a growth temperature of the spore-forming lactic acid-producing bacterium. The incubation time in the seed fermenter depends, inter alia, on the concentration of spores in the powder and the desired concentration of vegetative cells in the production fermenter upon inoculation. As described herein, the seed fermenter is incubated to obtain an amount of vegetative cells that provides at least IxlO5CFU / ml in the production fermenter after inoculation, preferably at least IxlO6CFU / ml, at least IxlO7CFU / ml, or between IxlO5to IxlO8CFU / ml in the production fermenter after inoculation. Each possibility represents a separate embodiment of the present invention.

[0058] As used herein, "after inoculation" or "upon inoculation" are interchangeable and refer to the CFU / ml immediately after the spores / bacteria are added into the fermenter, before further cultivation / fermentation are carried out.

[0059] Colony Forming Units (CFU) / ml may be determined by methods known in the art, for example using live counting from plates, also referred to as plate counting. To determine the concentration of viable microorganisms expressed as CFU / ml a sample is subjected to serial dilution in a sterile diluent, and a defined aliquot of one or more selected dilutions is plated onto a solid growth medium using a spread or pour plate technique. The plates are incubated under controlled conditions sufficient to permit colony development, after which colonies are enumerated on plates exhibiting counts within a predefined range that minimizes statistical error (typically 25-250 or 30-300 colonies per plate). The CFU / mL value is calculated according to the formula CFU / mL = (N x DF) / V, where N is the colony count (or mean of replicate counts), DF is the dilution factor corresponding to the plated dilution, and V is the plated volume in milliliters. This method provides a direct measure of viable cells capable of forming colonies under the specified conditions and may optionally include replicate plating, automated counting, and statistical averaging to enhance accuracy and reproducibility.

[0060] Alternative or additional methods and parameters can be used to evaluate the state of the bacteria that germinated from the spores and determine the correct timing for transferring the culture from the seed fermenter to the production fermenter, such as measuring base consumption rate and ODeoo. In some preferred embodiments, the culture is transferred when the cells are at the logarithmic growth phase.

[0061] "Base consumption rate” refers to the amount of a neutralizing agent (base) required to maintain the pH of a culture medium within a predetermined range during fermentation. For the purpose of cultivation in a seed fermenter according to the present invention, the term refers to the amount of neutralizing agent required to maintain the pH in the seed fermenter within a predetermined range. The bacteria that germinate from the spores in the seed fermenter produce lactic acid. The base consumption rate reflects the extent of acid generation and, therefore, serves as an indirect indicator of the metabolic activity of the culture. In some embodiments, an increase in base consumption rate corresponds to heightened metabolic activity and may indicate that the culture is in the logarithmic (exponential) growth phase, whereas a decrease or stabilization in the rate may suggest transition to the stationary phase. The base consumption rate may be determined by monitoring the quantity of base added to the culture medium over a defined time period while maintaining the pH at a setpoint. The base consumption rate may be expressed as a quantity of base (e.g., grams or moles) added per unit time, per unit volume of culture, or per unit of lactic acid produced.

[0062] “ODeoo” refers to the optical density of a bacterial culture measured at a wavelength of 600 nanometers, which provides an estimate of the cell concentration in the culture. ODeoo is commonly used as an indirect measure of biomass and growth phase. In some embodiments, an increase in ODeoo corresponds to active cell proliferation and may indicate that the culture is in the logarithmic growth phase, whereas stabilization or a plateau in ODeoo may indicate entry into the stationary phase.

[0063] ODeoo may be determined by withdrawing a sample from the fermentation medium and measuring its absorbance at 600 nm using a spectrophotometer or similar optical device. The measurement is typically performed on diluted samples to ensure readings fall within the linear range of the instrument. ODeoo values can be correlated to cell concentration (e.g., colony-forming units per mL or dry cell weight) using calibration curves prepared for the specific bacterial strain.

[0064] In some embodiments, the timing for transferring the culture from the seed fermenter to the production fermenter is determined based on at least one parameter selected from the group consisting of: target CFU / ml in the production fermenter upon inoculation of the production fermenter, base consumption rate in the seed fermenter prior to the transfer, and ODeoo in the seed fermenter prior to the transfer. Each parameter and each combination thereof represents a separate embodiment of the present invention.

[0065] In some embodiments, the target CFU / ml in the production fermenter upon inoculation is at least IxlO5CFU / ml, at least IxlO6CFU / ml, at least IxlO7CFU / ml or between IxlO5to IxlO8CFU / ml. Each possibility represents a separate embodiment of the present invention, including each value within the specified range.

[0066] In some embodiments, the base consumption rate in the seed fermenter prior to the transfer is in the range of 1.5-60 mmole E'1 11- 1(millimoles per liter per hour), including each value within the range, each possibility representing a separate embodiment of the present invention.

[0067] In some embodiments, the ODeoo in the seed fermenter prior to the transfer is in the range of 0.1-1.7 including each value within the range, each possibility representing a separate embodiment of the present invention. In additional embodiments, the ODeoo in the seed fermenter prior to the transfer is in the range of 0.1-5.0, including each value within the range, each possibility representing a separate embodiment of the present invention.

