Methods for reducing water consumption in bioethanol production processes

Abstract

The present invention provides a method for producing ethanol and protein feed or food products from starch-containing feedstocks, such as grains and grain-derived products, preferably in combination with CO2 capture to further increase yields. The method facilitates reduced water usage compared to conventional ethanol plants without affecting the quality and quantity of the end product.

Description

[Technical field]

[0001] The present invention preferably uses CO 2 In combination with capture, a method is provided for producing ethanol and protein feed or food products from grains and grain-derived products that facilitates reduced water usage compared to conventional ethanol plants without affecting the quality and quantity of the final product.

[0002] The present invention relates to a method for producing ethanol and high protein feed or food products from a feedstock containing starch, the main steps of which are biopreservation of the feedstock using probiotic treatment, raw starch hydrolysis, use of thin stillage for steam production, reuse of process water, CO2 extraction using carbonic anhydrase, and 2 Includes capture.

[0003] By combining several process steps in a new and innovative manner, the method of the present invention facilitates the conservation of moisture from the feedstock and associated water savings, reduction in overall water consumption in the ethanol production plant, reduced ethanol losses resulting from less evaporation, overall improved ethanol yield, removal of volatile acids in the backset water, and improved conditions for yeast fermentation. [Background technology]

[0004] International organizations agree that achieving carbon neutrality by 2050 requires increased adoption of sustainable biofuels such as bioethanol, which is produced from biological sources by fermentation using microorganisms such as yeast. In recent years, bioethanol has reduced the atmospheric CO2 emissions by more than 500 million tonnes. 2This is a factor in avoiding carbon dioxide emissions. This has encouraged bioethanol production, and its market as a biofuel is likely to grow further in the future. However, by 2030, the world will face a nearly 40% gap in freshwater supplies. This could pose a potential bottleneck to sustainable bioethanol production and bring new urgency to address water scarcity.

[0005] As per a DOE report published in 2019 (Wu M. & Xu. H, 2018), a typical well-optimized dry-grind first generation ethanol plant (i.e., starch-based ethanol plant) consumes 2.8 liters of water to produce 1 liter of ethanol (3.15 liters of water are evaporated in the cooling tower, while corn moisture contributes about 0.3 liters of water, resulting in 2.8 liters of water per liter of ethanol). Most of this water is required primarily for heating, washing, cooling, and drying. It is important to note that most of these ethanol plants have little or no wastewater discharge. The plants reuse most of their process water through backsets. Therefore, the water demand is primarily related to energy production, specifically the cooling tower and boiler systems. As per an analysis conducted by Pfromm.P in 2008, a conventional dry-grind ethanol plant consumes about 9194 BTU (British Thermal Units) of energy to produce 1 liter of ethanol. The main reason behind such high energy consumption is the high temperature liquefaction process where starch from cereals is hydrolyzed to sugars at 85°C. The thermal energy intake poses a challenge to further reduce the water consumption, due to the fundamental fact that the thermal energy intake of the plant must be balanced by an adequate heat sink such as water evaporation in cooling towers.

[0006] Another way to hydrolyze starch is by raw starch hydrolysis or low-temperature saccharification, where the liquefaction temperature is lower than the starch gelatinization temperature of about 45-50 °C. The low-temperature saccharification process (Lewis et. al. 2011; described in US Patent No. 874814) can potentially reduce the thermal energy intake. In the low-temperature saccharification process, starch is enzymatically hydrolyzed at ≦50 °C, resulting in a significant reduction in the energy intake (about 4732 BTU / ethanol, based on mass balance). As per thermodynamic principles, this significantly reduces the water consumption to 1.6 l water / 1 l ethanol (based on energy balance). However, the realized water consumption is about 2.2 l water / 1 l ethanol (1.6 l water evaporated in the cooling tower + 1.5 l fresh water required for the process - 0.3 l water from corn moisture - 0.6 l water stored in the boiler). The main limitation of the low temperature saccharification process is the inability to recycle the backset due to the high concentration of volatile acids (VA: lactic and acetic acids). Backset containing ≥ 5 g / l VA can inhibit yeast, resulting in slow / stopped fermentation. Furthermore, because low temperature saccharification avoids jet cooking (105-121°C for 5-10 minutes) and high temperature liquefaction (85°C for 2 hours), contamination with Lactic Acid Bacteria (LAB), Acetic Acid Bacteria (AAB) and molds is more likely. The possible presence of such potentially harmful contaminants is another reason why few ethanol plants use low temperature saccharification technology. Plants that use low temperature saccharification are aware of this, and ways to circumvent this challenge are by: a) Adjust the pH to within the range of 3.0-4.5 using an inorganic acid (e.g. sulfuric acid or phosphoric acid). b) Adding antibiotics or hop acids during the mash / fermentation stage c) Send the syn / whole stillage to an anaerobic digester (AD) facility.

[0007] Both conventional and low temperature saccharification techniques require the use of dry feedstock (about 16% moisture w / w). The typical moisture content of raw feedstock is about 30%, which can potentially sustain microbial contamination such as molds and LAB and AAB. Mold infection can produce mycotoxins, resulting in poor quality feed products (distillers dried grains with solubles, DDGS) unsuitable for consumption by livestock such as ruminants and pigs, and therefore a significant risk of revenue loss for bioethanol producers. LAB and AAB infection can also result in slow or stalled ethanol fermentation. Most ethanol producers avoid this by drying the feedstock (using hot air to remove moisture from the grains). Dry feedstock has a moisture content of about 16%, meaning that almost 14% of the water or about 0.3 l of water / l of ethanol is lost during the drying process (Pfromm. P, 2008). Apart from water loss, repeated harsh drying also causes a decline in grain quality in terms of starch retrogradation, lower oil and protein yields. Moreover, the drying process also increases the carbon footprint of the incoming feedstock.

[0008] To prevent the accumulation of LAB and AAB, plants operating in batch mode (using both conventional and low-temperature saccharification technologies) must clean their heat exchangers daily with hot (~90°C) sodium hydroxide (5% NaOH). However, hot caustic treatment is not a complete defense, and many times these contaminants form biofilms (Rich et al., 2015), rendering the cleaning-in-place (CIP) treatment ineffective and resulting in slow fermentation. Ethanol producers overcome this by extending the CIP treatment duration and / or using harsher chemicals such as nitric acid. To prevent air pollution from volatile organic compounds (VOCs) such as acetaldehyde, ethyl acetate, acrolein, and acetone, most ethanol plants install scrubbers at the fermenter vents. Fresh water is circulated through the scrubbers and mixed with a wide variety of additives to remove CO2. 2 This cleans the water, trapping VOCs and adding some of them to the water footprint. Summary of the Invention

[0009] The present invention relates to a method for producing ethanol and a protein feed or food product from a feedstock comprising starch and protein, the method comprising the steps of: a) providing a feedstock comprising starch; b) pre-treating the feedstock by applying probiotic microorganisms; c) adding an aqueous liquid to the pretreated feedstock followed by mixing to obtain a slurry; d) optionally adjusting the pH of the slurry to 5 to 7, for example by adding aqueous ammonia; e) enzymatically treating the slurry by addition of amylase enzyme to obtain a hydrolysate; f) fermenting the hydrolysate by addition of yeast; and g) i. Ethanol ii.CO 2 , and iii. Protein Separately collecting The present invention provides a method comprising:

[0010] In one embodiment, the present invention provides a method for producing ethanol and a protein feed or food product from a feedstock comprising starch and protein, comprising the steps of: (a) providing a feedstock comprising starch and protein; (b) pre-treating the feedstock by applying a culture of one or more probiotic species of Lactobacillus and / or spore-forming Bacillus to the feedstock; (c) adding an aqueous liquid to the pretreated feedstock followed by mixing to obtain a feedstock slurry; (d) optionally adjusting the pH of the feed slurry to a pH of 5-7; (e) adding an amylase enzyme to the feedstock slurry and incubating the slurry to obtain a feedstock hydrolysate; (f) Yeast is added to the feedstock hydrolysate to obtain a fermentation broth, and step 1 is carried out at a dilution rate of 0.10 to 0.55 h -1 Stage 2 has a dilution rate of 0.04 to 0.10 h -1 fermenting the fermentation broth by a continuous fermentation comprising two stages having a steady state ethanol concentration of 5-11% (w / w) in stage 1 and 10-13% (w / w) in stage 2; (g) i. ethanol, ii.CO 2 , and iii. Protein Separately collecting Including, CO 2 However, carbonic anhydrase and Zn + are captured in the fermentation broth during continuous fermentation by adding ethanol and CO 2 is separated and recovered in the distillation and concentration steps. A method is provided.

[0011] In one embodiment, the feedstock is a grain and / or one or more grain-derived products, such as a grain selected from wheat, rice, oats, barley, rye, barley, millet, corn, triticale, and sorghum seeds.

[0012] In one embodiment, the moisture content of the feedstock is at least 10% w / w, at least 15% w / w, or at least 20% w / w, or at least 30% w / w.

[0013] In one embodiment, the probiotic culture in step (b) is applied onto the surface of the feedstock by spraying and optionally mixing the probiotic culture onto the surface.

[0014] In one embodiment, the probiotic culture in step (b) comprises a Lactobacillus species and a spore-forming Bacillus species; for example, the Lactobacillus species is selected from Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus amylovorus, and Lactobacillus hammesii; for example, the spore-forming Bacillus species is selected from Bacillus licheniformis, Bacillus clausii, and the like. clausii, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus fusiformis and Bacillus megaterium.

[0015] In one embodiment, the continuous fermentation in step (f) is carried out at a dilution rate of 0.04 to 0.10 h -1 resulting in an ethanol concentration in stage 3 of 12-15% (w / w).

[0016] In one embodiment, step (f) of the process comprises maintaining the pH at 5-6 during fermentation stages 1 and 2.

[0017] In one embodiment, step (g) of the method comprises administering 0.3×10 -12 ~3.0×10 -12 mol Zn + This includes the addition of

[0018] In one embodiment, Zn + ZnSO 4 It is added in the form of

[0019] In one embodiment, ethanol and CO 2 The recovery and separation of ethanol and CO 2 The ethanol is evaporated, and the liquid is condensed, and CO 2 Ethanol and CO are released in gaseous form using a condenser. 2 For example, evaporating ethanol and CO2 is at a temperature of 70-90°C, and condensing ethanol is at a temperature of 5-30°C.

