Improved fermentation processes for making ethanol

Abstract

The present invention provides a probiotic and dextranase pretreatment method for improving microbial stability and yield of sugar syrups (e.g. molasses) without using thermal, chemical or antimicrobial pretreatment. Pretreated sugar syrup is applied in a continuous ethanol fermentation using at least two fermenters for dilution from one to next to maintain steady state ethanol concentration, resulting in ethanol productivity improvement as well as reduction in energy and water usage compared to traditional ethanol plants, without affecting quality and quantity of the end products.

Description

[0001] TITLE: IMPROVED FERMENTATION PROCESSES FOR MAKING ETHANOL

[0002] FIELD OF THE INVENTION

[0003] The present invention provides a method for making ethanol, preferably in combination with making a protein feed or food product, from a sugar rich feedstock. The method facilitates a reduction in energy and freshwater consumption, avoids the need to add antimicrobials, and improves ethanol yield compared to traditional ethanol plants, without affecting quality and quantity of the end products, such as protein for feed or food.

[0004] The present invention relates to a method for producing ethanol and a high protein feed or food product from a feedstock comprising sugars, where a key step of the method is bio-preservation of feedstock using probiotic microorganisms, thereby avoiding energy intensive thermal treatment to sterilize the feedstock, and further improving ethanol yield by avoiding formation of non-fermentable polysaccharides such as dextran, by controlling growth of contaminants which produce dextran, such as Leuconostoc mesenteroides. Additionally, any existing dextran in the feedstock (e.g. produced before the bio-preservation treatment) may preferably be enzymatically converted to monosaccharides, such as by addition of dextranase or a microorganism which produces dextranase, for example Lactobacillus bifidus.

[0005] By combining several process steps in a novel and innovative way, the method of the present invention facilitates reduction in energy consumption and related water savings, improved ethanol productivity, overall improved ethanol yields, and improved conditions for yeast fermentation.

[0006] BACKGROUND OF THE INVENTION

[0007] Molasses is the one of the main by-products of sugar processing of sugar containing crops such as cane sugar and sugar beets. Molasses primarily comprises sugars, with the most prominent sugar being sucrose. It further comprises other sugars and a variety of compounds, which give it its distinctive flavor and properties. The types of sugars found in molasses include: sucrose, glucose and fructose (produced when sucrose is broken down), and potentially small amounts of lactose and galactose. Molasses-derived bioethanol has emerged as a leading renewable material because it has many advantages; e.g. its production requires no saccharification, compared to grains it has lower carbon footprint, and it can avoid competition with sugar production. The efficiency of yeast in turning the sugars from the molasses into alcohol depends very much on the quality of the molasses to be fermented. In industrial production of ethanol, usually, the molasses substrate is not fully sterilized (due to scale of operation it is difficult to use traditional sterilization methods) prior to the fermentation process, which allows the entry of a large number of microbial contaminants in the process, which negatively affects efficiency and productivity. Contaminants may produce acetic and lactic acids, increase yeast flocculation, and reduce yeast viability.

[0008] One of the main contaminants found in molasses itself is Leuconostoc mesenteroides, a lactic acid bacterium which causes sucrose molecules to polymerize into non- fermentable dextran chains. If the contamination is very extensive, the molasses may look 'ropey' due to this polymerization process by Leuconostoc mesenteroides. The produced dextran is unfermentable and will result in reduced ethanol yields. Leuconostoc, being lactobacilli, produces lactic acid, which inhibits the yeast and thus further reduces ethanol yield. Apart from this, production of dextran also causes various problems such as an increase of viscosity which produces blockage in the process line, pumps and filters; lower heat exchange; evaporation diminution; blockages in centrifuges and sucrose losses to molasses.

[0009] Ethanol producers are aware of these molasses contamination challenges and use various methods to control contaminant populations during the ethanol fermentation process. Some of the commonly used methods include

[0010] (i) Acid treatment (Melle-Boinot process) where large amount of sulfuric acid is used to pretreat molasses to reduce bacterial contamination. However, it is not completely efficient and may select resistant bacteria and consequently promote the osmotic stress of the yeasts, and as a response, the yeasts produce greater amounts of glycerol, thereby reducing the ethanol yield.