[0068] The amount of spore powder depends, inter alia, on the concentration of spores in the powder, the volume of the media in the seed and production fermenters, and the desired concentration of spores in the seed and production fermenters upon inoculation. Exemplary concentrations of spores in the powder are typically in the range of 10A8- 10 12 CFU / gr, including each value within the range, for example about 5*10 10 CFU / gr. In some embodiments, about 50-300gr spore powder may be used to inoculate a seed fermenter containing 100-300 L medium, to provide at least l*10A5 CFU / ml in the seed fermenter after inoculation. In additional embodiments, about 5-50 gr spore powder may be used to inoculate a seed fermenter containing 100-300 L medium, to provide at least l*10A5 CFU / ml in the seed fermenter after inoculation. In some embodiments, the seed fermenter is inoculated to provide at least 1* 10A6 CFU / ml in the seed fermenter after inoculation. In some embodiments, the seed fermenter is inoculated to provide at least l*10A7 CFU / ml in the seed fermenter after inoculation. In some embodiments, the seed fermenter is inoculated to provide at least l*10A8 CFU / ml in the seed fermenter after inoculation. Each possibility represents a separate embodiment of the present invention. The seed fermenter may be incubated for 1-15 hours, for example, 2-12 hours, 5-15 hours or 5-10 hours, e.g., 5, 6, 7, 8, 9 or 10 hours, including each value within the range, with each possibility representing a separate embodiment, to provide at least IxlO5CFU / ml in a production fermenter containing 8000 -10,000 L after inoculation, or even at least IxlO6CFU / ml in the production fermenter after inoculation, or at least IxlO7CFU / ml in the production fermenter after inoculation. Each possibility represents a separate embodiment of the present invention. In some embodiments, the CFU / ml in seed fermenter prior to transfer to the production fermenter is in the range of lxl0A8 - 2xlOA9, including each value within the range.

[0069] It was surprisingly found that such an inoculum preparation process significantly shortens the time needed for the bacteria to begin lactic acid production - over 40% reduction in lag time and in some embodiments even over 50% reduction in lag time - and also significantly shortens the time needed to complete the fermentation. In addition, altogether, the inoculum preparation takes only several hours and requires only a single seed fermenter prior to inoculation into the production fermenter. Thus, the use of an inoculum of germinated spores according to the present invention reduces operation costs and improves overall yield of lactic acid production, as more lactic acid can be produced per a given period of time.

[0070] The growth conditions in the seed and production fermenters are selected and adjusted according to the selected spore-forming lactic acid-producing bacterium. In some embodiments, incubating in the seed (first) and production (second) fermenters is carried out at a pH in the range of 5-7. In some particular embodiments, the incubating is carried out at a pH in the range of 5.5 - 6.5.

[0071] In some embodiments, incubating in the seed (first) and production (second) fermenters is carried out at a temperature in the range of 45 - 60°C. In some particular embodiments, the incubating is carried out at a temperature in the range of 50-55°C.

[0072] "Fermenting" or "incubating" in the production fermenter is carried out until substantially no further lactic acid is produced. Typically and preferably, this corresponds to substantially complete conversion / utilization of fermentable sugars in the medium. The present invention advantageously provides for a shortened time between inoculation of the bacteria into the production fermenter and reaching a plateau such that substantially no further lactic acid is produced.

[0073] Organic waste management facilities handle collection, transport, processing, recycling / disposal and monitoring of waste materials. In order to recycle the waste into useful chemicals such as lactic acid, namely, utilize the organic waste as a substrate for industrial fermentation processes, an on-site fermentation system is typically required. The conventional method of inoculating industrial fermenters utilizes a wet inoculum of vegetative bacteria (wet seed train). This method has many disadvantages that makes it difficult to implement in waste management facilities, including the need to (i) tightly synchronize the wet seed preparation with the exact inoculation time of the production fermenter, (ii) have an on-site seed train production line which includes a few smaller scale fermenters for the production of the wet seed train (typically a ratio of 1: 10 down to few liters flasks).

[0074] The wet seed train is a time-consuming and resource-exhausting process. It increases the production time, which consequently limits the number of fermentation cycles that can be performed per a given time period.

[0075] The present invention advantageously allows simple integration of lactic acid production into organic waste management facilities, for on-site production of lactic acid from the organic waste. The compositions of dried spores as disclosed herein can be easily transported to the waste management site, stored and removed from storage upon need.

[0076] Advantageously, a need for seed line is eliminated by the present invention.

[0077] Using a dried composition of spores have major advantages over the conventional wet inoculum, including: (i) avoiding the need to tightly synchronize the seed preparation with the inoculation time of the production fermenter; (ii) avoiding the need to have an on-site seed train production line which includes a few small scale fermenters for the production of a wet seed (typically a seed train of a ratio of 1 : 10 down to few liters flasks); (iii) an extended shelf-life, e.g. several months or longer, with a minimal effect on spores viability (in effect, a wet seed does not have any shelf-life); (iv) ease of transportation without special containers and conditions since the dried composition of spores is much more resilient to uncontrolled transportation conditions; and (v) significantly reduced seed weight (e.g., over 95% weight reduction compared to a wet inoculum), due to water removal during its preparation, which significantly reduces transportation costs.

[0078] Importantly, the preparation of the dried composition of spores can be done at a site that is separated by time and location from the waste management facility, thereby reducing the need for a skilled biotechnology engineer that is dedicated to prepare the seed at the waste management facility.

[0079] In addition, the fact that the dried composition of spores can be prepared weeks or months ahead, put in storage and be available promptly for use in lactic acid fermentation, significantly shortens the lactic acid production process.

[0080] Lactic acid production from organic waste typically comprises (i) degradation of polysaccharides that are present in the waste using one or more polysaccharide-degrading enzyme in order to release soluble reducing sugars that are suitable for fermentation (“saccharification”); and (ii) fermentation of reducing sugars to lactic acid by a lactic- acid producing microorganism (e.g., Bacillus coagulans as disclosed herein).