[0020] In one embodiment, step (g) of the process further comprises recovering aqueous liquid resulting from one or more previous steps of the process, e.g., a distillation step; the aqueous liquid added to the feedstock in step (c) comprises the aqueous liquid recovered in step (g), e.g., more than 50% of the aqueous liquid added to the feedstock in step (c) is the aqueous liquid recovered in step (g). [Brief description of the drawings]

[0021] [Figure 1A] Schematic of a typical dry grind bioethanol production plant, including features and process steps: (a) slurry mixing tank, (b) jet cooker, (c) liquefaction tank, (d) yeast growth tank, (e)-(h) battery of batch fermenters, (i) beer well tank, (j) distillation column, (k) molecular sieve, (l) ethanol storage tank, (m) CO2 purification system, battery of three compressors, (n) CO2 storage tank, (o) decanter / centrifuge, (p) thin stillage storage tank, (q) evaporator effect, (r) syrup tank, and (s) DDGS dryer. [Figure 1B] G2B's SustainMax process, innovation steps 1-7 (marked with stars), result in significant reductions in energy and water consumption. Innovation step 1: Pretreatment of grain material with probiotic cultures; Innovation step 2: Adjustment of slurry pH and low-temperature liquefaction (raw starch hydrolysis) with aqueous ammonia is performed; Innovation step 3: Continuous fermentation process, dosing of carbonic anhydrase and addition of ammonium nitrate; Innovation step 4: Release of CO2 during distillation stage; Innovation step 5: Boiling of stillage to create steam for distilling ethanol; Innovation step 6: Separation of fiber from whole stillage; Innovation step 7: Hydrocyclone-based separation of insoluble proteins from the filtrate. The process features and steps include (A) slurry mixing and low temperature saccharification tank, (B) yeast propagation tank, (C)-(E) continuous reactors for stages 1, 2, and 3, respectively, (F) stillage boiling tank, (G) distillation column, (H) condenser, (I) CO2 purification system compressor, (J) CO2 storage tank, K) molecular sieve, (L) ethanol storage tank, (M) separation, (N) filtrate, (O) hydrocyclone, and (P) drying. [Diagram 2]Diagram of a three-stage continuous fermentation system. Pump P1 is used to continuously feed liquefied slurry from the liquefaction (hydrolysis) tank to the stage 1 fermenter. The flow rate is adjusted to maintain the dilution rate within the range of 0.1-0.55 h-1. A pH probe is used to continuously measure the pH in the stage 1 fermenter. A base is used to maintain the pH between 5.5 and 7.0. Pump P2 continuously pumps fermented mash from the stage 1 fermenter to the stage 2 fermenter. The flow rate is adjusted to maintain the dilution rate within the range of 0.04-0.1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Abbreviations, Terms, and Definitions: As used herein, the phrase "low temperature saccharification" refers to a process for converting starch to ethanol using alpha-amylase for saccharification of starch and without heat treatment for gelatinization. Generally, in the process of the present invention, "low temperature saccharification" refers to maintaining a temperature below the starch gelatinization temperature such that enzymatic saccharification occurs and directly converts raw native insoluble starch to soluble glucose while avoiding traditional starch gelatinization conditions. Starch gelatinization temperatures are typically in the range of 50°C to 93°C, depending on the type of starch source and polymer. In the process of the present invention, dextrinization of starch using traditional liquefaction techniques is not necessary for efficient fermentation of grain carbohydrates.

[0023] As used herein, the phrase "feedstock" refers to all raw materials, including starch (e.g., cereal grains, bread, bakery products). The feedstock further comprises protein. Preferably, the feedstock is a grain and / or a grain-derived product. Suitable feedstocks include bakery products such as bread, croissants, dough, biscuits, cakes, and grains such as corn (maize, e.g., whole corn), sorghum (milo), barley, wheat, rye, rice, and millet; and starchy root vegetables, tubers, or roots, such as sweet potato, cassava, etc. The feedstock may be a mixture of such materials.

[0024] As used herein, the term "probiotics" refers to bacteria and yeasts that are generally considered safe for consumption. Suitable probiotics include lactic acid bacteria such as Lactobacillus plantarum 1A7, W. anomalus LCF1695, Lactobacillus amylovorus, and Lactobacillus hammesii. Other suitable probiotics include species of Bacillus, such as Bacillus licheniformis, Bacillus clausii, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus fusiformis, and Bacillus megaterium (also called Priestia megaterium). The probiotic can be a mixture of bacteria and yeast.

[0025] As used herein, the phrase "baseline" refers to existing state-of-the-art bioethanol production processes.

[0026] As used herein, the phrase "high protein" refers to a protein co-product obtained from the ethanol fermentation process, said "high protein" product containing at least 50% protein (with about 10% moisture).

[0027] As used herein, the term "culls" refers to grains and / or grain-derived products that have been declared unfit for human consumption.

[0028] The term "liquefact" as used herein refers to the grain hydrolysate resulting from the hydrolysis step. The term "CIP" as used herein refers to clean in place.

[0029] The term "whole stillage" as used herein refers to the aqueous fraction remaining after removal of ethanol during the distillation stage.

[0030] The term "wet cake" as used herein refers to the insoluble portion obtained after centrifugation of the hall stillage.

[0031] The term "thin stillage" as used herein refers to the aqueous fraction remaining after removal of the wet cake following centrifugation of the whole stillage.

[0032] As used herein, the term "DDGS" refers to distillers dried grains with solubles added.

[0033] The terms "backset" or "backset water" or "backset liquor" as used herein refer to the mixture of thin stillage and water produced during DDGS drying, molecular sieve and evaporator operations.

[0034] As used herein, the term dilution rate (D) is usually expressed in units per hour (h -1 ) and the flow of medium into the fermenter (liters h -1 The term "aqueous ammonia" as used herein refers to the relationship between the amount of ammonia in the fermenter and the fermentation volume in liters in the fermenter. 3 Denoted by (aq).

[0035] Detailed description of the invention: The present invention facilitates reduced water and energy consumption compared to conventional ethanol plants without affecting the quality and quantity of the final product. The present invention preferably utilizes CO2 to further increase yields. 2Combined with capture, it provides a novel method for producing ethanol and protein feed or food products from grains and grain-derived products. The entire process is highly sustainable as it has very low GHG (greenhouse gas) emissions and is robust to large variations in the moisture content of the feedstock.

[0036] The present invention relates to a method for producing ethanol and high protein feed or food products, the major steps of which include biopreservation of the feedstock using probiotic treatment, raw starch hydrolysis, a continuous fermentation process, use of thin stillage for steam production, reuse of process water, and close to 100% recovery and recycling of backset and process water.

[0037] By combining several process steps in a new and innovative way, the method of the present invention promotes the conservation of moisture from the raw materials and associated water savings by avoiding drying of the feedstock, reducing water consumption in cooling towers, significant inclusion of backset, such as up to 100%, significantly reducing CIP (clean in place) cycles, avoiding mold contamination in the feedstock, avoiding antibiotic addition during saccharification and / or fermentation stages, and improving conditions for yeast fermentation.

[0038] I. METHODS FOR PRODUCING ETHANOL AND PROTEIN FEED OR FOOD PRODUCTS The present invention provides a method for producing ethanol and protein feed or food products, which further includes the step of converting CO 2 Further combination with capture is possible.

[0039] In one embodiment, the method comprises the steps of: a) providing grain and / or grain-derived products; b) pre-treating the cereal and / or cereal-derived product by applying probiotic microorganisms to the cereal and / or cereal-derived product; c) adding an aqueous liquid to the pretreated grain and / or grain-derived products followed by mixing to obtain a grain slurry; d) optionally adjusting the pH of the grain slurry to ≧5.5 by adding aqueous ammonia to the grain slurry; e) enzymatically treating the grain slurry by adding an amylase enzyme to the grain slurry to obtain a grain hydrolysate; f) fermenting the cereal hydrolysate by adding yeast to the cereal hydrolysate; and g) i. Ethanol ii. a protein, and iii. In some cases, CO 2 Separately collecting Includes.

[0040] In one embodiment, the method comprises the steps of: a) providing a feedstock comprising starch; b) pre-treating the feedstock by applying probiotic microorganisms; c) adding an aqueous liquid to the pretreated feedstock followed by mixing to obtain a slurry; d) optionally adjusting the pH of the slurry to 5 to 7, for example by adding aqueous ammonia; e) enzymatically treating the slurry by addition of amylase enzyme to obtain a hydrolysate; f) fermenting the hydrolysate by addition of yeast; and g) i. Ethanol ii.CO 2 , and iii. Protein Separately collecting Includes.

[0041] The present invention improves water reclamation in ethanol production plants, particularly dry grind ethanol production plants. The present invention provides a novel process for producing bioethanol, which is based on a conventional low-temperature saccharification process, but which is first treated with a probiotic microorganism to ensure the absence of unwanted bacterial or fungal growth, and then preferably hydrolyzed using a low-temperature saccharification process, and finally used to ferment ethanol in a continuous mode, preferably with a specific stage-associated dilution rate. In this way, the total water consumption is significantly reduced, which can be achieved without the need for either advanced chemical separation techniques or ion-exchange resin-based processes. The applicability of said probiotic-treated feedstock is further improved by adding fermentation residues (ammonia water) from an anaerobic digester (biogas) plant before the low-temperature saccharification process. Running the ethanol fermentation process in a continuous mode while maintaining the stages at a specific dilution rate results in pollution prevention and ethanol yields similar to the baseline. Finally, CO 2 A capture enzyme is preferably added at the fermentation step to further reduce water loss. In addition to recovering ethanol from the process, a protein product is also recovered that is essentially "pure" and therefore can be used as feed or in food applications.

[0042] The various process steps are described in more detail below. The invention is illustrated graphically in FIG. 1B. This illustration should not be considered as a limitation of the invention, but merely an illustration of the process for producing ethanol, protein feed / food products, and CO2 while reducing energy and water consumption. 2 The present invention is merely provided as an illustration of one means of practicing the present invention, for producing ethanol from a "baseline" typical dry-grind bioethanol production plant. For comparison, FIG. 1A illustrates a "baseline" typical dry-grind bioethanol production plant.

[0043] II. Feedstocks - e.g. grains and grain-derived products The present invention provides a method for producing ethanol and protein feed or food products from a feedstock comprising starch. In a preferred embodiment, the feedstock is a grain and / or grain-derived product.

[0044] A "grain" is an edible seed of a plant. There are two main types of grain crops: cereals and legumes. Cereal grains are members of the grass family and have a high carbohydrate content. Some examples of cereal grains are wheat, rice, oats, barley, rye, barley, millet, corn, triticale, and sorghum. Grains also include pseudocereals such as chia, quinoa, and buckwheat. The edible seeds of legumes or legumes are members of the legume family and have a higher protein content than cereal grains. Some further examples of cereal grains are chickpeas, mung beans, soybeans, kidney beans, lentils, and lima beans.

[0045] "Cereal-based products" refers to products made using the above-mentioned cereals, which may have a high starch content. Flour made from the above-mentioned cereals is an example of a cereal-based product. Some further examples of cereal-based products are bread, cakes, dough, chocolate, biscuits and breakfast cereals.

[0046] "Grain material" is used herein as a common term for grains and grain-derived products.

[0047] In one embodiment the cereal material has a starch content of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even at least 95%. In one embodiment the cereal material has a starch content of 5-95%, 10-95%, 15-95%, 20-95%, 25-95%, 30-95%, 35-95%, 40-95%, 45-95%, 50-95%, 55-95%, 60-95%, 65-95%, 70-95%, 75-95%, 80-95% or 90-50%.

[0048] In one embodiment, the cereal material is selected from wheat, rice, oats, barley, rye, barley, millet, corn, triticale, and sorghum seeds, and any products derived from these cereals. In a preferred embodiment, the cereal material is wheat-based bread.

[0049] I.ii Pretreatment with probiotic bacteria and / or yeast In one embodiment of the present invention, probiotic microorganisms are applied to raw materials (e.g., cereal materials) to enhance microbial stability in the raw materials and reduce unwanted mold contamination. The term pretreatment means that this step is carried out before the hydrolysis and fermentation steps. In Figure 1B, this is illustrated as "Innovation Step 1" (star 1). The positive effect of probiotic microorganism pretreatment is further demonstrated in Example 1.

[0050] Conventionally, the feedstock is dried or its moisture content reduced by other means to ensure a less favorable environment for unwanted contaminants. However, in the present invention, such drying steps may be omitted or at least significantly reduced, since the probiotic microorganisms prevent the growth of other unwanted microbial contaminants. The potential problem of contamination most often arises when the raw material supply includes a "storage step", such as the time required to transport the raw material from its origin to the ethanol plant, or during other common "storage" conditions before use in the process.

[0051] In one embodiment, the probiotic microorganisms are added to raw materials having a moisture content (i.e. water content) of more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or even more than 45%. In one embodiment, the probiotic microorganisms are added to raw materials having a water content of 10-50%, 15-50%, 20-50%, 25-50%, 30-50%, 40-50%, or 45-50% (w / w). In one embodiment, the probiotic microorganisms are added to raw materials having a water content of 10-15%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, or 10-45% (w / w). In one embodiment, the probiotic microorganisms are added to a raw material having a moisture content of 10-40%, preferably 15-40%, more preferably 20-30% (w / w).

[0052] An advantage of the present invention is that it provides a method that allows the grain material to be used "as is" without reducing the moisture content of the raw material. Microbial pretreatment is a solution to extend the shelf life of the feedstock without reducing its water activity and without compromising its nutritional properties.