[0011] (ii) Another common way to control bacterial contamination in distilleries is the use of antibiotics, but drug resistance has been a limiting factor in contamination control efficiency. Some sugar factories use Busan, a commercial name for L12-C14 A / Kyldimethyle benzyl ammonium chloride or Benzalkonium chloride (50% aqueous solution) to inhibit Leuconostoc mesenteroides. However, the big limitation is, it is well known that Benzalkonium chloride also has proven efficacy against yeast, limiting its use when the treated molasses is used for ethanol production.

[0012] (iii) Thermal treatment like jet cooker which is commonly used in grain-based ethanol production process and can be used to reduce contamination load in molasses, but this is expensive with respect to energy requirement, further, high viscosity from molasses has negative impact on jet cooker operations due to blockages resulting in frequent shutdowns. (iv) Another way of sterilizing molasses is by using electron beams to irradicate contaminants. Electron beams results in damaging microbial replication machinery (DNA.RNA), resulting in cell death. Although the technology is sophisticated and advanced, it is less effective against spores and particulate media.

[0013] (v) Besides molasses, water used for dilution of the molasses is the second biggest source of contamination. The dissolved solids concentration in molasses is ~ 60-80 Brix (Brix is a measure of the amount of dissolved solids in a liquid via its specific gravity) and is very viscous. The mixture of molasses and water is made to provide the ideal sugar concentration for the fermentation process. Some of the most used methods to control contamination in water are chlorination, physical filtration, membrane filtration and thermal pretreatment. Chlorine treatment is effective, but it can also have a damaging effect on yeast later in the fermentation process, membrane filtration can be expensive and may not be able to prevent contamination 100%, thermal pretreatment is effective but can be expensive in terms of energy requirement.

[0014] Another challenge is inhibitory volatile compounds present in the molasses. Approximately 1-3% butyric acid is formed during production of beet molasses which is enough to inhibit yeast growth.

[0015] SUMMARY OF THE INVENTION

[0016] The present invention concerns an improved fermentation process, using a sugar syrup (e.g. molasses) as a substrate in the fermentation process or a as booster to the fermentation process.

[0017] In a first aspect, the present invention concerns a fermentation process, using a pretreated sugar syrup as a substrate in a fermentation process. Specifically, the present invention provides a method for making ethanol from a sugar syrup, said method comprising the steps of: a) providing a sugar syrup, b) pretreating the sugar syrup by applying a microbial and enzyme cocktail to the sugar syrup, said cocktail comprising (i) a first culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants and (ii) a dextranase enzyme or a second culture of one and / or more dextranase producing probiotic microorganisms, c) adding an aqueous liquid to the pretreated sugar syrup, followed by mixing to obtain a diluted sugar solution, d) fermenting the diluted sugar solution by addition of yeast Saccharomyces cerevisiae, and e) recovering ethanol from the fermentation.

[0018] In one embodiment, the sugar syrup is selected from molasses, fructose, maltose, glucose, and sucrose syrup or a mixture of one or more of these sugar syrups. The sugar syrup may originate from a starch-comprising material, e.g. corn, wheat, rye, malt, barley, triticale, rice, potato, and cassava. Alternatively, the sugar syrup may originate from cellulose-comprising material, e.g. wheat straw, corn stover, and switchgrass. Preferably the sugar syrup is molasses, such as originating from sugar cane or sugar beet.

[0019] In one preferred embodiment, the culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants in the sugar syrup comprises a species of Lactobacillus and / or a species of Fusarium, preferably a combination of Lactobacillus sake! and Fusarium venenatum. The Lactobacillus sake! helps avoid / control contaminant growth of unwanted microbes in the sugar syrup, especially Leuconostoc mesenteroides, while the Fusarium venenatum helps avoid contamination from potential airborne contaminants.

[0020] The dextranase enzyme helps hydrolyze any dextran polymer present in the sugar syrup. In one embodiment, the dextranase enzyme in microbial and enzyme cocktail is a purified or partly purified enzyme. In another embodiment, the dextranase enzyme in microbial and enzyme cocktail is produced and secreted by a probiotic microorganism, preferably Lactobacillus bifidus. Hence, in such embodiment, the culture of one or more probiotic microorganisms producing a dextranase enzyme comprises Lactobacillus bifidus.