[0081] Renewable carbohydrate sources for lactic acid production typically include varied ratios of reducing sugars (glucose, fructose, lactose, etc.), but also large amounts of polysaccharides such as starch and optionally also lignocellulosic material. Typically, lactic acid-producing microorganisms can utilize reducing sugars like glucose and fructose, but do not have the ability to degrade polysaccharides like starch and cellulose. Thus, to utilize such polysaccharides the process requires adding polysaccharidedegrading enzymes, optionally in combination with chemical treatment, to degrade the polysaccharides and release reducing sugars. The integration of polysaccharidedegrading enzymes into the process may be sequentially, such that the substrate is treated with one or more polysaccharide-degrading enzymes and subsequently the lactic acidproducing microorganism is added and ferments the reducing sugars, or simultaneously, where the one or more polysaccharide-degrading enzymes and the lactic acid-producing microorganism are mixed together to perform simultaneous saccharification and fermentation.

[0082] According to some embodiments, the methods of the present invention employ simultaneous saccharification and fermentation. Polysaccharide-degrading enzyme(s) are added to the organic waste together with an inoculum of germinated spores as described herein, to obtain simultaneous degradation of polysaccharides present in the waste and production of lactic acid.

[0083] When saccharification and fermentation are carried out as separate sequential steps, each step may take between about 18 -24 hours. Conducting the two steps simultaneously significantly shortens the process, which results in improved productivity, as more organic waste can be converted to lactic acid per a given time period.

[0084] Bacillus coagulans spore compositions

[0085] Bacillus coagulans is a Gram-positive, thermophilic, facultative anaerobic, sporeforming bacterium that produces lactic acid, particularly L-lactic acid. B. coagulans has been proposed for industrial fermentation processes to produce L-lactic acid. B. coagulans has also been shown to maintain normal intestinal microflora and improve digestibility, and is commonly marketed as a probiotic to maintain the ecological balance of the intestinal microflora and normal gut function. For example, LactoSpore® is a Bacillus coagulans (MTCC 5856) spore preparation intended for use as a probiotic, containing a spray-dried powder of B. coagulans spores mixed with maltodextrin.

[0086] Yadav et al. (2009) Indian Journal of Chemical Technology, 16: 519-522 examined calcium lactate, calcium gluconate, Spirulina and maltodextrin as probiotic protectants of Bacillus coagulans during spray drying.

[0087] Bacillus coagulans strains that may be used according to the present invention include but are not limited to: B. coagulans ATCC 8038 DSM 2312, B. coagulans ATCC 23498 DSM 2314, B. coagulans MTCC 5856, B. coagulans PTA-6086 (GBL30, 6086), B. coagulans SNZ 1969. Each possibility represents a separate embodiment of the present invention.

[0088] Spores may be prepared, for example, as follows: in the first step, a pure culture of B. coagulans is inoculated to a sterile seed medium and incubated on shaker at 50-55°C for 12-24 hours. The seed culture is then transferred to a sporulation medium and incubated at 50-55°C for 24-48 hours. Induction of sporulation requires stress conditions, for example, lack of nutrients, a relatively rich nitrogen source, such as yeast extract, along with limitation of the carbon and phosphor, presence of Mn2+and Ca2+ions, pH in the range of 5-6.5, incubation of 24-48 hours (preferably 24 hours), and combinations of the aforementioned stress-inducing factors. The spore concentration in the obtained spore culture is preferably at least 10A7 spores / ml, more preferably at least 10A8 spores / ml. Each possibility represents a separate embodiment.

[0089] Following incubation, the broth is harvested, centrifuged and the pellet is collected. In some embodiments, the harvested pellet, referred to herein as “semi-dried” or “partially-dried” preparation of the spores (moisture content in the range of 15%-30% w / w), is collected and kept as a partially-dried composition of spores for further use.

[0090] In some embodiments, the harvested pellet is weighed and subsequently mixed with a magnesium lactate solution to obtain a composition comprising the harvested spores and 15-25% magnesium lactate (w / w of the total weight of the composition). In some embodiments, the concentration of magnesium lactate in the composition comprising the harvested spores (prior to drying) is in the range of 15-20% (w / w), for example, 15%, 16%, 17%, 18%, 19% or 20% (w / w) of the total weight of the composition. Each possibility represents a separate embodiment of the present invention.

[0091] The harvested pellet is subsequently dried, for example, spray-dried or heat-dried at 80°C, to obtain a dried spore composition in a powder form. The moisture content of a dried spore composition according to the present invention is up to 15% (w / w) or any amount therebetween, preferably up to 10% (w / w) or any amount therebetween, typically between 4% - 10% (w / w) or any amount therebetween. Each possibility represents a separate embodiment of the present invention.

[0092] As provided herein, the moisture content of a dried composition comprising B. coagulans spores refers to the amount of water outside the spores (namely, “moisture content” as used herein does not include water found inside the spores). The moisture content is provided as a percentage out of the total weight of the composition.

[0093] In some embodiments, heat selection at a temperature of 70°C - 80°C is carried out following incubation and prior to drying.