[0053] As explained in Example 8, the water contribution in the feedstock to ethanol process can increase by as much as 50% because the grain material does not need to be dried in the drying process (see further calculation details in Example 8).

[0054] In one embodiment, the water contribution from the feedstock is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or even 50% in the ethanol process compared to conventional processes where a step of drying the feedstock is required. The probiotic bacteria will not only reduce and / or prevent bacterial and mold contamination of the initial raw materials, but also reduce contamination in the subsequent ethanol fermentation. In one embodiment, the probiotic microorganism is a microbial cell or cell culture selected from bacteria or yeast, or a combination of bacteria and yeast.

[0055] In one embodiment, the probiotic microorganism applied to the feedstock comprises one or more probiotic bacteria - i.e. one or more probiotic bacterial cells or probiotic bacterial cell cultures. In a preferred embodiment, the probiotic bacteria comprises one or more lactic acid bacteria. In one embodiment, the probiotic bacteria comprises one or more homofermentative lactic acid bacteria (HoLAB). In one embodiment, the lactic acid bacteria is one or more strains of Lactobacillus. In one embodiment, the lactic acid bacteria is selected from Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus plantarum (e.g., strain 1A7), Lactobacillus amylovorus, and Lactobacillus hammesii. In one embodiment, the probiotic bacteria is one or more strains of Pediococcus. In one embodiment, the probiotic bacteria is selected from Pediococcus acidilactici (e.g., strain KTU05-7), Pediococcus pentosaceus (e.g., strain KTU05-8) and Pediococcus pentosaceus (e.g., strain KTU05-10).

[0056] In one embodiment, the probiotic microorganisms applied to the feedstock include one or more yeast species. In one embodiment, Pichia anomala (e.g., strain SKM-T) is used in the pretreatment of the raw material.

[0057] In a particular embodiment, a combination of the probiotic microorganisms Lactobacillus plantarum and Wickerhamomyces anomalus is used in the pretreatment of raw materials. In particular, the Lactobacillus plantarum 1A7 strain and the Wickerhamomyces anomalus LCF1695 strain may be used in the pretreatment of raw materials.

[0058] In one particular embodiment, a combination of the spore-forming probiotic microorganisms Bacillus licheniformis, Bacillus clausii, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus fusiformis and Bacillus megaterium (also called Priestia megaterium) may be used in the pre-treatment of the raw material.

[0059] In one preferred embodiment, the probiotic microorganisms applied in the methods of the present invention comprise a combination of Bacillus and Lactobacillus species.

[0060] In a preferred embodiment, a culture / suspension of one or more probiotic species of Lactobacillus and / or spore-forming Bacillus is applied to the feedstock.

[0061] In one such embodiment, the probiotic bacteria is (i) for example Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus plantarum (e.g., strain 1A7), Lactobacillus amylovorus, Lactobacillus hammesii, Pediococcus acidilactici (e.g., strain KTU05-7), Pediococcus pentosaceus (e.g., strain KTU05-8) and Pediococcus pentosaceus (e.g., strain KTU05-9). pentosaceus (e.g., strain KTU05-10), and (ii) one or more strains of Bacillus species disclosed herein, for example, selected from the spore-forming Bacillus species Bacillus licheniformis, Bacillus clausii, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus fusiformis, and Bacillus megaterium.

[0062] In one important embodiment, microbial pretreatment of the raw materials is carried out by applying probiotic microorganisms to the surface of the raw materials, for example the surface of cereal materials, to avoid contamination with other unwanted microbial contaminants.

[0063] In one embodiment, the probiotic microorganisms are applied by spraying onto the surface of the raw materials. As an example, the probiotic microorganisms may be sprayed onto bread to avoid contamination before the bread ingredients undergo further processing steps in the ethanol production facility.

[0064] In one embodiment the probiotic microorganisms are applied to the surface of freshly harvested cereals and / or freshly discarded cereal-derived products, for example by spraying the microorganisms onto the surface.

[0065] In one embodiment, probiotic bacteria and / or yeasts are applied and then the grains and grain materials are ground to flour in a milling process step prior to the low temperature saccharification step.

[0066] I.iii Hydrolysis The grain material is hydrolyzed in an aqueous solution. In FIG. 1B, this is illustrated as "Innovation Step 2" (star 2). Specifically, this is a pretreated feedstock (probiotic pretreatment discussed in section I.ii), preferably a pretreated grain material, which is enzymatically hydrolyzed to obtain monosaccharides for ethanol fermentation.

[0067] The feedstock may be chopped, ground, broken / reduced in size by known processes prior to the addition of the aqueous solution. In a preferred embodiment, the feedstock is ground to flour prior to the hydrolysis step.

[0068] Depending on the initial moisture content of the cereal material, the aqueous solution is added to the cereal material to obtain a cereal slurry having the desired dry matter percentage. In one embodiment, the hydrolysis is carried out at a dry matter content of 20-40%, preferably 25-35%, more preferably 28-33%.

[0069] In one embodiment, the method includes recovering process water, and the aqueous liquid added to the feedstock to obtain the feedstock slurry includes the recovered process water. In one embodiment, the aqueous liquid added to the grain material includes backset liquid from within the ethanol processing plant. This reclaimed water may originate from the ethanol distillation process described as "Innovation Step 5" (star 5) in FIG. 1B. In one embodiment, the aqueous liquid includes a mixture of backset and fresh water.

[0070] The pH of the feedstock slurry is optionally adjusted to pH > 5.5 prior to hydrolysis. In one embodiment, the feedstock is a grain or grain-derived product and the pH of the feedstock is adjusted to pH > 5.5 prior to hydrolysis. In one embodiment, the pH of the feedstock is adjusted to pH 5-7, for example pH 5.5-7, prior to hydrolysis. In one such embodiment, the pH of the hydrolysis solution is adjusted by the use of an alkaline solution, for example potassium hydroxide, sodium hydroxide, or any alkali, such as aqueous ammonia. In a preferred embodiment, the pH is adjusted using aqueous ammonia, as disclosed in section I.iv.

[0071] In the present process the cereal material is hydrolyzed at a temperature of 30-65° C., preferably at a temperature of 35-65° C., more preferably at a temperature of 40-55° C., most preferably at 40-45° C., even more preferably at 50-55° C. The temperature of the cereal material may be controlled by the addition of process water as described above.

[0072] An advantage of the present invention is that it provides a method in which the traditional liquefaction for conversion of starch to sugars is instead carried out by a low temperature saccharification process, where the temperature is maintained below the starch gelatinization temperature and the enzymatic hydrolysis is carried out directly on the raw (ungelatinized) starch material. An example of such a low temperature saccharification process is given in Example 3.

[0073] The grain material is hydrolyzed at a pH of 4 to 7, preferably at a pH of 5 to 6.50, more preferably at 5.5 to 6.0. In some embodiments, pH adjustment is required at one or more points during the hydrolysis to maintain the pH within these pH ranges.

[0074] The mixing is preferably carried out during the hydrolysis process.

[0075] A starch hydrolase enzyme is used to hydrolyze the starch of the grain. One or more amylase enzymes are used to hydrolyze the starch of the grain. In one embodiment, the amylase enzyme comprises an alpha- and / or beta-amylase belonging to the EC 3.2.1 enzyme classification.

[0076] In a further embodiment, a mixture of one or more glucoamylases, acid amylases and cellulases is used in the hydrolysis step; these may also be used in combination with an alpha-amylase.

[0077] In one particular embodiment, the commercially available enzymes Liquoflow® GO 2X and Saczyme® (from Novozymes A / S) are used, either alone or in combination.

[0078] Based on the feedstock for hydrolysis, the enzyme dosage can be optimized, as will be recognized by one of skill in the art.

[0079] In one embodiment, an amylase enzyme is added to a feedstock slurry and the slurry is incubated to obtain a feedstock hydrolysate.

[0080] I.iv Use of aqueous ammonia for pH adjustment As will be appreciated by those skilled in the art, various process steps may require pH adjustment for optimal performance. In one embodiment, aqueous ammonia is used for pH adjustment in the process of the present invention. The term "aqueous ammonia" refers to an aqueous solution containing ammonia. An example of pH adjustment using aqueous ammonia is provided in Example 2.

[0081] One of the by-products of biogas plants is ammonia-containing water. Disposing of this water poses a major challenge for biogas plants; the main reason for this is the high ammonia emissions. Currently available ammonia extraction technologies are not only expensive but also have a high carbon footprint. Therefore, in a preferred embodiment, ammonia water is generated from the biogas plant, e.g. originating from a biogas waste stream.

[0082] Thus, the present invention uses this aqueous ammonia in the slurry mixing step of the ethanol process, which is illustrated as part of "Innovation Step 2" (star 2) in Figure 1B. The aqueous ammonia adds value beyond just being a means of pH adjustment - it also serves as a suitable nitrogen source for yeast in the fermentation step, as compared to simply using hydroxide compounds for pH adjustment, for example.

[0083] IV Fermentation The hydrolyzed raw materials are fermented by yeast, such as baker's yeast, Saccharomyces cerevisiae. In Figure 1B, this is illustrated as "Innovation Step 3" (star 3).

[0084] In one embodiment, the fermentation is carried out at a dry matter content of 20-60%, preferably 20-50%, more preferably 25-40%, most preferably 28-32%.

[0085] Mixing is preferably carried out during the fermentation process.

[0086] In one embodiment the hydrolyzed cereal material is fermented at a temperature of 20-40°C, preferably at a temperature of 28-36°C, more preferably at 30-32°C.

[0087] In one embodiment, the fermentation is carried out at a pH of 4 to 7, preferably 4.5 to 6.5, more preferably 5.0 to 6.0, most preferably 5.0 to 5.5; for example, 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5. Maintaining the pH above 5.0 ensures that lactic and acetic acids present in the solution do not inhibit the yeast.

[0088] pKa (acid dissociation constant) plays an important role in yeast growth. Among all the contaminations reported in bioethanol processes, contamination with acetic acid and lactic acid is the most harmful to yeast health. Volatile acids (VA) are in proton form below their pKa (pKa of acetic acid is 4.76; pKa of lactic acid is 3.86). In the protonated state, these VA can pass through the yeast cell membrane and lower the pH of the cytoplasm. Yeast will excrete these VA at the expense of ATP. However, because of the proton form, these VA continue to pass through the yeast cell membrane barrier. The large energy waste by the continuous excretion of VA will result in the inhibition of fermentation (i.e., fermentation stop / slow fermentation). The advantage of the present invention is that maintaining the pH above the pKa of these VA as the most ideal method makes the bioethanol fermentation process robust against VA inhibition.

[0089] The fermentation may be carried out in several steps, e.g., one, two, three or more steps, to optimize the process, such as to allow for a continuous process rather than a batch fermentation. In Figure 1B, a three-stage fermentation is shown as one embodiment of the present invention, but should not be considered as limiting the invention.

[0090] In one embodiment, the fermentation is carried out in three stages of fermentation with stage-specific dilution rates to ensure a specific ethanol concentration in each stage.

[0091] In one embodiment, the fermentation is carried out in a three-stage continuous mode, with the ethanol concentration (w / w) in the first stage being maintained between 1% and 10%, preferably between 4% and 9%, more preferably between 5% and 7%, and most preferably between 5.5 and 6.0%; for example, 5.5%, 5.6%, 5.7%, 5.8%, 5.0%, or 6.0%. The steady-state concentration of ethanol in stage 1 is maintained by increasing the dilution rate (D) between 0.10 and 0.55 h -1 , preferably 0.10 to 0.30 h -1 , more preferably 0.10 to 0.20 h -1 This is accomplished by continuously flowing the hydrolyzed feedstock into the fermentation broth in the stage 1 fermentor and continuously flowing the fermented material from the stage 1 fermentor into the stage 2 fermentor, while maintaining the ethanol concentration within this desired range, particularly 5.5-6.0%, in the first fermentor. The advantage of maintaining the ethanol concentration in this desired range, particularly 5.5-6.0%, in the first fermentor is that potential contaminants such as lactic acid bacteria (LAB), acetic acid bacteria (AAB), etc. are inhibited, but yeasts are not. The residence time in the first fermentor is 5-80 hours, preferably 10-30 hours, more preferably 12-15 hours.