[0021] Preferably, the fermentation is a continuous fermentation, using diluted sugar syrup as feed into the fermentation. Most preferably, the continuous fermentation comprises at least two fermenters, wherein the dilution rate of the first fermenter is between 0.10- 0.55 IT1, resulting in a steady state ethanol concentration between 5-11 % (w / w) in the first fermenter, and wherein the dilution rate of the second fermenter is between 0.04- 0.10 IT1, resulting in a steady state ethanol concentration between 10-13% (w / w) in the second fermenter.

[0022] In one embodiment of the first aspect, the method may further produce a protein feed or food product, by providing a sugar syrup comprising protein. Sugar beet-derived sugar syrups are traditionally known to comprise protein.

[0023] In one embodiment, one or more chemicals suitable for limiting growth of unwanted contaminants is added to the sugar syrup. The one or more chemicals may be selected from ethanol, lactic acid and sodium propionate. In such embodiment when ethanol is added, it is preferably added to reach a concentration of 1-10 wt% in the sugar syrup, more preferably 4-9 wt%, most preferably 6-8 wt%. In such embodiment when lactic acid is added, it is preferably added to reach a concentration of 0.2-2 wt% in the sugar syrup, more preferably 0.3-1.5 wt%, most preferably 0.5-1.0 wt%. In such embodiment when sodium propionate is added, it is preferably added to reach a concentration of 0.05-1 wt% in the sugar syrup, more preferably 0.2-0.8 wt%, most preferably 0.3-0.6 wt%.

[0024] In a second aspect, the present invention concerns a fermentation process, using a pretreated sugar syrup as a sugar booster in the fermentation process. Specifically, the present invention provides a method for making ethanol and optionally a protein feed or food product from a feedstock comprising glucose or glucose-derived compound, wherein said method comprises fermenting said feedstock by continuous fermentation using yeast Saccharomyces cerevisiae, wherein said method further comprises providing a sugar syrup and pretreating the sugar syrup by applying a microbial and enzyme cocktail to the sugar syrup, said cocktail comprising (i) a first culture of one or more probiotic microorganism(s) suitable for limiting growth of unwanted contaminants in the sugar syrup and (ii) a dextranase enzyme and / or a second culture of one or more dextranase producing probiotic microorganism(s), and wherein said continuous fermentation comprises at least two fermenters, wherein the first fermenter is continuously fed with a combination of the feedstock and pretreated sugar syrup, and wherein the dilution rate of the first fermenter is between 0.10-0.55 IT1, resulting in a steady state ethanol concentration between 5-11 % (w / w) in the first fermenter, and wherein the dilution rate of the second fermenter is between 0.04-0.10 IT1, resulting in a steady state ethanol concentration between 10-13% (w / w) in the second fermenter, and optionally wherein pretreated sugar syrup is also continuously added to the second fermenter.

[0025] A glucose-derived compound refers to a compound comprising glucose, either as glucose dimer, oligomer, or polymer, or as glucose in combination with other sugar molecules - all which may enzymatically can be hydrolyzed to obtain a monomer or dimer which Saccharomyces cerevisiae is capable of fermenting to ethanol, e.g. glucose, maltose or sucrose. Preferably, the glucose-derived compound is starch.

[0026] In one preferred embodiment, the continuous fermentation comprises at least three fermenters, wherein the dilution rate of the third fermenter is between 0.02-0.06 h1, resulting in a steady state ethanol concentration between 12-15 % (w / w) in the third fermenter, and optionally wherein pretreated sugar syrup is also continuously added to the third fermenter.

[0027] In a most preferred embodiment of the second aspect, the feedstock is a feedstock comprising starch.