[0094] In some embodiments, following drying, a dried composition in a powder form according to the present invention includes at least 10A8 spores / g powder, for example, 10A8 - 10 10 spores / g powder. In some embodiments, a dried composition according to the present invention includes, for example at least 10A8, at least 10A9, at least 10 10 spores / g powder. Each possibility represents a separate embodiment of the present invention. A dried composition according to the present invention may further include magnesium lactate, at a concentration of 40-60% (w / w), for example, 45%-55% (w / w), 40%-50% (w / w), 50%-60% (w / w). Each possibility represents a separate embodiment of the present invention.

[0095] In some embodiments, a dried composition according to the present invention does not require cold storage prior to use thereof. Thus, in some embodiments, a need for cold storage of the lactic-acid producing microbe is eliminated by the methods of the present invention.

[0096] According to the present invention, un-immobilized spores are used.

[0097] According to embodiments of the present invention, activation of the spores prior to inoculation into the fermenter is not required. For example, heat activation prior to inoculation into the fermenter is not required. As a further example, acid activation is not required prior to, or following, inoculation into the fermenter.

[0098] In some embodiments, following seeding into a seed fermenter as described herein, at least 90% of the spores germinate and produce vegetative cells, for example between 90%-100% of the spores germinate and produce vegetative cells, including each value within the range.

[0099] Lactic acid production from organic waste

[0100] As used herein, the term "lactic acid" refers to the hydroxycarboxylic acid with the chemical formula CH3CH(OH)CO2H. The terms 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 to a combination thereof.

[0101] For most industrial applications, L-lactic acid monomers with high purity (optical purity) are required in order to produce polylactic acid (PLA) with suitable properties. Thus, the methods and systems of the present invention are directed, in particular, to processes for the production of L-lactic acid or L-lactate salts at high yields.

[0102] Organic waste suitable for use according to the present invention is typically a complex organic waste comprising solid and non-solid materials. A complex 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, lipids, proteins, color components, inert materials and more. Organic waste for use with the present invention may also comprise inorganic solid components such as plastics, glass and the like. Examples of organic wastes for use according to the present invention include, but is not limited to, food waste, organic fraction of municipal waste, agricultural waste, plant material, and a mixture or combination thereof. Each possibility represents a separate embodiment.

[0103] In some particular embodiments, the organic waste for use with the present invention is food waste.

[0104] Food waste in accordance with the present invention encompasses waste of food 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. Each possibility represents a separate embodiment. The organic food waste may originate from vegetable and fruit residues, plants, cooked food, protein residues, slaughter waste, and / or combinations thereof. Each possibility represents a separate embodiment. 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). Industrial organic food waste also encompasses whey. Commercial organic food waste may include waste from shopping malls, restaurants, supermarkets, etc.

[0105] 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). In some embodiments, 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).

[0106] Food waste typically comprises 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. In some embodiments, 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.

[0107] Plant material in accordance with the present invention encompasses agricultural waste and manmade products such as paper waste. Typically, organic waste comprises endogenous D-lactic acid, L-lactic acid or both L- and D- lactic acid, originating, for example, from natural fermentation processes, e.g., in dairy products.

[0108] Organic waste for use with the methods and systems of the present invention typically comprises complex polysaccharides including starch, cellulose, hemicellulose and combinations thereof. The organic waste also comprises soluble reducing sugars, and / or is saccharified with one or more polysaccharide-degrading enzyme to obtain soluble reducing sugars (fermentable carbohydrates). As used herein, the term "fermentable carbohydrates" refers to carbohydrates which can be fermented by the spore-forming lactic acid-producing bacterium to lactic acid during a fermentation process. The reducing sugars typically comprise C5 sugars (pentoses), C6 sugars (hexoses) or a combination thereof. In some embodiments, said reducing sugars comprise glucose. In some embodiments, said reducing sugars comprise xylose.

[0109] Organic waste according to the present invention typically comprises complex polysaccharides and reducing sugars at varying ratios. The composition depends on the source of the waste, where some organic wastes may be more starch-rich (e.g., food waste from bakeries, mixed food waste of municipalities) and others may be rich with lignocellulosic material (e.g., agricultural waste). In some embodiments, the organic waste includes a combination of wastes from different sources.

[0110] In some embodiments, the percentage of at least one of starch, cellulose and hemicellulose in the organic waste is determined prior to treatment with one or more polysaccharide-degrading enzyme. In some embodiments, the percentage of soluble reducing sugars is determined prior to the fermentation.

[0111] Organic waste typically includes nitrogen sources and other nutrients needed for bacterial growth and lactic acid production, but such nutrients may also be supplied separately to the lactic acid production fermenter if needed.

[0112] Pretreatment of the organic waste according to the present invention typically includes decreasing particle size and increasing surface area, and also inactivating endogenous bacteria within the waste. In some embodiments, the pretreatment comprises shredding, mincing and sterilization.

[0113] Pretreatment typically begins by forming a slurry of the organic waste. As used herein, 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. In some embodiments, forming a slurry of organic waste according to the present invention, particularly a slurry of food waste, comprises subjecting the waste to depackaging, namely, removal of packaging material, including plastic, metal and glass packaging material. In some embodiments, the organic waste may naturally be in the form of a slurry.

[0114] 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. In some embodiments, 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. In some embodiments, 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.

[0115] In some embodiments, 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 30 present invention is characterized by a water content in the range of 65%-85%, including each value within the range.

[0116] In some embodiments, the organic waste for use with the present invention (e.g. food waste) naturally contains the aforementioned solid / liquid / water content. In other embodiments, water is added to the organic waste (e.g., food waste) to obtain a slurry characterized by the aforementioned solid / liquid / water content.

[0117] 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.