[0092] The overflow from the first fermenter is transferred to a second fermenter and the ethanol concentration (w / w) is maintained at 6%-18%, preferably 8%-16%, more preferably 9%-12%; for example, 9%, 9.5%, 10%, 10.50%, 11%, or 12%. The steady state concentration of ethanol is maintained at a dilution rate (D) of 0.04-0.10 h. -1 , preferably 0.04 to 0.08 h -1 , more preferably 0.4 to 0.06 h -1 This is accomplished by continuously flowing fermented material from the stage 1 fermentor into the stage 2 fermentor and continuously flowing fermented material from the stage 2 fermentor into the stage 3 fermentor while maintaining the temperature within the range of 5-80 hours, 5-60 hours, preferably 10-40 hours, more preferably 25-30 hours.

[0093] The overflow from the second fermentor is transferred to a third fermentor and the ethanol concentration (w / w) is maintained at 10%-15%, preferably 12%-15%. The residence time in the third fermentor is 3-8 hours, preferably 4-7 hours, more preferably 5-6 hours. A steady state ethanol concentration is achieved by continuously flowing fermented material from the stage 2 fermentor into a stage 3 fermentor and continuously flowing fermented material from the stage 3 fermentor into a distillation unit.

[0094] In one embodiment, yeast is added to the feedstock hydrolysate to obtain a fermentation broth. The fermentation broth (both) is fermented by a continuous fermentation comprising at least two stages, preferably three stages. In one embodiment, the liquefact (feedstock hydrolysate) resulting from the enzymatic hydrolysis step is appropriately dosed to the stage 1 fermentor to maintain the ethanol concentration in the stage 1 fermentor at 5-11% (w / w); this ethanol concentration in stage 1 is maintained by adjusting the dilution rate to 0.1 h in stage 1. -1- ~0.55h -1 In a further embodiment, the fermented mash from the stage 1 fermentor is then continuously fed to a stage 2 fermentor to maintain the ethanol concentration in the stage 2 fermentor at 10-13% (w / w); this ethanol concentration in stage 2 can be achieved by maintaining a dilution rate of 0.04 h -1 ~0.1h -1 In a further embodiment, the fermented mash from the stage 2 fermentor is then fed to a stage 3 fermentor and the ethanol concentration is maintained at 12-15% (w / w) by continuously feeding the fermented mash from stage 3 to a distillation unit.

[0095] The advantage of the continuous mode is that the fermentation does not need to be stopped, thus saving a significant amount of CIP cycles, and thus reducing the use of water, energy and chemicals. Furthermore, reducing the CIP also improves the downtime of the entire process. In ethanol production, typically about 8-10 hours are lost in cleaning and restarting the batch fermentation process (downtime). In conventional ethanol plants, such a mode of operating the process in a continuous mode is avoided due to the risk of contamination. The present invention overcomes this potential limitation. In one embodiment, ammonium nitrate is added during the fermentation. Without wishing to be bound by theory, it is speculated that the nitrate salt of ammonium nitrate is converted to ammonium ion during the fermentation (nitrate respiration). The ammonium produced for nitrate respiration maintains the fermentation pH above the pKa of the volatile acid. Nitrate → Nitrite → Ammonium. The ammonium formed is utilized by the yeast as a nitrogen source (similar to aqueous ammonia). Overall, a two-tiered safety net is created to prevent the pH from dropping below the pKa of the volatile acids: 1) by adjusting the pH with aqueous ammonia, and 2) by maintaining the pH adjusted with nitrates.

[0096] In one embodiment, the ammonium nitrate concentration during fermentation is 50-500 ppm, more preferably 100-350 ppm, most preferably 150-250 ppm, e.g., 160 ppm, 170 ppm, 190 ppm, 200 ppm, 210 ppm, 230 ppm, 250 ppm. The addition of ammonium nitrate ensures that the pH is maintained at ≧5.00, thereby preventing the inhibitory effect from VA on the yeast.

[0097] The process of the present invention benefits from maintaining the pH at about pH 5 (or higher) since it maintains the VA in their unprotonated state, which reduces the VA inhibition of the yeast, thus creating the most favorable conditions for yeast fermentation and ensuring less stress on the yeast, thereby avoiding potential fermentation stalls. Furthermore, the higher ethanol concentration obtained in the first stage of fermentation results in suppression of potential contaminants, which allows well-adapted yeast to be sent to the second stage of fermentation, leading to an overall improved ethanol yield. Apart from this, the continuous fermentation mode also results in a significant reduction in the downtime (time required for a new fermentation to start), thus improving the overall productivity of the process.

[0098] I.vi Ethanol recovery The first major product of the process of the present invention is ethanol. Ethanol can be recovered by distilling the output from the fermentation tank. In Figure 1B, this is illustrated as "Innovation Step 4" (star 4).

[0099] In one example, as will be appreciated by those of skill in the art, the fermented slurry can be heated to about 60° C., for example, by using a heat exchanger, and then injected into a distillation column where the temperature can be maintained at about 80° C., for example, by using steam generated from a stillage boiler tank (illustrated in FIG. 1B as “Innovation Step 5” (star 5) and further described in Section I.viii). Ethanol is evaporated while the resulting whole stillage is sent for further separation and filtration for protein recovery (illustrated in FIG. 1B as “Innovation Step 6” (star 6) and further described in Section I.vii).

[0100] The evaporated ethanol can be further rectified to an ethanol concentration of about 95%. Molecular sieves are further applied to dehydrate the ethanol product to a concentration of 99.99%. Finally, the dehydrated ethanol can be denatured by adding a suitable denaturing agent.

[0101] I.vii Protein recovery The second major product of the process of the present invention is a protein feed or food product. In one embodiment, the whole stillage from the distillation process is processed to produce a high protein product, for example a protein product that contains at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80% protein in the final composition. Protein products with high protein content (w / w) are highly desirable.

[0102] To obtain such high protein products, the whole stillage must undergo separation and / or purification steps. In Figure 1B, this is illustrated as "Innovation Steps 6 and 7" (stars 6 and 7).

[0103] In one embodiment, the whole stillage is separated into two fractions: fiber and filtrate. Separation can be achieved by using any of the existing methodologies, such as decanters, centrifuges, filter presses, pressure screens, and hydrocyclones, either alone or in combination. In one example, the whole stillage is passed through a pressure screen (e.g., a sieve size of 75 μm). The resulting fiber-free filtrate can be further separated into high density and low density slurry fractions. Separation can be achieved by using any of the existing methodologies, such as decanters, centrifuges, filter presses, pressure screens, and hydrocyclones, either alone or in combination. In one example, the fiber-free filtrate is passed through a hydrocyclone, resulting in two streams: a high density stream with insoluble solids (about 40% dry matter) and a low density overflow thin stillage stream containing soluble solids (about 5% dry matter). The dense stream is further dehydrated in a spray dryer column using hot air (about 150° C.), resulting in a high protein powder product. In one embodiment, the protein recovered in the process is spray dried.

[0104] An advantage of the process is that the final protein product is either fiber-free or contains very little fiber, while a separate fiber side-stream can be sold as a co-product or further valorized into a higher value product (such as soluble dietary fiber).

[0105] In one embodiment, the whole stillage is separated into two fractions: thin stillage, which contains soluble fiber, fat, salts, and protein, and wet cake, which consists of insoluble fiber and protein. The oil or fat content of the thin stillage is further removed using separation techniques such as disc stack centrifuges or decanters. The defatted or deoiled thin stillage is boiled (step I.viii) and returned for reuse. The wet cake is dried using conventional dryers such as tray, ring, or DDGS dryers to obtain high protein.

[0106] In one embodiment, the oil or fat content of the wet cake is treated with supercritical CO 2 Alternatively, it may be removed using an organic solvent such as hexane or octanol.

[0107] I.viii Boiling of low density stillage In one embodiment, low density stillage from the protein recovery process (see Section I.vii) is boiled using existing technologies such as natural gas fired boilers, energy recovery from a jet cooker, or other process steps. In Figure 1B, this is illustrated as "Innovation Step 5" (star 5).

[0108] As an example, low density stillage is boiled using jacket heating where steam from a boiler is used. The steam vapor generated from the low density stillage is used to distill ethanol (see Section I.vi Ethanol Recovery). This low density stillage contains volatile acids (originating from the liquefaction (low temperature saccharification) stage where the backset is mixed with the ground feedstock to create a slurry). During the boiling process, these VA are evaporated and the resulting backset stream with low VA concentration (≦0.2%) is (re)used to mix with the feedstock in an earlier step of the overall process (see Section I.iii Hydrolysis). This use of backset water from the boiling of low density stillage reduces the overall fresh water consumption of the process, which is illustrated in Example 7, where it is observed that the fresh water consumption is reduced from 2.8 L water per L ethanol to 0.6 L water per L ethanol. The reduced concentration of VA in the backset ensures that the use of this backset water will not adversely affect the ethanol yield. Boiling of low density stillage also reduces the risk of contamination coming from the thin stillage. Furthermore, boiling of low density stillage ensures maximum concentration of soluble proteins and other moieties present in the stillage, as steam (water) is lost from the stillage, and also increases the dry matter of the backset due to evaporation of the thin stillage.

[0109] I.ix CO 2 Collection of The third major product of the process of the present invention is CO 2 In FIG. 1B, this is illustrated as "Innovation Step 4" (star 4). In one embodiment, 2 is captured by the addition of carbonic anhydrase in the fermentation process. Carbonic anhydrase is an enzyme that aids in the rapid interconversion of carbon dioxide and water to carbonic acid, protons and bicarbonate ions. Carbonic anhydrase belongs to the EC 4.2.1.1 enzyme classification. One example of such a carbonic anhydrase is carbonic anhydrase from bovine erythrocytes, cas number 9001-03-0, sold by Merck.

[0110] Carbonic anhydrase may be added to one of more fermentation tanks. In a preferred embodiment, carbonic anhydrase is added to all of the fermentation tanks (innovation step 3 (star 3) in FIG. 1B). As previously disclosed, fermentation may be carried out in multiple steps. In a preferred embodiment, carbonic anhydrase is added in fermentation tanks 1, 2 and 3 (numbers refer to the first, second and third fermenters mentioned in section IV), more preferably carbonic anhydrase is added in fermentation tanks 1 and 2. In a further embodiment, carbonic anhydrase is added only in the first fermentation tank, but is carried over to the next tank(s) as the fermentation broth is transferred to the next tank as part of a continuous operation.

[0111] In one embodiment, CO 2 is captured during fermentation by the addition of carbonic anhydrase to the fermentation broth, for example, preferably by adding it directly to the feedstock hydrolysate together with the addition of yeast or at a later time. 4 is further added to the fermentation broth. The term fermentation broth refers to the feed slurry in the fermenter, which may include initial feed hydrolysate in combination with fermented feed broth as the fermentation process progresses.

[0112] In one embodiment, the amount of carbonic anhydrase added to the fermentation is 0.1-2 kg / tonne of dry matter, preferably 0.1-1.5 kg / tonne of dry matter, more preferably 0.2-1 kg / tonne of dry matter, and most preferably 0.3-0.5 kg / tonne of dry matter.

[0113] In one embodiment, the amount of carbonic anhydrase added to the fermentation is 200-4000 units / gram dry matter, preferably 200-3000 units / gram dry matter, more preferably 400-2000 units / gram dry matter, and most preferably 600-1000 units / gram dry matter. Enzyme units refer to Wilbur-Anderson units (WA units). One W-A unit will lower the pH of 0.02 M Trizma buffer at 0° C. from 8.3 to 6.3 per minute.