[0028] Specifically, in one preferred embodiment, the present invention provides a method for making ethanol and a protein feed or food product, from a feedstock comprising starch and protein, said method comprising the steps of:

[0029] (a) providing the feedstock comprising starch and protein,

[0030] (b) adding an aqueous liquid to the feedstock, followed by mixing to obtain a feedstock slurry,

[0031] (c) optionally adjusting pH of the feedstock slurry to pH 5-7,

[0032] (d) adding one or more starch hydrolyzing enzyme(s) to the feedstock slurry, and incubating to obtain a feedstock hydrolysate,

[0033] (e) adding yeast Saccharomyces cerevisiae to the feedstock hydrolysate to obtain a fermentation broth,

[0034] (f) fermenting the fermentation broth by continuous fermentation comprising at least two fermenters, wherein the dilution rate of the first fermenter is between 0.10-0.55 IT1, resulting in a steady state ethanol concentration between 5-11 % (w / w) in the first fermenter, and wherein the dilution rate of the second fermenter is between 0.04-0.10 IT1, resulting in a steady state ethanol concentration between 10-13% (w / w) in the second fermenter, and

[0035] (g) separately recovering ethanol and protein, said method further comprising the steps of:

[0036] (h) providing a sugar syrup,

[0037] (i) pretreating the sugar syrup by applying a microbial and enzyme cocktail to the sugar syrup, said cocktail comprising (i) a first culture of one or more probiotic microorganism(s) suitable for limiting growth of unwanted contaminants in the sugar syrup and (ii) a dextranase enzyme and / or a second culture of one or more dextranase producing probiotic microorganism(s),

[0038] (j) adding pretreated sugar syrup to one or more of the fermenters during the continuous fermentation.

[0039] In one preferred embodiment, the continuous fermentation comprises at least three fermenters, wherein the dilution rate of the third fermenter is between 0.02-0.06 h1, resulting in a steady state ethanol concentration between 12-15 % (w / w) in the third fermenter. In one further preferred embodiment, the feedstock comprising starch and protein is pretreated to limit growth of unwanted contaminants in the feedstock. Specifically, in one such further preferred embodiment, the present invention provides a method for making ethanol and a protein feed or food product, from a feedstock comprising starch and protein, said method comprising the steps of:

[0040] (a) providing the feedstock comprising starch and protein,

[0041] (b) pretreating the feedstock by applying to the feedstock a third culture of one or more probiotic microorganism(s) suitable for limiting growth of unwanted contaminants in the feedstock,

[0042] (c) adding an aqueous liquid to the pretreated feedstock, followed by mixing to obtain a feedstock slurry,

[0043] (d) optionally adjusting pH of the feedstock slurry to pH 5-7,

[0044] (e) adding one or more starch hydrolyzing enzyme(s) to the feedstock slurry, and incubating to obtain a feedstock hydrolysate,

[0045] (f) adding yeast to the feedstock hydrolysate to obtain a fermentation broth,

[0046] (g) fermenting the fermentation broth by continuous fermentation comprising at least two fermenters, wherein the dilution rate of the first fermenter is between 0.10-0.55 IT1, resulting in a steady state ethanol concentration between 5-11 % (w / w) in the first fermenter, and wherein the dilution rate of the second fermenter is between 0.04-0.10 IT1, resulting in a steady state ethanol concentration between 10-13% (w / w) in the second fermenter, and

[0047] (h) separately recovering ethanol and protein, said method further comprising the steps of:

[0048] (i) providing a sugar syrup,

[0049] (j) pretreating the sugar syrup by applying a microbial and enzyme cocktail to the sugar syrup, said cocktail comprising (i) a first culture of one or more probiotic microorganism(s) suitable for limiting growth of unwanted contaminants in the sugar syrup and (ii) a dextranase enzyme and / or a second culture of one or more dextranase producing probiotic microorganism(s),

[0050] (k) adding pretreated sugar syrup to one or more of the fermenters during the continuous fermentation.

[0051] In one preferred embodiment, the continuous fermentation comprises at least three fermenters, wherein the dilution rate of the third fermenter is between 0.02-0.06 h1, resulting in a steady state ethanol concentration between 12-15 % (w / w) in the third fermenter. In one embodiment, the feedstock is grain and / or one or more grain derived products, such as grain selected from wheat, rice, oats, barley, rye, barley, millet, corn, triticale, and sorghum grain.

[0052] In one embodiment, the probiotic culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants in the feedstock comprises a species of Lactobacillus and / or a species of spore forming Bacillus, preferably a combination of Lactobacillus and Bacillus,- such as a species of Lactobacillus selected from Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus amylovorus, and Lactobacillus hammesii; such as a species of spore forming Bacillus is selected from Bacillus licheniformis, Bacillus clausii, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus fusiformis and Bacillus megaterium.