[0118] Sterilization may be carried out by methods known in the art, including for example, high pressure steam, UV radiation or sonication.

[0119] The pretreatment may include, for example, shredding and sterilization. Pretreatment may also include mincing with an equal amount of water using a waste mincer, such as, e.g., an extruder, sonicator, shredder or blender.

[0120] Lactic acid fermentation according to the present invention is typically carried out under anaerobic or microaerophilic conditions, using batch, fed-batch, continuous or semi-continuous fermentation. Each possibility represents a separate embodiment of the present invention.

[0121] In batch fermentation, the carbon substrates and other components are loaded into the reactor, and, when the fermentation is completed, the product is collected. Except for an alkaline compound for pH control, other ingredients are not added to the reaction before it is completed. The fermentation is kept at substantially constant temperature and pH, where the pH is maintained by adding the alkaline compound.

[0122] In fed-batch fermentation, 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.

[0123] In continuous fermentation, the substrate is added to the reactor continuously at a fixed rate, and the fermentation products are taken out continuously.

[0124] In semi-continuous processes, a portion of the culture is withdrawn at intervals and fresh medium is added to the system. Repeated fed-batch culture, which can be maintained indefinitely, is another name of the semi-continuous process.

[0125] Fermentations that produce acidic products such as organic acids etc. are typically performed in the presence of an alkaline compound, such as a metal oxide, a carbonate or a hydroxide. The alkaline compound is added to adjust the pH of the fermentation broth to a desired value, typically in the range of 4 - 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. During fermentation the pH in the fermenter decreases due to the production of the lactic acid, which adversely affects the productivity of the spore-forming lactic acid-producing bacterium. Adding bases such as magnesium-hydroxide / oxide, sodiumhydroxide, potassium-hydroxide, or calcium-hydroxide adjusts the pH by neutralizing the lactic acid thereby resulting in the formation of a lactate salt.

[0126] 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.

[0127] After fermentation is completed, the broth may be clarified by centrifugation or passed through a filter press to separate solid residue from the fermented liquid. The filtrate may be concentrated, e.g., using a rotary vacuum evaporator.

[0128] The fermentation broth according to the present invention may contain D-lactic acid originating from the organic waste. The D-LA is undesired in the production of L-LA for polymerization as it results in the formation of more D,D-lactide and meso-lactide, which adversely impact the quality of the PLLA final product. In some embodiments, the methods and systems of the present invention advantageously eliminate D-lactic acid by employing a D-lactic acid degrading enzyme or a D-lactic acid utilizing microorganism to the organic waste prior to lactic acid production, or to the fermentation broth during and / or following fermentation. Each possibility represents a separate embodiment.

[0129] Currently preferred is the use of a D-lactate oxidase as a D-lactic acid degrading enzyme. A D-lactate oxidase is an enzyme that catalyzes the oxidation of D-lactate to pyruvate and H2O2 using O2 as an electron acceptor. The enzyme uses flavin adenine dinucleotide (FAD) as a co-factor for its catalytic activity. A D-lactate oxidase according to the present invention is typically a soluble D-lactate oxidase (rather than membranebound). Advantageously, the enzyme works directly in organic wastes and also in fermentation broths, to eliminate the D-lactic acid. In some embodiments, the D-lactate oxidase is from Gluconobacter sp. In some embodiments, the D-lactate oxidase is from Gluconobacter oxydans (see, for example, GenBank accession number: AAW61807). Elimination of D-lactate from fermentation broths derived from organic wastes using a D-lactate oxidase is described in WO 2020 / 208635 assigned to the Applicant of the present invention.

[0130] Suitable D-lactic acid-utilizing microorganisms within the scope of the present invention include, but are not limited to, an Escherichia coli lacking all three L-lactate dehydrogenases. As used herein, "elimination", when referring to D-lactic acid / D-lactate, refers to reduction to residual amounts such that there is no interference with downstream processes of producing L-lactic acid and subsequently polymerization to poly(L-lactic acid) that is suitable for industrial applications. "Residual amounts" indicates less than 1% (w / w) D-lactate, and even more preferably less than 0.5 % (w / w) D-lactate, out of the total lactate (L+D) in a treated mixture of a fermentation broth at the end of fermentation. In some particular embodiments, elimination of D-lactate is reduction to less than 0.5 % (w / w) D-lactic acid out of the total lactate in a fermentation broth at the end of fermentation.

[0131] According to further aspects and embodiments, L-lactate monomers are further purified. The L-lactate monomers may be purified as L-lactate salts. Alternatively, a reacidification step with, e.g., sulfuric acid, may be carried out in order to obtain crude L-lactic acid, followed by purification steps to obtain a purified L-lactic acid.

[0132] The purification processes may include distillation, extraction, electrodialysis, adsorption, ion-exchange, crystallization, and combinations of these methods. Several methods are reviewed, for example, in Ghaffar et al. (2014) Journal of Radiation Research and Applied Sciences, 7(2): 222-229); and Lopez-Garzon et al. (2014) Biotechnol Adv., 32(5):873-904). Alternatively, recovery and conversion of lactic acid to lactide in a single step may be used (Dusselier et al. (2015) Science, 349(6243):78-80).

[0133] Saccharide-degrading enzymes

[0134] "Saccharide-degrading enzymes" as used herein refers to hydrolytic enzymes (or enzymatically-active portions thereof) that catalyze the breakdown of saccharides, including 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 of the present invention. The saccharide-degrading enzymes for use with the present invention are selected from those that are active towards saccharides (such as polysaccharides) found in organic wastes, including food waste and plant material. In some embodiments, the saccharide-degrading 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 modification may include one or more mutations that result in improved activity of the enzyme. In some embodiments, the saccharide-degrading enzymes are wild type (WT) enzymes.