[0114] In one embodiment, zinc sulfate is added at a concentration of 0.6 to 6.0 mmol Zn per kg carbonic anhydrase in the fermentation broth. + In one embodiment, the amount of Zn in the fermentation broth is + The amount of Zn is 0.6 to 6.0 mmol per kg of carbonic anhydrase. + , 0.6-4.8 mmol of Zn per kg of carbonic anhydrase + , 0.6-3 mmol of Zn per kg of carbonic anhydrase + , 1.2-2.4 mmol of Zn per kg of carbonic anhydrase + or approximately 1.8 mmol of Zn per kg of carbonic anhydrase. + It is.

[0115] In one embodiment, zinc sulfate is present at a concentration of 0.3×10 per unit of carbonic anhydrase in the fermentation broth. -9 ~3.0×10 -9 mmol Zn + In one embodiment, the amount of Zn in the fermentation broth is + The amount of carbonic anhydrase is 0.3 × 10 -9 ~3.0×10 -9 mmol Zn + , 0.3 × 10 per unit of carbonic anhydrase -9 ~2.4×10 -9 mmol Zn + , 0.3 × 10 per unit of carbonic anhydrase -9 ~1.5×10 -9 mmol Zn + , 0.6 × 10 per unit of carbonic anhydrase-9 ~1.2×10 -9 mmol Zn + , or approximately 0.9 × 10 per unit of carbonic anhydrase -9 mmol Zn + It is.

[0116] In one embodiment, zinc sulfate is present in the fermentation broth at a concentration of 0.1 to 1.0 grams of ZnO per 100 grams of carbonic anhydrase. 4 In one embodiment, ZnSO in the fermentation broth is added to ensure a concentration of 4 The concentration of ZnO was 0.1–1.0 g per 100 g of carbonic anhydrase. 4 , 0.1-0.8 grams of ZnO per 100 grams of carbonic anhydrase 4 , 0.1-0.5 grams of ZnO per 100 grams of carbonic anhydrase 4 , 0.2-0.4 grams of ZnO per 100 grams of carbonic anhydrase 4 or approximately 0.3 grams of ZnSO per 100 grams of carbonic anhydrase. 4 In one embodiment, the ZnSO in the fermentation broth 4 The amounts are 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, or 1.0 g / 100 g carbonic anhydrase.

[0117] In one embodiment, zinc sulfate is present at a concentration of 0.05×10 per unit of carbonic anhydrase in the fermentation broth. -8 ~0.5×10 -8 g ZnSO 4 In one embodiment, ZnSO in the fermentation broth is added to ensure a concentration of 4 The concentration of is 0.05 × 10 per unit of carbonic anhydrase. -8 ~0.5×10 -8 g ZnSO 4 , 0.05 × 10 per unit of carbonic anhydrase -8 ~0.4×10 -8 g ZnSO 4 , 0.05 × 10 per unit of carbonic anhydrase -8 ~0.25×10 -8g ZnSO 4 , 0.1 × 10 per unit of carbonic anhydrase -8 ~0.2×10 -8 g ZnSO 4 , or approximately 0.15 × 10 per unit of carbonic anhydrase -8 g ZnSO 4 In one embodiment, the ZnSO in the fermentation broth 4 The amount of is 0.05 × 10 -8 g, 0.1×10 -8 g, 0.15×10 -8 g, 0.2 × 10 -8 g, 0.25×10 -8 g, 0.3 × 10 -8 g, 0.35×10 -8 g, 0.4×10 -8 g, 0.45×10 -8 g, 0.5×10 -8 g / unit of carbonic anhydrase.

[0118] In a preferred embodiment, Zn+ is added together with carbonic anhydrase, the amount of carbonic anhydrase added to the fermentation is 0.3-0.5 kg / tonne dry matter, the amount of Zn+ is 1.2-2.4 mmol / kg carbonic anhydrase, and the enzyme activity is 2000 units / mg enzyme.

[0119] CO 2 Use of carbonic anhydrase to capture and store trapped CO 2 The use of heat to release is illustrated in Example 5.

[0120] CO 2 Carbonic acid formed from the capture of CO by carbonic anhydrase 2 The conversion of CO2 to CO2 reduces the pH of the fermentation medium. 2 However, for continuous pH adjustment, for example by using aqueous ammonia as described in sections I.iv and Iv, CO 2 is bicarbonate (HCO3 -) thus avoiding its release during fermentation. In one embodiment, the continuous pH adjustment as described above is performed in the first stage of fermentation. In one embodiment, the continuous pH adjustment is performed only in the first stage of fermentation and not in any of the subsequent stages of fermentation. In particular, the continuous pH adjustment is performed to ensure that the pH is between 5.5 and 6.5 during fermentation.

[0121] Captured CO 2 CO is released during the distillation stage. 2 The capture of CO reduces the ethanol concentration during fermentation. 2 Since no CO is lost, the fermentation volume remains more or less constant during fermentation, thus decreasing the ethanol concentration. Ethanol concentrations above 6% induce stress in yeast. As the ethanol concentration increases, the stress increases. In this process, the ethanol-induced stress is reduced, improving conditions for the yeast, resulting in a smoother fermentation. Furthermore, CO 2 Due to minimal losses, evaporation is also significantly reduced, resulting in more water in the fermenter and diluting the ethanol concentration. Due to the continuous mode, the ethanol concentration remains at around 10%, thus reducing yeast stress.

[0122] In addition, CO 2 The capture of CO reduces the loss of ethanol through evaporation. Approximately 0.5% of the ethanol is lost during evaporation. In ethanol fermentation, approximately 40% of the ethanol is in the form of vapor, and some of this ethanol is converted to CO2 by the evaporation process. 2 Lost as a result of the draft (CO 2 leakage).

[0123] A typical ethanol plant uses a "primary scrubber system" in the vents and other process gas emissions connected to the fermentation vessel to minimize vapor ethanol emissions and other volatile organic compounds ("VOCs") escaping to the atmosphere along with carbon dioxide and other vapors. As will be recognized by those skilled in the art, VOCs can include compounds such as acetaldehyde, ethyl acetate, acrolein, and acetone. These scrubbers use fresh water with a wide variety of additives to increase the solubility of ethanol and VOCs. The scrubber precipitate is water with a low concentration of ethanol. This water mixture is then reintroduced into the process as make-up water and then goes through a heating cycle with the mash. This is often in this region where the temperature is higher than the flash point of ethanol, allowing ethanol to be lost in the vent system and destroyed in the combustion oxidizer as well. As a result, both ethanol and VOCs are vented to the atmosphere, lowering ethanol yields and polluting the environment.

[0124] In one embodiment of the present invention, a fermentor scrubber is not required, thereby preventing ethanol loss, greatly reducing fresh water consumption, avoiding the addition of additives, and reducing energy consumption by avoiding heating cycles.

[0125] CO 2 A typical ethanol plant uses an "absorption-desorption column" to capture and release the clean CO from the primary scrubber system. 2 CO is captured in an absorption column by a solvent such as monoethanolamine (MEA). 2 The -MEA composite passes through a "desorption" column, where the temperature is raised to approximately 120 °C and CO 2 is released, the MEA is regenerated, and it is recycled back into the absorption column.

[0126] In one embodiment of the present invention, an MEA-based absorption / desorption system is not required, with the advantages that the use of chemicals such as MEA is avoided and energy consumption is reduced by avoiding the desorption step.

[0127] In one embodiment, the CO produced and captured during fermentation 2 is the CO in the distillation step. 2 In one embodiment, CO captured during fermentation may be released in the form of steam. 2 Both the ethanol produced during fermentation and distillation are vaporized by applying heat in a distillation step and separated using a condenser, where the ethanol is condensed into a liquid form and CO 2 remains gaseous, and therefore CO 2 can be recovered separately from the ethanol.

[0128] In an exemplary embodiment, CO 2 and the distillation temperature for the vaporization of ethanol is about 70-90°C, preferably about 80°C, and the condensation temperature is about 5-30°C, preferably about 5°C. Finally, the released CO from the condenser step 2 can be further refined using a conventional compression-expansion system.

[0129] In one embodiment, the process involves three, two or one compression-expansion cycles; in a preferred embodiment, the purification process involves only one compression-expansion cycle, resulting in a significant reduction in energy requirements.

[0130] Ix Reuse of process water An important aspect of the present invention is the reduced fresh water requirement / use as compared to conventional ethanol production processes. Several of the process steps of the present invention contribute to this significant reduction in water requirements.

[0131] In one embodiment, water consumption is reduced by 50%, 55%, 60%, 65%, 70%, 75% or even 80% compared to state-of-the-art ethanol production processes (FIG. 1A vs. FIG. 1B). An illustrative example is provided in Example 7 showing the calculation of reduced water consumption.

[0132] For example, the moisture contribution from the raw materials can be significantly increased, for example by up to 50% (see Example 7), eliminating the need to dry the raw materials.

[0133] Furthermore, the reuse of process water within the process contributes greatly to the overall reduction of water consumption. The process water can be reused anywhere related to the process. An example of the reuse of process water is given in Example 2.

[0134] The process water for reuse in the process may or may not be subjected to further purification step(s) to remove impurities prior to its reuse. Preferably, the process water is not subjected to any purification step, e.g., chemical or physical water purification processes, prior to its reuse in the process. The microbial pretreatment discussed in section I.ii helps to ensure that the process water is clean and free of unwanted contaminants that are undesirable in themselves, but can also produce products such as VAs that are undesirable in the process water.

[0135] The process of the present invention significantly reduces the amount of process water classified as waste. As illustrated in Figure 1B, the process water is recycled for feed conditioning prior to the hydrolysis step.

[0136] In one embodiment, the condensate is returned to the boiler (illustrated as "Innovation Step 5 (star 5)" in FIG. 1B), reducing water consumption to 0.6 liters of water per liter of ethanol produced.

[0137] In one embodiment, the method further comprises recovering aqueous liquid from one or more process steps of the method. This aqueous liquid is also referred to herein as process water. In a preferred embodiment, the aqueous liquid recovered from the process is reused within the process by adding it to the feedstock (to obtain a feedstock slurry for subsequent hydrolysis as discussed in section I.iii). Thus, in one embodiment, the aqueous liquid added to the feedstock comprises recovered process water. In one embodiment, more than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or even more than 50% of the aqueous liquid added to the feedstock is process water recovered from one or more other steps of the method.

[0138] In one embodiment, process water is recovered from the distillation step and this recovered water is reused in other process steps of the method as disclosed herein.

[0139] II. Products of the Invention II.i Ethanol One of the products obtained from the present method is ethanol. As mentioned above, the ethanol yield is improved compared to conventional ethanol processes.

[0140] In one embodiment, the ethanol purity is at least 99.3%; the density at 20° C. is at most 0.7915 kg / liter; the water content is at most 0.5% (by weight); the methanol is at most 0.5% (by weight), and the total acid (as acetic acid) is at most 0.007% (by weight).

[0141] II.ii Protein feed or food products One product resulting from this process is a protein.

[0142] The term "protein feed product" refers to a protein product, which, based on its purity, can be used as a feed, e.g., for animal consumption.

[0143] The term "protein food product" refers to a protein product, which, based on its purity, can be used as a food product, e.g., for human consumption.

[0144] In the present context, purity is measured as the amount of toxins, such as deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV), and zearalenone (ZON). By applying probiotics to the raw material as an initial step of the present invention, the protein product obtained by the method of the present invention is essentially pure in the sense that it is free of toxins, since other potentially toxic microbial contamination cannot occur in the raw material due to the presence of probiotic microorganisms. Probiotic microorganisms do not themselves produce toxins.

[0145] Fungal contamination is the biggest challenge in ethanol fermentation because of its toxins. For protein products destined for human food or animal feed, the presence of fungal toxins such as aflatoxins is a huge disadvantage and is not permitted in many jurisdictions. However, the presence of lactic acid bacteria (dead cells, as they are all killed in the distillation column) is considered an added value because it is a non-GMO probiotic.