[0053] In one embodiment, the probiotic culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants in the feedstock comprises a species of Aspergillus, preferably a spore forming species of Aspergillus , such as a spore forming species of Aspergillus selected from Aspergillus luchuensis, Aspergillus sojae, Aspergillus awamori and Aspergillus oryzae n one embodiment, one or more chemicals suitable for limiting growth of unwanted contaminants is added to the feedstock slurry. The one or more chemicals may be selected from ethanol, lactic acid and sodium propionate. In such embodiment when ethanol is added, it is preferably added to reach a concentration of 1-10 wt% in the slurry, more preferably 4-9 wt%, most preferably 6- 8 wt%. In such embodiment when lactic acid is added, it is preferably added to reach a concentration of 0.2-2 wt% in the slurry, more preferably 0.3-1.5 wt%, most preferably 0.5-1.0 wt%. In such embodiment when sodium propionate is added, it is preferably added to reach a concentration of 0.05-1 wt% in the slurry, more preferably 0.2-0.8 wt%, most preferably 0.3-0.6 wt%.

[0054] In one embodiment, the sugar syrup is selected from molasses, fructose, maltose, glucose, and sucrose syrup or a mixture of one or more of these sugar syrups. The sugar syrup may originate from a starch-comprising material, e.g. corn, wheat, rye, malt, barley, triticale, rice, potato and cassava. Alternatively, the sugar syrup may originate from cellulose-comprising material, e.g. wheat straw, corn stover, and switchgrass. Preferably the sugar syrup is molasses, such as originating from sugar cane or sugar beet.

[0055] In one preferred embodiment, the culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants in the sugar syrup comprises a species of Lactobacillus and / or a species of Fusarium, preferably a combination of Lactobacillus sake! and Fusarium venenatum. The Lactobacillus sake! helps avoid / control contaminant growth of unwanted microbes in the sugar syrup, especially Leuconostoc mesenteroides,- while the Fusarium venenatum helps avoid contamination from potential airborne contaminants.

[0056] The dextranase enzyme helps hydrolyze any dextran polymer present in the sugar syrup. In one embodiment, the dextranase enzyme in microbial and enzyme cocktail is a purified or party purified enzyme. In another embodiment, the dextranase enzyme in microbial and enzyme cocktail is produced by a probiotic microorganism, preferably Lactobacillus bifidus. Hence, in such embodiment, the culture of one or more probiotic microorganisms producing a dextranase enzyme comprises Lactobacillus bifidus.

[0057] In one embodiment, one or more chemicals suitable for limiting growth of unwanted contaminants is added to the sugar syrup. The one or more chemicals may be selected from ethanol, lactic acid and sodium propionate. In such embodiment when ethanol is added, it is preferably added to reach a concentration of 1-10 wt% in the sugar syrup, more preferably 4-9 wt%, most preferably 6-8 wt%. In such embodiment when lactic acid is added, it is preferably added to reach a concentration of 0.2-2 wt% in the sugar syrup, more preferably 0.3-1.5 wt%, most preferably 0.5-1.0 wt%. In such embodiment when sodium propionate is added, it is preferably added to reach a concentration of 0.05-1 wt% in the sugar syrup, more preferably 0.2-0.8 wt%, most preferably 0.3-0.6 wt%.

[0058] DESCRIPTION OF THE INVENTION

[0059] Brief description of the figures:

[0060] Figure 1: Illustration of a three fermenter continuous fermentation system according to the invention.