[0135] 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).

[0136] In some embodiments, the saccharide-degrading enzymes used in the present invention are polysaccharide-degrading enzymes. In some embodiments, the polysaccharide-degrading enzymes are enzymes that degrade polysaccharides selected from starch and non-starch plant polysaccharides.

[0137] In some embodiments, the polysaccharide-degrading enzymes are glycoside hydrolases.

[0138] In some embodiments, the polysaccharide-degrading enzymes are selected from amylases, cellulases and hemicellulases. Each possibility represents a separate embodiment of the present invention.

[0139] A cellulase may be selected from, but not limited to: endo-(l ,4)- -D-glucanase, s%o- (1 ,4)-P-i)-glucanase, P-glucosidases, Carboxymethylcellulase (CMCase); endoglucanase; cellobiohydrolase; avicelase, celludextrinase, cellulase A, cellulosin AP, alkali cellulase, and pancellase SS. Each possibility is a separate embodiment.

[0140] A hemicellulase may be a xylanase. Non-limiting examples of 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. Each possibility represents a separate embodiment of the present invention.

[0141] An amylase may be selected from, but not limited to: glucoamylase, a -amylase; (1,4-a-D-glucan glucanohydrolase; 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 and 1 ,4-a-D-glucan glucohydrolase. Each possibility is a separate embodiment.

[0142] In some embodiments, the saccharide-degrading enzymes used in the present invention are disaccharide-degrading enzymes. In some embodiments, the disaccharidedegrading enzymes are selected from lactases and invertases. Each possibility represents a separate embodiment of the present invention.

[0143] The saccharide-degrading enzymes according to the present invention may be from a bacterial source. In some embodiments, 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. Typically, 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 fusccr, Amylases - Bacillus sp. (e.g. Bacillus stearothermophilus), Geobacillus sp. (e.g. Geobacillus thermoleovorans), Chromohalobacter sp., Rhodothermus marinus. Each possibility is a separate embodiment.

[0144] In additional embodiments, the bacterial source of the saccharide-degrading enzymes is a mesophilic bacterium. The term "mesophilic bacterium" as used herein 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. A person of skill in the art understands that some mesophilic bacteria (e.g., several Bacillus sp.) produce thermostable enzymes.

[0145] The saccharide-degrading enzymes according to the present invention may also be from a fungal source. Non-limiting examples of fungal sources for saccharide-degrading enzymes include: Cellulases and hemicellulases - Trichoderma reesei, Humicola insolens, Fusarium oxysporunv, Amylases (e.g., glucoamylases) - Aspergillus niger Aspergillus oryzae, Penicilliumfellutanum, Thermomyces lanuginosu.

[0146] Additional sources for saccharide-degrading enzymes for use in accordance with the present invention can be found, for example, at the CAZy server mentioned above.

[0147] The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0148] EXAMPLES

[0149] EXAMPLE 1

[0150] Lactic acid fermentation employing a spore inoculum versus an inoculum of germinated spores according to the present invention

[0151] Mixed food waste collected from supermarket logistic returns and surplus and rejects from cafeteria, containing expired bakery and dairy products, beverages, and fruits and vegetables, was pretreated by grinding, sterilization, saccharification and clarification by solid-liquid separation. Next, two 1000ml production fermenters were each filled with 800 ml of the pretreated waste. The glucose content of the substrate, as determined by HPLC analysis, was 96 g / kg.

[0152] One production fermenter was inoculated with a spore inoculum prepared as follows:

[0153] 1) 0.02 gr of a dried composition of Bacillus coagulans spores in a powder form was suspended in 8ml of water at room temperature. CFU of the powder: 5*10 10 CFU / gr.

[0154] 2) The suspension from the previous step was inoculated at a ratio of 1: 100 into the production fermenter. CFU after inoculation was 1.5*10A6 CFU / ml. CFU / ml was measured by live counting from plates.

[0155] Another production fermenter was inoculated with an inoculum of germinated spores according to the present invention, prepared as follows:

[0156] 1) 0.5 gr of the spore powder described above (CFU of the powder: 5*10A10 CFU / gr) was suspended in 8ml of water.

[0157] 2) The suspension from the previous step was added at a ratio of 1: 100 into a seed fermenter containing a synthetic medium with glucose as a carbon source (CFU / ml of 2.2 xlOA7 upon inoculation), and incubated for 6 hours at 52°C, pH 6.2. CFU at end of this step was 1.2 *10A9 CFU / ml.

[0158] 3) The medium with bacteria obtained at the end of the previous step were inoculated at a ratio of 1:36 into the production fermenter. CFU after inoculation was 4*10A7 CFU / ml.

[0159] Fermentations were carried out at 52°C, pH 6.2. Lactate production was observed 9:40 hours after inoculation of the spore inoculum, and only 3:40 hours after inoculation of the inoculum of germinated spores. Total fermentation time was 18:40 hours for the spore inoculum and only 10:50 hours for the inoculum of germinated spores, with a similar yield.

[0160] As can be seen from the above, the use of an inoculum of germinated spores according to the present invention significantly shortens the time needed for the bacteria to begin lactic acid production - over 50% reduction in lag time - and also significantly shortens the time needed to completely ferment the substrate into lactic acid. Thus, the use of an inoculum of germinated spores according to the present invention reduces operation costs and improves overall yield of lactic acid production, as more lactic acid can be produced per a given period of time.