[0146] In one embodiment, the protein product has 90% dry matter; crude protein > 60%; crude fat > 8%; and essential amino acids (eg, lysine) > 2%.

[0147] II.iii CO 2 One of the products obtained from this process is CO 2 It is.

[0148] In one embodiment, CO 2The purity is 99.9%; moisture is 20 ppmv max; oxygen is 30 ppmv max; carbon monoxide is 10 ppmv max; ammonia is 2.5 ppmv max; methanol is 10 ppmv max; acetaldehyde is 0.2 ppmv max; sulfur dioxide (SO2) is 1 ppmv max; solid CO 2 No off-flavor (snow); solid CO 2 No strange appearance (snow); no strange odor or flavor in the water; no color or cloudiness in the water.

[0149] III. Advantages and Commercial Use As disclosed above, the method of the present invention has many advantages over conventional ethanol fermentation and has direct commercial applications. The following list summarizes some advantages of the process: Reduce water consumption in the ethanol process. In Example 7, it is calculated that the water consumption is reduced from 2.8 liters of fresh water per liter of ethanol in the conventional ethanol process to 0.6 liters of fresh water per liter of ethanol in the present process. A reduction in CIP cycles from 120 to 40 per year (each CIP cycle is approximately 5 hours), resulting in a reduction of approximately 400 hours / year of downtime from CIP-related activities. Produce a pure high protein product that can be used as food or feed. Reduce energy needs · Reduce ethanol GHG emissions. · Improve ethanol fermentation productivity by eliminating or reducing downtime. Eliminates the grain drying step · Improve the microbial stability of waste grain and grain materials allowing for better upcycling. Energy efficient CO 2 capture.

[0150] Preferred Numbered Embodiments of the Invention Numbered embodiment 1. A method for producing ethanol and protein feed or food products from grains and / or grain-derived products, comprising the steps of: (a) providing grain and / or grain-derived products; (b) pre-treating the grain and / or grain-derived product by applying a probiotic microorganism to the grain and / or grain-derived product; (c) adding an aqueous liquid to the pretreated grain and / or grain-derived product followed by mixing to obtain a grain slurry; (d) optionally adjusting the pH of the grain slurry to ≧5.5 by adding aqueous ammonia to the grain slurry; (e) enzymatically treating the grain slurry by adding an amylase enzyme to the grain slurry to obtain a grain hydrolysate; (f) fermenting the grain hydrolysate by adding yeast to the grain hydrolysate; and (g) i. Ethanol ii. a protein, and iii. In some cases, CO 2 Separately collecting A method comprising:

[0151] Numbered embodiment 2. The method according to numbered embodiment 1, wherein the probiotic microorganisms in step (b) are applied to the surface of the cereal and / or gran-derived product, for example by spraying the microorganisms onto the surface.

[0152] Numbered Embodiment 3. The method of numbered embodiment 1 or 2, wherein step (g) further comprises recovering process water; and the aqueous liquid added to the grain and / or grain-derived products in step (c) comprises recovered process water.

[0153] Numbered Embodiment 4. The method of any one of numbered embodiments 1-3, wherein the grain in step (a) is selected from wheat, rice, oats, barley, rye, barley, millet, corn, triticale, and sorghum seeds.

[0154] Numbered embodiment 5. The method according to any one of numbered embodiments 1 to 4, wherein the probiotic microorganism in step (b) is a lactic acid bacterium selected from, for example, Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus amylovorus, and Lactobacillus hammesii.

[0155] Numbered Embodiment 6. The method of any one of numbered embodiments 1 to 5, wherein the aqueous ammonia in step (d) is derived from a biogas plant waste stream.

[0156] Numbered embodiment 7. The method according to any one of numbered embodiments 1 to 6, wherein the enzymatic treatment in step (e) is carried out at a temperature in the range of 50 to 55°C.

[0157] Numbered Embodiment 8. The method of any one of numbered embodiments 1-7, wherein the ethanol in step (g) is recovered by distillation.

[0158] Numbered Embodiment 9. The method of any one of numbered embodiments 1 to 8, wherein the protein recovered in step (g) is spray dried.

[0159] Numbered embodiment 10. CO in step (g) 2The method of any one of numbered embodiments 1 to 9, wherein the is recovered by use of carbonic anhydrase.

[0160] Working Example Example 1A: Pretreatment of cereal material with probiotic bacteria: Lactobacillus plantarum 1A7 and W. anomalus LCF1694 Microbial stability on laboratory-scale bread was performed using Lactobacillus plantarum 1A7. Lactobacillus plantarum 1A7 was grown for 24 h at 30°C in MRS (nonselective medium for the growth of lactic acid bacteria, Oxoid Laboratories, Hampshire, UK) supplemented with live yeast extract (5% [vol / vol]) and 28 mM maltose to a final pH of 5.6 (mMRS). W. anomalus LCF1694 was grown for 48 h at 30°C in YEPG (10 g / liter yeast extract, 10 g / liter peptone, 20 g / liter glucose). The antifungal activity of Lactobacillus plantarum 1A7 and W. anomalus LCF1694 was investigated by an in vitro challenge test to control spoilage of bread samples caused by Mucor species VBBM7 and Aspergillus fumigatus MTCC2796. Bread samples were cut into small pieces (4.0 ± 0.3 cm / 8.2 ± 0.4 cm), sterilized in Petri dishes and divided into six sets. The first and second sets were treated only with cell suspensions of Lactobacillus plantarum 1A7 and W. anomalus LCF1694, respectively. The third and fourth sets were treated with Mucor species (3.7 × 10 4 spores / ml) and Aspergillus fumigatus MTCC2796 (4.6 × 104 The bread pieces were inoculated separately with 1000 spores / ml. In the fifth set, cell suspensions of Lactobacillus plantarum 1A7 and W. anomalus LCF1694 were added before treatment with Mucor and Aspergillus species. The sixth set was kept where the bread pieces were not treated with either organism. The same procedure (sterilization and inoculation) was repeated with grains from wheat, corn, triticale and rice. Care was taken to adjust the moisture of the grains to about 30% with sterile water.

[0161] All samples were kept at room temperature (26.0 ± 2.0°C) for up to 30 days and observed for the development of spoilage.

[0162] [Table 1]

[0163] Bread treated with probiotics remained microbiologically stable even after a challenge where spores from Aspergillus fumigatus and Mucor were sprinkled onto the probiotic-treated bread. Controls challenged with Aspergillus fumigatus or Mucor and not receiving any probiotic treatment showed heavy growth of Aspergillus fumigatus and Mucor. Interestingly, controls not treated with probiotics and not challenged with Aspergillus fumigatus or Mucor also became contaminated within 7 days as evidenced by the bread cavity.

[0164] Conclusion: Lactobacillus plantarum 1A7 and W. anomalus LCF1694 prevented mold contamination in cereals and bread for almost one week.

[0165] Example 1B: Pretreatment of bread with probiotic bacteria: Bacillus species Microbial stability of bread made from laboratory-scale wheat flour was carried out using the Bacillus species; Bacillus licheniformis, Bacillus clausii, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus fusiformis and Bacillus megaterium.

[0166] All Bacillus cultures were maintained on agar plates in 3% trypticase soy broth (TSB) and 0.5% yeast extract and inoculated into test tubes containing 3.5 mL of 3% TSB and 0.5% yeast extract. After 24 h of incubation at 37°C, the cultures were transferred to 1 L or 2 L volumes of 0.3% TSB in a ratio of 1 L per tube. Spores were collected after 6 days of incubation at 37°C. The antifungal activity of Bacillus species was investigated by an in vitro challenge test to control spoilage of bread samples caused by Mucor species VBBM7 and Aspergillus fumigatus MTCC2796. The bread samples were cut into small pieces (4.0±0.3 cm / 8.2±0.4 cm), sterilized in Petri dishes and divided into nine sets.

[0167] The first set was treated with a cell suspension of Bacillus spp. only. The second and third sets were treated with Mucor spp. (3.7 × 10 4spores / ml) and Aspergillus fumigatus MTCC2796 (4.6 × 10 4 In the fourth set, a cell suspension of Bacillus licheniformis was added before treatment with Mucor and Aspergillus species. In the fifth set, a cell suspension of Bacillus clausii was added before treatment. In the sixth set, a cell suspension of Bacillus amyloliquefaciens was added before treatment. In the seventh set, a cell suspension of Bacillus subtilis was added before treatment. In the seventh set, a cell suspension of Bacillus megaterium was added before treatment. Finally, in the eighth set, all bacilli species were used to make a suspension and added prior to treatment with Mucor and Aspergillus species. The ninth set was kept where the bread pieces were not treated with any organisms.

[0168] The same procedure (sterilization and inoculation) was repeated with grains from wheat, corn, triticale and rice, taking care to adjust the grain moisture to approximately 30% with sterile water.

[0169] All samples were kept at room temperature (30.0 ± 2.0°C) for a maximum of 30 days and observed for the occurrence of spoilage.

[0170] [Table 2]

[0171] Conclusion: Most Bacillus species are able to control mold growth for up to 4-6 days. Interestingly, a consortium of bacilli can prevent mold contamination for more than 8 days.

[0172] Example 2: Reuse of process water and pH adjustment using aqueous ammonia A laboratory-scale simulation of untreated backset containing ≥ 1 g / l volatile acid for ground flour was carried out using shake flasks (starting weight of 75 g), where white bread was ground and mixed with water to 30% slurry dry matter. Acetic and lactic acid solutions were added to shake flasks 3, 4, 5 and 6 to 1% and 0.5% concentrations, respectively. The pH was adjusted to 6.20 in flasks no. 5 and 6 using 20% ​​ammonia solution. Alpha amylase, Novozymes Thermostable Alpha Amylase Liquoflow GO 2X (dosage; 0.2 kg / tonne of bread), was added and liquefaction was carried out at 85 °C for 2 h. It is noted that the optimum temperature range for this thermostable alpha amylase is 75-86 °C. The resulting liquefied mash was cooled to 32 °C. An appropriate amount of urea was added to all shake flasks to achieve an inorganic nitrogen concentration of 250 ppm. Approximately 1 g of dried yeast Innova Fit from Novozymes was added to all shake flasks. Finally, glucoamylase and cellulose-containing Saczyme yield from Novozymes (dosage; 0.5 kg / ton of pan) was added to all flasks. Note that this enzyme is different from the alpha amylase used in liquefaction. The main purpose of this enzyme is to produce glucose. Moreover, the optimum temperature range for this enzyme is 30-40°C. All flasks were kept in an incubator shaker (32°C at 80 RPM). After 72 hours, samples were taken from the flasks to measure key parameters such as pHh, glucose, and ethanol.

[0173] [Table 3]

[0174] As can be seen from the above results, adjusting the pH to about 6.20 with aqueous ammonia is confirmed to prevent inhibition of acetic and lactic acids without affecting the ethanol titer.

[0175] Example 3: Raw Starch Hydrolysis (Low Temperature Saccharification) Process Lab-scale experiments were carried out using shake flasks (starting weight of 75 g) in which white bread was ground and mixed with water to a slurry dry matter of 30%. The pH was measured (but not adjusted). Liquoflow GO 2X (thermostable alpha-amylase, dosage; 0.2 kg / tonne of bread) was added to shake flasks 1 and 2 and liquefaction was carried out at 85°C for 2 hours. Saczyme cold mash, consisting of raw starch hydrolyzing enzymes glucoamylase, acid amylase and cellulose from Novozymes, was added at dosage; 1 kg / tonne of bread to shake flasks 3 and 4 and liquefaction was carried out at 45°C for 30 minutes. The resulting liquefied mash was cooled to 32°C. An appropriate amount of urea was added to all shake flasks to achieve an inorganic nitrogen concentration of 250 ppm. Approximately 1 g of dry yeast (Innova Fit) was added to all shake flasks. Finally, glucoamylase, Saczyme yield (dosage; 0.5 kg / tonne of bread) was added to all flasks. There is some glucoamylase activity in the Saczyme cold mash, and the reason for adding another glucoamylase, Saczyme yield, later in the fermentation is to enhance the saccharification process. All flasks were kept in an incubator shaker (32°C at 80 RPM). After 72 hours, samples were taken from the flasks to measure key parameters such as pH, glucose, and ethanol.