[0061] (A) Three fermenter continuous fermentation process. Pretreated (diluted) sugar syrup (e.g. pretreated (diluted) molasses) is the main substrate in the fermentation. The pretreated (diluted) sugar syrup is used as input in fermenter 1, and fermentation starts with addition of yeast and optionally addition of an appropriate nitrogen source (e.g. urea or ammonium sulfate or di ammonium Phosphate). By continuously pumping pretreated (diluted) sugar syrup into fermenter 1, and pumping fermented mash from fermenter 1 into fermenter 2, the flow rate is preferably adjusted to maintain dilution rate in the range of 0.1 -0.55 h1in fermenter 1. Further, by continuously pumping fermented mash from fermenter 2 into fermenter 3, the flow rate is preferably adjusted to maintain dilution rate in the range of 0.04-0.1 h1in fermenter 2. Finally, by continuously pumping fermented mash from fermenter 3 to an ethanol distillation unit, the flow rate is preferably adjusted to maintain dilution rate in the range of 0.02-0.06 h1in fermenter 3. Finally, ethanol is recovered from fermenter 3. (B) Three fermenter continuous fermentation process with sugar booster. The main fermentation substrate is (pretreated) feedstock comprising starch. Pretreated sugar syrup is used as sugar booster during the fermentation. Feedstock is fed into fermenter 1. Fermentation starts with addition of yeast and optionally addition of appropriate nitrogen source (e.g. urea or ammonium sulfate or di ammonium phosphate) in fermenter 1. At an ethanol concentration of between 5-11%, probiotic pretreated sugar syrup (e.g. pretreated molasses) is continuously added to fermenter 1. The flow rate in fermenter 1 is preferably adjusted to maintain dilution rate in the range of 0.1 -0.55 IT T Fermented mash is continuously pumped from fermenter 1 into fermenter 2. The flow rate in fermenter 2 is preferably adjusted to maintain dilution rate in the range of 0.04- 0.1 IT1. Fermented mash from fermenter 2 is continuously pumped into fermenter 3. The flow rate in fermenter 3 is preferably adjusted to maintain a dilution rate in the range of 0.02-0.06 IT1. Probiotic pretreated sugar syrup may also be continuously added to fermenter 2 and / or 3. Finally, ethanol is recovered from fermenter 3.

[0062] Figure 2: Ethanol concentration in a continuous fermentation process according to the invention mentioned in Figure IB. Ethanol concentration was measured using HPLC throughout the fermentation (~ 10 days). Probiotic pretreated molasses dosing was started at 5 ml / h in fermenter 2 from day 3 and stopped on day 8. The dosing rate was maintained between 5-50 ml / h, keeping the weight of the fermentation broth constant throughout the trial. As seen from the graph, ethanol concentration increased by ~ 1% in fermenter 2 and 3 during molasses dosing, demonstrating that unsterilized, probiotic pretreated molasses does not negatively affect the fermentation and that it may be added as a booster to the fermentation to further increase ethanol yields. All the three fermentations were ran in fermenters with 5 liter capacity.

[0063] Abbreviations, terms, and definitions:

[0064] As used herein, the term "sugar syrup" refers to an aqueous liquid comprising dissolved sugar, such as a sugar selected from fructose, maltose, glucose, and sucrose, or a mixture of two or more of these sugars. The sugar syrup has at least 50 Brix value. One example of a sugar syrup is molasses, such as sugarcane molasses or sugar beet molasses. The Brix value, expressed as degrees Brix (°Bx), is how many grams of sugar are present per 100 grams of liquid solution measured on a scale of one to 100.

[0065] The term "diluted sugar syrup" as used herein refers to dilution of sugar syrup by aqueous solution to adjust the Brix value to between 5- 30Brix.

[0066] As used herein, the term "feedstock" refers to a composition comprising glucose or glucose-derived compound(s). Preferably the feedstock comprises starch, (e.g. cereal grain, bread, bakery products). The feedstock may further comprise protein. The feedstock may be a grain and / or grain-derived product. Suitable feedstocks include bakery products like bread, croissant, dough, biscuits, cake, and grains such as maize (corn, e.g., whole ground corn), Sorghum (milo), barley, wheat, rye, rice, and millet; and starchy root crops, tubers, or roots such as potato, cassava. The feedstock can be a mixture of two or more of such materials.

[0067] As used herein, the term "feedstock slurry" refers to the composition obtained by adding an aqueous liquid to a pretreated feedstock, followed by mixing, thereby obtaining the feedstock slurry. Such feedstock slurry typically comprises 20-40 wt% solids.

[0068] As used herein, the term "probiotics" refers to bacteria, yeast, and fungi, which are considered generally safe for human consumption.

[0069] The term "unwanted contaminants" includes microorganisms which are not considered safe for human consumption - e.g. pathogens, such as toxin producing microorganisms. It may further refer to microorganisms which affect ethanol fermentation process by inhibiting the fermentation, such as by inhibiting yeast growth and ethanol production.