[0161] EXAMPLE 2

[0162] Comparative - industrial lactic acid fermentation employing a seed train

[0163] 8000L of pretreated mixed food waste substantially as described in Example 1 were filled into a 15-ton fermenter. A seed train of Bacillus coagulans was prepared as follows:

[0164] 1) Five frozen cryovials were thawed, each containing 0.5ml of frozen Bacillus coagulans cells. CFU in the cryovials = ~ l*10A8 CFU / ml.

[0165] 2) The thawed bacteria from each cryovial were added to a IL shake flask containing 0.5L of LB medium (total of five shake flasks, providing 2.5L of medium with bacteria). The flasks were incubated for 24 hours at 52°C without pH monitoring, with shaking of 120 rpm, 2.5 cm orbit. CFU at end of this step = 5.5*10A6 CFU / ml.

[0166] 3) The 2.5L medium with bacteria obtained at the end of the previous step was added into a 1500L fermenter containing 750L of LB medium. The fermenter was incubated for 24 hours with mixing at 52°C, pH 6.2. CFU at end of this step = 2.1*10A8 CFU / ml.

[0167] 4) The 750L medium with bacteria obtained at the end of the previous step was inoculated into the 15-ton fermenter containing the 8000L of pretreated food waste described above. CFU after inoculation was = 3.3*10A6 CFU / ml.

[0168] Fermentation was carried out at 52°C, pH 6.2. Lactate production was observed 10 hours after inoculation. Total fermentation time was approximately 25 hours.

[0169] As exemplified in Example 1 above, the use of an inoculum of germinated spores according to the present invention avoids the need of a lengthy and laborious seed train and provides a high titer of live bacterial cells after only a few hours of incubation and using a single, small-scale, seed fermenter. Furthermore, the use of an inoculum of germinated spores according to the present invention significantly shortens the lag time for lactic acid production and the total fermentation time, thus saving operation costs and improving overall yield of lactic acid production, as more lactic acid can be produced per a given period of time.

[0170] EXAMPLE 3

[0171] Spore inoculum versus an inoculum of germinated spores prepared using a lower initial amount of spores

[0172] A further comparison was carried out between a spore inoculum and an inoculum of germinated spores, substantially as described in Example 1, except that the inoculum of germinated spores was prepared using a lower initial amount of spores.

[0173] Specifically, two 1000 ml production fermenters - Fermenter A and Fermenter B - were each filled with 800 ml of pretreated mixed food waste substantially as described in Example 1. Fermenter A was inoculated with a spore inoculum prepared as described in Example 1. CFU upon inoculation of Fermenter A was 6xl0A5 CFU / ml.

[0174] Fermenter B was inoculated with an inoculum of germinated spores, prepared as follows:

[0175] 0.02 gr of the spore powder described in Example 1 were suspended in 8ml of water and the suspension was added at a ratio of 1:100 into a seed fermenter containing a synthetic medium with glucose as a carbon source. CFU / ml upon inoculation was 7xlOA5. The seed fermenter was incubated for 8.5 hours at 52°C, pH 6.2. CFU at end of this step was 1.70 xl0A8 CFU / ml.

[0176] The medium and bacteria from the seed fermenter were inoculated at a ratio of 1 : 36 into Fermenter B. CFU upon inoculation was 6x10A5 CFU / ml.

[0177] Fermentations were carried out at 52°C, pH 6.2. Lactate production was observed 12:30 hours after inoculation of the spore inoculum, and only 07:00 hours after inoculation of the inoculum of germinated spores. Total fermentation time was 34:00 hours for the spore inoculum and only 15:30 hours for the inoculum of germinated spores, with a similar yield.

[0178] In this example, the inoculum of germinated spores was prepared using a lower initial amount of spores (0.02 g of spore powder compared to 0.5 g used in Example 1). Despite the reduced spore amount, the inoculum of germinated spores successfully completed the fermentation and significantly reduced process times. Specifically, the lag phase prior to lactic acid production was reduced by over 44%, and the time required for complete substrate fermentation into lactic acid was reduced by over 54%. Accordingly, the use of an inoculum of germinated spores, even when based on a lower initial spore amount, can reduce operational costs and improve overall lactic acid production efficiency.

[0179] EXAMPLE 4

[0180] Lactic acid fermentation at elevated glucose levels using an inoculum of germinated spores

[0181] In this example, fermentations were conducted on pretreated mixed food waste having a high glucose content. Elevated glucose concentrations can inhibit glucose-based fermentations by causing osmotic and metabolic stress, which may slow or interrupt glycolytic and fermentative pathways. This example compares the performance of a spore inoculum and an inoculum of germinated spores under these conditions.

[0182] Specifically, two 1000 ml production fermenters - Fermenter A and Fermenter B - were each filled with 800 ml of pretreated mixed food waste with a glucose content of 124 gr / kg, as determined by HPLC analysis. Fermenter A was inoculated with a spore inoculum prepared as described in Example 1. CFU upon inoculation of Fermenter A was lxlOA6 CFU / ml.

[0183] Fermenter B was inoculated with an inoculum of germinated spores, prepared as follows:

[0184] 0.02 gr of the spore powder described in Example 1 was suspended in 8ml of water and the suspension was added at a ratio of 1: 100 into a seed fermenter containing a synthetic medium with glucose as a carbon source. CFU / ml upon inoculation was 4xlOA6. The seed fermenter was incubated for 6.33 hours at 52°C, pH 6.2. CFU at end of this step was 3.4 x 10A8 CFU / ml. The medium and bacteria from the seed fermenter were inoculated at a ratio of 1:36 into Fermenter. CFU upon inoculation was 1.2 x 10A7 CFU / ml.