[0176] [Table 4]

[0177] As can be seen from the above results, it is confirmed that similar ethanol titers can be achieved using a low temperature saccharification process.

[0178] Example 4: Ammonium nitrate as an inorganic nitrogen source A lab-scale experiment was carried out using shake flasks (starting weight of 75 g) in which white bread was ground and mixed with water to a slurry dry matter of 30%. The pH was measured (but not adjusted). Saczyme cold mash, a live starch hydrolase (dosage; 1 kg / tonne of bread), was added to all shake flasks and cold liquefaction (also called live starch hydrolysis) was carried out at 45°C for 30 min. The resulting liquefied mash was cooled to 32°C. An appropriate amount of urea was added to shake flasks 1 and 2 to achieve an inorganic nitrogen concentration of 250 ppm. An appropriate amount of ammonium nitrate was added to shake flasks 3 and 4 to achieve an inorganic nitrogen concentration of 250 ppm. Approximately 1 g of dry yeast (Innova Fit) was added to all shake flasks. Finally, Saczyme yield, an aminoglucosidase (dosage; 0.5 kg / tonne of bread), was added to all flasks. All flasks were kept in an incubator shaker (32° C. at 80 RPM). After 72 hours, samples were taken from the flasks to measure key parameters such as pH, glucose, and ethanol.

[0179] [Table 5]

[0180] As can be seen from the above results, it is confirmed that the use of ammonium nitrate as the inorganic nitrogen source maintained the pH at approximately the starting pH.

[0181] It was observed that in the batches containing ammonium nitrate, the pH did not drop below 5. It is considered that besides nitrate respiration, deamination may also be one of the reasons for the same.

[0182] Example 5A:CO 2Carbonic anhydrase to capture Lab-scale experiments were carried out using shake flasks (starting weight of 75 g), in which white bread was ground and mixed with water to a slurry dry matter of 30%. Saczyme cold mash, a raw starch hydrolase (dosage; 1 kg / tonne of bread), was added to all shake flasks and liquefaction was carried out at 45 °C for 30 min. The resulting liquefied mash was cooled to 32 °C. An appropriate amount of urea was added to all shake flasks to achieve an inorganic nitrogen concentration of 250 ppm. Approximately 1 g of dry yeast (Innova Fit) was added to all shake flasks. Saczyme yield, an aminoglucosidase (dosage; 0.5 kg / tonne of bread), was added to all flasks. Carbonic anhydrase (dosage: 0.3 kg / tonne of bread) was added to flasks no. 3–6. Finally, ZnSO 4 was added to flasks 5 and 6 to add approximately 0.3 grams of ZnSO 4 100 grams of carbonic anhydrase was ensured. All flasks were kept in an incubator shaker (32°C at 80 RPM). pH was measured twice daily in all flasks. In flasks 3-6, pH was adjusted to approximately 5.5-6.0 using aqueous ammonia. In all flasks, a few ml of CO 2 Indicator fluid (Aqua Rebell CO 2 Check with your local aquarium shop) and add CO 2 The concentration was monitored qualitatively and the color was monitored twice daily. After 72 hours, samples were taken from the flasks to measure weight loss (escaped CO 2 ), pH, glucose, and ethanol were measured as key parameters.

[0183] [Table 6]

[0184] As can be seen from the above results, carbonic anhydrase is 2 Capture and store CO from shake flasks 2It is confirmed that carbonic anhydrase prevents loss / leakage. The weight loss was significantly reduced in the fermentations with added carbonic anhydrase (shake flasks no. 3-6). Surprisingly, the addition of zinc (Zn+) to the fermentation medium reduced the CO 2 As can be seen from the results, flasks 5 and 6 have the lowest weight loss, hence the ethanol concentration (due to less evaporation losses). By observing the color of the reagent, it is clear that the CO 2 The capture was further confirmed by the reagent used, which consisted of a pH indicator, bromothymol blue, in water. 2 After contact with CO, the solution turns yellow due to the formation of acidic carbonic acid. In shake flasks 1 and 2, the color changes from blue to yellow, and CO 2 This suggests that CO was released from the vent during fermentation. In shake flasks 5 and 6, where Zn+ was added, the color of the reagent remained unchanged from blue, indicating that CO 2 It is interesting to note that the color of the reagent changed from blue to green in shake flasks 3 and 4, suggesting no or minimal CO 2 This suggested that there was some release of , but it was still less compared to shake flasks 1 and 2.

[0185] Example 5B: CO 2 Effect of temperature on emission The shake flasks from section 5A were kept in an incubator shaker and the shaker temperature was increased to 80 °C in 10 °C intervals every hour. The increased temperature allowed for the release of CO from the fermentation broth. 2 The optimum temperature for CO release was found. 2 To qualitatively monitor CO emissions, 2 The color of the check reagent was observed throughout the experiment.

[0186] [Table 7]

[0187] As the results show, the captured CO 2 CO from2 The optimum temperature for release (shake flasks 3 to 6) is about 80°C, at which the color of the reagent turns yellow.

[0188] The results from 5A and 5B verify our hypothesis. Carbonic anhydrase converts CO into CO in the fermentation broth while the fermentation is in progress. 2 When the temperature is increased to about 80 °C, such as that applied in the distillation stage, the captured CO 2 is released.

[0189] Example 6: Probiotic-treated feedstock, pH adjustment, ammonium nitrate and carbonic anhydrase Finally, laboratory-scale simulations were performed to determine the integrated impact of all the above-mentioned techniques on ethanol fermentation.

[0190] For shake flasks 1 and 2, white bread was ground and mixed with water to a slurry dry matter of 30%. The pH was measured (but not adjusted). Liquoflow GO 2X (dosage; 0.2 kg / tonne bread) was added to both shake flasks and liquefaction was carried out at 85°C for 2 hours. The resulting liquefied mash was cooled to 32°C. An appropriate amount of urea was added to all shake flasks to achieve an inorganic nitrogen concentration of 250 ppm. Approximately 1 g of dry yeast (Innova Fit) was added to all shake flasks. Finally, Saczyme yield, an aminoglucosidase (dosage; 0.5 kg / tonne bread), was added to all flasks.

[0191] For shake flasks 3–6, the following procedure was used: Lactobacillus plantarum 1A7 and Lactobacillus fermentum were grown for 24 h at 30°C in MRS (Oxoid Laboratories, Hampshire, UK) supplemented with live yeast extract (5% [vol / vol]) and 28 mM maltose to a final pH of 5.6 (mMRS). W. anomalus LCF1694 was grown for 48 h at 30°C in YEPG (10 g / liter yeast extract, 10 g / liter peptone, 20 g / liter glucose). Appropriate amounts of cultures from both species were added to strong flour to give approximately 10^3 cells per gram of strong flour. The resulting mixture was mixed with water to give a slurry dry matter of 30%.

[0192] For shake flasks 7-8, the following procedure was used: All Bacillus cultures were maintained on agar plates in 3% trypticase soy broth (TSB) and 0.5% yeast extract and inoculated into test tubes containing 3.5 mL of 3% TSB and 0.5% yeast extract. After 24 hours of incubation at 37°C, the cultures were transferred to a volume of 1 L or 2 L of 0.3% TSB in a ratio of 1 L per test tube. After 6 days of incubation at 37°C, spores were harvested. An appropriate amount of Bacillus spores was added to the flour to give approximately 10^3 spores per gram of flour. The resulting mixture was mixed with water to give a slurry dry matter of 30%.

[0193] In shake flasks no. 3 and 4, the pH was measured (but not adjusted). In shake flasks 5-8, the pH was adjusted to 6.20 using 20% ​​ammonia solution. Saczyme cold mash, a live starch hydrolase (dosage; 1 kg / tonne of pan), was added to shake flasks 3-6 and liquefaction was carried out at 45°C for 30 min. The resulting liquefied mash was cooled to 32°C.

[0194] An appropriate amount of urea was added to shake flasks Nos. 1-4 to achieve an inorganic nitrogen concentration of 250 ppm. An appropriate amount of ammonium nitrate was added to shake flasks Nos. 5-8 to achieve an inorganic nitrogen concentration of 250 ppm.

[0195] Carbonic anhydrase (dosage: 0.3 kg / tonne) was added to flasks no. 5-8 6.

[0196] Approximately 1 g of dry yeast (Innova Fit) was added to all shake flasks. Finally, aminoglucosidase, Saczyme yield (dosage; 0.5 kg / tonne of pan) was added to all flasks. All flasks were kept in an incubator shaker (32°C at 80 RPM). After 72 hours, samples were taken from the flasks to measure weight loss (escaped CO 2 ), pH, glucose, ethanol and glycerol were measured as main parameters.

[0197] [Table 8]

[0198] [Table 9]

[0199] From the above results, the following is confirmed: In shake flasks 3 and 4, uncontrolled growth of Lactobacillus and W. anomalus resulted in lactic and acetic acid production of ≥1%, which also stressed the yeast. The glycerol:ethanol ratio (g / g) is a good indicator of yeast stress; a ratio of approximately 0.07-0.09 is considered a healthy ethanol fermentation (Brumm & Hebeda, 1988). As can be seen in the table, compared to shake flasks 1 and 2, the glycerol:ethanol ratio is about three times higher in shake flasks 3 and 4, suggesting yeast stress (due to the strong inhibitory effect of volatile acids on yeast), which is also reflected in the higher unutilized glucose concentration and less weight loss. Surprisingly, the amount of ethanol produced (g) improved in shake flasks 5 and 6, despite the growth of Lactobacillus and W. anomalus (confirmed by microscopy). Another interesting observation is the “reduced weight loss” in shake flasks 5-8 compared to the controls (shake flasks 1 and 2). The reduced weight loss is due to the CO 2 The effect of CO capture and conversion to carbonic acid by carbonic anhydrase. Furthermore, on an industrial scale, CO 2 Note that a significant amount of water is lost along with the CO. 2 They succeeded in converting it into carbonic acid, thereby reducing the amount of CO in the atmosphere. 2 and appears to inhibit water loss. Another observation based on the lactate and acetate concentrations from shake flasks 5 and 6 is that initially, both Lactobacillus and W. anomalus outnumbered yeast, but eventually yeast appeared to prevail. A plausible explanation could be that the yeast was not stressed because the pH was maintained at ≥ 5.50 for nitrate respiration and bicarbonate formation (influence of carbonic anhydrase). Another observation based on the concentration of Bacillus from shake flasks 7 and 8 is that Bacillus species did not grow at all fermentation stages and yeasts appeared to predominate. A plausible explanation could be that Bacillus is an aerobic organism, whereas ethanol fermentation is strictly anaerobic. Another observation based on the glycerol to ethanol ratio in shake flasks 7 and 8 appears to be that yeast cells are not stressed in the presence of Bacillus, which is also reflected in the slightly higher ethanol concentration compared to shake flasks 5 and 6. A plausible explanation could be that Lactobacillus species are able to grow in anaerobic environments utilizing some sugars, whereas Bacillus cannot.

[0200] Example 7: Three-stage continuous fermentation Figure 2 illustrates the experimental setup of the three-stage fermentation system. The substrate feed bottles contained a mixture of bread crumb, Saczyme C mash (raw starch hydrolase), Saczyme yield (mixture of cellulase and glucoamylase), Viscoferm (xylanase) and urea. Liquefaction was carried out by raising the slurry temperature to about 45°C. After 2 hours, about 1.2 liters of the resulting liquefaction was transferred to fermenter 1 (stage 1). Innova Fit (yeast) was inoculated to kickstart the fermentation after adjusting the pH to 6.20 using aqueous ammonia.