[0070] As used herein, the phrase "in situ hydrolysis of dextran" refers to hydrolysis of dextran polymer to monomer sugars in a sugar syrup (e.g. molasses), in a diluted sugar solution, during cold mashing, and / or during fermentation.

[0071] As used herein, the phrase "in situ production of dextranase" refers to the dextranase enzyme being produced by probiotic microorganism, e.g. by Lactobacillus bifidus (Bifidobacterium bifidum) during pretreatment of a sugar syrup (e.g. molasses).

[0072] As used herein, the term "cold mashing" refers to a process of enzymatically converting starch to ethanol (e.g. using alpha-amylase for saccharification of the starch) at temperatures < 55°C.

[0073] As used herein, the term "High Protein" refers to the protein co-product obtained from an ethanol fermentation process, wherein said "high protein" product comprises at least 50% protein (at ~ 10% moisture).

[0074] The term "liquefact" as used herein refers to the feedstock hydrolysate obtained from the hydrolysis step.

[0075] The term "whole stillage" as used herein refers to the aqueous fraction left after removal of ethanol during the distillation stage.

[0076] The term "wet cake" as used herein refers to insoluble portion obtained after centrifugation of the whole stillage.

[0077] The term dilution rate (D) as used herein, usually expressed in units per hour (h-1), refers to the relationship between the total inflow into a fermenter (liters-h-1), and the fermentation volume within the fermenter (liters). The term "base" as used herein refers to any chemicals which can be used to increase pH, examples include ammonia water (NHsaq), sodium hydroxide (NaOH) and potassium hydroxide (KOH).

[0078] Detailed description of the invention:

[0079] The present invention facilitates a reduction in water usage and energy consumption compared to traditional ethanol plants, without affecting quality and quantity of the end products. The present invention concerns an improved fermentation process, using a sugar syrup (e.g. molasses) as a substrate in the fermentation process or as a booster to a fermentation process which is based on a different feedstock as main substrate. It provides a novel method for making ethanol from a pretreated sugar syrup. It further provides a novel method for making ethanol and a protein feed or food product from starch feedstocks (e.g. grain and grain-derived products), using pretreated sugar syrups (e.g. molasses) as a sugar booster. The overall process is highly sustainable with ultralow GHG (green-house-gas) emissions and is robust against fluctuations in the composition of both the feedstock (e.g. moisture content) and the sugar syrup (e.g. dextran content).

[0080] In one aspect, the present invention relates to a method for producing ethanol from a sugar syrup (the sugar syrup being the main substrate of the fermentation process), where key steps of the method comprise bio-preservation of the sugar syrup using probiotic microorganism for limiting growth of unwanted contaminants, enzyme assisted hydrolysis of dextran polymer to produce fermentable sugars, and a continuous fermentation process for producing ethanol.

[0081] In a further aspect, the present invention relates to a method for producing ethanol and a high protein feed or food product from a feedstock comprising starch and protein, applying pretreated sugar syrups (e.g. molasses) as a sugar booster; where key steps of the method comprise bio-preservation of feedstock using probiotic treatment for limiting growth of unwanted contaminants, starch hydrolysis, and a continuous fermentation process, in combination with bio-preservation of the sugar syrup using probiotic microorganism for limiting growth of unwanted contaminants, enzyme assisted hydrolysis of dextran polymer to produce fermentable sugars, and continuously dosing the pretreated sugar syrup in the continuous fermentation process.

[0082] By combining several process steps in a novel and innovative way, the methods of the present invention facilitates water and energy savings by avoiding thermal pretreatment of the feedstock and sugar syrup; reducing water consumption in cooling tower; controlling naturally occurring unwanted contaminant microbial population(s) present in the feedstock and sugar syrup without using antibiotic, thermal or chemical treatment, or without using antibiotic or thermal treatment; avoiding airborne contamination in the feedstock and sugar syrup prior to and during usage in the fermentation process; avoiding antibiotic addition in saccharification and / or fermentation stage; improving ethanol yield by keeping yeast healthy and making more sugars available during fermentation.