[0185] Fermentations were carried out at 52°C, pH 6.2.

[0186] In this example, the substrate contained a high glucose concentration (124 g / kg), which can inhibit fermentation. The inoculum of germinated spores demonstrated improved performance compared to the spore inoculum. The lag phase prior to lactic acid production was 6:30 hours for the spore inoculum and only 3:45 hours for the inoculum of germinated spores. Total fermentation time was 17:00 hours for the spore inoculum and only 13:45 hours for the inoculum of germinated spores, with comparable lactic acid yield.

[0187] EXAMPLE 5

[0188] Lactic acid fermentation employing an inoculum of germinated spores - analysis of base consumption rates and ODeoo

[0189] Four 1000 ml production fermenters - A, B, C, D - were each filled with 800 ml of pretreated mixed food waste substantially as described in Example 1. Fermenter A was inoculated with a spore inoculum prepared as described in Example 1. CFU upon inoculation of Fermenter A was 1.1 x 10A6 CFU / ml.

[0190] Fermenter B-D were each inoculated with an inoculum of germinated spores, prepared as described in Table 1. As shown in the table, each of Fermenter B-D was inoculated with bacteria that were taken from the seed fermenter at different time points. The CFU / ml, base consumption rate and ODeoo just before the transfer from the seed fermenter to the production fermenters were measured and the results are summarized in the table.

[0191] Table 1 -Inoculum of germinated spores

[0192] Medium with bacteria from the seed fermenter was inoculated at a ratio of 1:36 into Fermenters B-D. CFU / ml in each fermenter upon inoculation are specified in Table 1.

[0193] Fermentations were carried out at 52°C, pH 6.2. Lactate production was observed 8:45 hours after inoculation of the spore inoculum (Fermenter A), and only 3:00, 3:30 and 3:45 hours after inoculation of germinated spores in Fermenters B, C, D, respectively. Total fermentation time was 16:35 hours for the spore inoculum (Fermenter A) and only 13:00, 12:30, 13:25 hours for Fermenters B, C, D, respectively, inoculated with germinated spores. All fermenters ended with a similar yield.

[0194] Analyses of base consumption rates and ODeoo were conducted on more than twenty successful lactic acid fermentations utilizing an inoculum of germinated spores. Base consumption rates in the seed fermenter, prior to the transfer to the production fermenter, were in the range of 1.5-60 mmole L'1h’1, and ODeoo values were in the range of 0.1-1.7.

[0195] Further analyses of fermentations performed in industrial-scale fermenters demonstrated ODeoo values ranging from 0.1 to 5.0.

[0196] The data established that on average, inoculation with germinated spores results in a reduction in lag time of approximately 40-50% compared to inoculation with spores. Additionally, the use of germinated spores provides a substantial reduction in total process time, exceeding 20% in the majority of fermentations and even exceeding 50% in certain fermentations.

[0197] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose 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 invention.

Claims

CLAIMS1. A method for producing lactic acid or a salt thereof from organic waste, the method comprising:(i) providing a dried composition of spores of a spore-forming lactic acidproducing bacterium, optionally suspended in an aqueous medium to obtain a suspension of spores;(ii) providing a first fermenter containing therein a first fermentation medium suitable for growth of the spore-forming lactic acid-producing bacterium, and a second fermenter containing therein a second fermentation medium comprising pretreated organic waste;(iii) inoculating the dried composition or suspension of spores into the first fermentation medium to obtain at least IxlO5CFU / ml upon inoculation, and incubating to induce spore germination and obtain vegetative cells of the sporeforming lactic acid-producing bacterium;(iv) inoculating vegetative cells obtained in step (iii) into the second fermentation medium, wherein the volume of the inoculum corresponds to 0.5-30% of the volume of the second fermentation medium, and wherein the incubating in step (iii) is carried out to obtain an amount of vegetative cells that provides at least IxlO5CFU / ml in the second fermentation medium after inoculation;(v) fermenting the pretreated organic waste to produce lactic acid; and(vi) recovering the produced lactic acid or a salt thereof.

2. The method of claim 1, wherein step (iv) comprises inoculating the entire first fermentation medium with vegetative cells obtained in step (iii) into the second fermentation medium.

3. The method of claim 1, wherein the volume of the inoculum corresponds to 1-10% of the volume of the second fermentation medium.

4. The method of claim 1, wherein the volume of the inoculum corresponds to 1-5% of the volume of the second fermentation medium.

5. The method of any one of claims 1-4, wherein the dried composition of spores ischaracterized by a spore concentration in the range of l*10A8-l*10 12 CFU / gr.

6. The method of any one of the preceding claims, wherein the first fermentation medium comprises pretreated organic waste that is the same as the pretreated organic waste in the second fermentation medium.

7. The method of any one of the preceding claims, wherein the organic waste is selected from the group consisting of food waste, municipal waste, agricultural waste, plant material and a mixture or combination thereof.

8. The method of any one of claims 1-6, wherein the organic waste is food waste.

9. The method of any one of the preceding claims, wherein the incubating in step (iii) is carried out for a period of time in the range of 2-12 hours.

10. The method of any one of the preceding claims, wherein the spore-forming lactic acid-producing bacterium is Bacillus coagulans.

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