[0201] A minimum ethanol concentration of about 6% was maintained to be bactericidal. To keep the ethanol in that concentration range, pumps P1 and P2 were started at a rate of 150 ml / h when the ethanol concentration in fermenter 1 was about 6% ethanol (w / w; about 20 hours). This was achieved with a dilution rate (D) of 0.15 h. -1 The temperatures in stages 1 and 2 were maintained at 35°C and 30°C, respectively, using cold water. When the ethanol concentration in fermenter 2 was approximately 10% ethanol (w / w; approximately 36 hours), pump P3 was started at a rate of 150 ml / h. This corresponds to a dilution rate (D) of 0.05 h -1The fermented mash was continuously harvested in the final fermentation tank (also called the beer well tank) where it was further fermented for another 6-7 hours to a final ethanol concentration of about 12-15% (w / w). The fermenter level and pump speed were continuously monitored to avoid any changes in dilution rate. Samples were taken at regular intervals to measure ethanol, sugars, glycerol and biomass. The continuous process was run for 10 days to check robustness and ensure steady state in stages 1 and 2.

[0202] The same procedure (as described above) was repeated using the probiotic pretreated feedstock (described in Example 6). Note that in this experiment, the feedstock was treated with Lactobacillus plantarum 1A, Lactobacillus fermentum, W. anomalus and bacilli species.

[0203] Finally, a challenge test was performed, where Aspergillus niger spores and Lactobacillus fermentum were introduced into the stage 1 fermenter to mimic contamination. The main reason behind the addition of both Aspergillus niger spores and Lactobacillus fermentum is to examine the effectiveness of the continuous fermentation. Aspergillus niger is chosen to represent a mold contaminant. Lactobacillus fermentum is chosen to represent a bacterial contaminant of lactic acid; it is well known that fermentation arrest can be associated with acetate production, especially by obligate heterofermentative species, e.g., L. fermentum and L. mucosae. Samples were taken at regular intervals and microscopic examination was performed.

[0204] The results from the continuous fermentation can be seen in FIG.

[0205] Conclusion: As can be seen from Figure 3, despite contamination (addition of pollutants), the ethanol concentration (%w / w) was maintained very well within the range of 5-8% in stage 1 and 10.5-12% in stage 2. Furthermore, the probiotics (lactobacilli and bacilli species) did not show any detrimental effect on the ethanol fermentation process.

[0206] As can be seen from Figure 3, in stage 1, the ethanol concentration drops slightly but remains at ≥ 5% and, interestingly, performance recovers in stage 2, suggesting robustness of the yeast cells against contaminants. During the challenge test, in some batches, fungal spores were observed on day 1 in samples taken from stage 1 (due to the addition); however, none were observed on day 2. Similar observations were made with Lactobacillus fermentum (based on microscopy). A possible reason is that unfavorable growth conditions (high concentration of native yeast population means competition for substrate, limited high ethanol concentration and anaerobic conditions) lead to a reduced growth (≤ 0.15 h). -1 ), or no growth (for Lactobacillus) and no spore germination (for Aspergillus). -1 , these had no growth or very low growth rates resulting in "washout." It is well known that in continuous processes, populations that manage to grow at high dilution rates can persist, while low growth rates result in washout.

[0207] The advantage of a continuous system with phase-specific growth / dilution rates is that it mimics the natural selection mechanism of "survival of the fittest". The continuous system ensures two selection pressures, high growth rate and high ethanol concentration to prevent contamination. Furthermore, even in a phase-specific continuous system, a healthy yeast population is always maintained in phase 1 (the yeast cell growth rate is constant at the set dilution rate (0.15 h -1 As soon as the pH drops below 0.1, it is guaranteed to be washed out. Maintaining yeast health plays an important role in overcoming possible contamination.

[0208] In stage 2, the dilution rate is designed to promote ethanol fermentation. Aspergillus niger spores and Lactobacillus fermentum cells were observed the same day after addition in stage 1, confirming the "washout" phenomenon. Furthermore, it was observed that these contaminants were washed out into the beer well tank the next day, where they remained inactive.

[0209] Stage 3 is the beer well tank, whose main purpose is to ensure complete conversion of the starch before it is sent to the distillation column. The ethanol concentration was measured and found to be in the range of 10-13% w / w.

[0210] Example 8: Sustain max Overview of energy and water consumption of technologies A model-based simulation was performed to show the possible water savings from the cold mash / raw starch hydrolysis step. A thermodynamics-based water balance for a state-of-the-art bioethanol plant was used as a baseline (Pfromm. 2008). The water balance is based on the fundamental principal that the thermal energy intake of a state-of-the-art bioethanol facility needs to be balanced by an appropriate heat sink, here assumed to be the evaporation of water in cooling towers. The model is further extended to include water savings in the boiler (resulting in condensate being returned to the boiler, thereby reducing the fresh water required for boiler operation).

[0211] The model assumed that the average thermal energy consumption in a bioethanol plant is about 9280 British Thermal Units (BTU) per liter of ethanol, via a boiler where natural gas is burned and 77% of the released heat is transferred to the process steam. Therefore, about 7146 BTU goes into the process per liter of ethanol produced. This energy needs to be released to the environment by evaporation of water in cooling towers, estimated at 3.15 liters of water per liter of ethanol produced (see equation number 1 below). However, there is about 0.32 liters of water per liter of ethanol, given the moisture from the grain, so the actual water consumption turns out to be 2.8 liters of water per liter of ethanol produced. Interestingly, the G2B Sustain max The process has the potential to reduce water consumption to 0.6 l water / l ethanol, which is almost five times higher than conventional processes.

[0212] This is probably because: a) Reduced energy requirements to 4732 BTU / L of ethanol, which translates into a significant reduction in water needed for heat balance. b) The water provided by the grain material is greater than that of grains used in conventional bioethanol processes (as drying is avoided). c) Condensate is returned to the boiler, further reducing fresh water consumption.

[0213] In this calculation, it was assumed that the ethanol yield and process water remained unchanged at both process conditions.

[0214] Equation to estimate water consumption in an ethanol plant: Formula Number 1: E total,thermal * ε nat.gas * V EtOH = V H20 *рH20 (Δh latent,H20 + ΔT Cp Where: E total,thermal is the specific heat energy from burning natural gas in joules per liter of ethanol = 9692376 J / L of ethanol ε nat.gas is the process thermal efficiency for steam production from the combustion of natural gas = 0.77 V EtOH is the volume of ethanol produced in liters V H20 is the volume of cooling water in liters рH20 is the density of water = 1kg / l ΔT is 25KCp, and the average specific heat capacity of water = 4178.2J / Kg.K Δh latent,H20 is the average enthalpy of vaporization of water = 2264000 J / Kg

[0215] [Table 10]

[0216] References Brumm PJ and Hebeda RE. Glycerol production in industrial alcohol fermentations. Biotechnology Letters. 1988. Vol 10, No 9, 677-682. Lewis S.M et.al., Methods and systems for producing ethanol using raw starch. 2011. US007919291B2 Pfromm. P. The Minimum Water Consumption of Ethanol Production via Biomass Fermentation. The Open Chemical Engineering Journal, 2008, 2, 1-5 Rich, J. O., Leathers, T. D., Bischoff, K. M., Anderson, A. M., & Nunnally, M. S. (2015). Biofilm formation and ethanol inhibition by bacterial contaminants of biofuel fermentation. Bioresource Technology, 196, 347-354. Wu, May, and Xu, Hui. Consumptive Water Use in the Production of Ethanol and Petroleum Gasoline - 2018 Update. United States: N. p., 2018.

Claims

1. 1. A process for producing ethanol and a protein feed or food product from a feedstock comprising starch and protein, comprising: (a) providing said feedstock comprising starch and protein; (b) pre-treating the feedstock by applying to the feedstock a culture of one or more probiotic species of Lactobacillus and / or spore-forming Bacillus; (c) adding an aqueous liquid to the pretreated feedstock followed by mixing to obtain a feedstock slurry; (d) optionally adjusting the pH of the feedstock slurry to a pH of 5-7; (e) adding an amylase enzyme to the feedstock slurry and incubating the slurry to obtain a feedstock hydrolysate; (f) adding yeast to the feedstock hydrolysate to obtain a fermentation broth, and step 1 is performed at a dilution rate of 0.10 to 0.55 h －１ and stage 2 has a dilution rate of 0.04 to 0.10 h －１ fermenting the fermentation broth by a continuous fermentation comprising two stages having a steady state ethanol concentration of 5-11% (w / w) in stage 1 and 10-13% (w / w) in stage 2; (g) i. ethanol, ii. CO ２ , and iii. Protein Separately collecting Including, CO ２ However, carbonic anhydrase and Zn ＋ to the fermentation broth during the continuous fermentation; ２ is separated and recovered in a distillation and concentration step.

2. 2. The method of claim 1, wherein the feedstock is a grain and / or one or more grain-derived products.

3. 3. The method of claim 2, wherein the grain is selected from wheat, rice, oats, barley, rye, barley, millet, corn, triticale, and sorghum seeds.

4. 4. The method of any one of claims 1 to 3, wherein the moisture content of the feedstock is at least 10% w / w, at least 15% w / w, at least 20% w / w, or at least 30% w / w.

5. 5. The method of claim 1, wherein the probiotic culture in step (b) is applied onto the surface of the feedstock by spraying the probiotic culture onto the surface and optionally mixing.

6. 8. The method of claim 1, wherein the probiotic culture in step (b) comprises Lactobacillus species and spore-forming Bacillus species.

7. 7. The method according to any one of claims 1 to 6, wherein the species of Lactobacillus is selected from Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus amylovorus, and Lactobacillus hammesii.

8. 8. The method according to any one of claims 1 to 7, wherein the species of spore-forming Bacillus is selected from Bacillus licheniformis, Bacillus clausii, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus fusiformis and Bacillus megaterium.

9. The continuous fermentation in step (f) is carried out at a dilution rate of 0.04 to 0.10 h －１ wherein the ethanol concentration in step 3 is 12-15% (w / w).

10. 10. The method of any one of claims 1 to 9, wherein step (f) comprises maintaining a pH of 5 to 6 in stages 1 and 2.

11. Step (g) is 0.3 × 10 per unit of carbonic anhydrase －１２ ~3.0 x 10 －１２ mol of Zn ＋ 11. The method of claim 1 , further comprising the addition of

12. The Zn ＋ But ZnSO ４ The method according to any one of claims 1 to 11, wherein the compound is added in the form of

13. The ethanol and the CO ２ The recovery and separation of the ethanol and CO ２ evaporating the ethanol, the ethanol being condensed in liquid form, and the CO ２ The ethanol and the CO are separated using a condenser in which CO is released in gas form. ２ 13. The method of claim 1, further comprising separating said vapor from said mixture.

14. 14. The method of claim 13, wherein the temperature for evaporating the ethanol and CO2 is 70-90°C and the temperature in the condenser is 5-30°C.

15. 15. The method of any one of claims 1 to 14, wherein step (g) further comprises recovering aqueous liquid resulting from one or more previous steps of the method; and the aqueous liquid added to the feedstock in step (c) comprises the aqueous liquid recovered in step (g).

16. 16. The method of claim 15, wherein the aqueous liquid recovered in step (g) is recovered from the distillation step.

17. 17. The method of claim 15 or 16, wherein greater than 50% of the aqueous liquid added to the feedstock in step (c) is the aqueous liquid recovered in step (g).

18. 18. The method of any one of claims 1 to 17, wherein the pH in step (d) is adjusted to pH 5.5 to 7.

19. 19. The method of any one of claims 1 to 18, wherein the pH in step (d) is adjusted using aqueous ammonia, such as aqueous ammonia from a biogas plant waste stream.

20. 20. The method of any one of claims 1 to 19, wherein step (e) is carried out at a temperature in the range of 45 to 55°C.

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