[0083] I. Method for making ethanol from a sugar syrup

[0084] In one aspect, the present invention provides a method for making ethanol from a sugar syrup, said method comprising the steps of: a) providing a sugar syrup, b) pretreating the sugar syrup by applying a microbial and enzyme cocktail to the sugar syrup, said cocktail comprising (i) a first culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants and (ii) a dextranase enzyme or a second culture of one and / or more dextranase producing probiotic microorganisms, c) adding an aqueous liquid to the pretreated sugar syrup, followed by mixing to obtain a diluted sugar solution, d) fermenting the diluted sugar solution by addition of yeast Saccharomyces cerevisiae, and e) recovering ethanol from the fermentation.

[0085] In the first aspect, the invention provides a novel process for producing ethanol, characterized in the sugar syrup initially being treated with probiotic microorganisms to ensure (a) controlling existing unwanted contamination, (b) preventing further unwanted bacterial or fungal contaminant growth during storage, and (c) hydrolyzing dextran to fermentable sugars during storage and during fermentation process. The pretreated sugar syrup is fermented to ethanol preferably in continuous mode. In this way, the microbial stability of the sugar syrup is significantly increased without using thermal or antibiotic pretreatment. The avoidance of thermal pretreatment also results in reducing energy and freshwater consumption. The applicability of said probiotic treated sugar syrup is further improved by running ethanol fermentation process in continuous mode while maintaining reactor(s) at specific dilution rates, resulting in contamination prevention and improved ethanol yield. In addition to recovering ethanol from the process, a protein product is preferably also recovered, which is essentially 'pure' and can therefore be used as feed or in food applications.

[0086] In a second aspect, the invention provides a novel process for producing ethanol, characterized in the feedstock and sugar syrup initially being treated with probiotic microorganisms to ensure (a) controlling existing unwanted contamination, (b) preventing further unwanted bacterial or fungal contaminant growth during storage, and (c) hydrolyzing dextran to fermentable sugars during storage and during fermentation process. The pretreated feedstock and sugar syrup are fermented to ethanol preferably in continuous mode. In this way, the microbial stability of the feedstock is significantly increased without using thermal, chemical, or antibiotic pretreatment, or without using thermal or antibiotic pretreatment. The avoidance of thermal pretreatment also results in reducing energy and freshwater consumption. The applicability of said probiotic treated feedstock and sugar syrup is further improved by running ethanol fermentation process in continuous mode while maintaining reactor(s) at specific dilution rates, resulting in contamination prevention and improved ethanol yield. In addition to recovering ethanol from the process, a protein product is preferably also recovered, which is essentially 'pure' and can therefore be used as feed or in food applications.

[0087] I.i Sugar syrup - such as molasses and other sugar syrups

[0088] In one aspect, the present invention provides a method for making ethanol from a sugar syrup. In another aspect, the invention provides a method for making ethanol from a feedstock, wherein a sugar syrup is used as a sugar booster.

[0089] "Sugar syrup" refers to a thick, viscous liquid comprising or mostly consisting of a solution of sugar in aqueous solution, containing a large amount of dissolved sugar(s), but showing little tendency to deposit crystals. The type of sugar in the sugar syrup may be one of more sugars selected from sucrose, glucose, maltose, fructose, lactose, galactose, and combinations thereof.

[0090] The sugars in the sugar syrup may originate from different sources. The sugar syrup may originate from a starch-comprising material, e.g. corn, wheat, rye, malt, barley, triticale, and cassava. In one embodiment, the sugar in the sugar syrup is derived from starchy grains such as wheat, rice, oats, barley, rye, millet, corn, triticale, and sorghum, or is from grain derived products such as bread, cakes, dough, chocolates, biscuits, breakfast cereals. The type of sugar in these sugar syrups is primarily glucose. Alternatively, the sugar syrup may originate from cellulose-comprising material, e.g. wheat straw, corn stover, and switchgrass.

[0091] In one preferred embodiment, the sugar syrup is molasses. Molasses is the viscous substance, principally obtained from the refining of sugarcane or sugar beet juice into sugar. Molasses varies in the amount of sugar, the method of extraction and the age of the plant. The types of sugars found in molasses include: sucrose, glucose and fructose (produced when sucrose is broken down), and potentially small amounts of lactose and galactose, with the most prominent sugar being sucrose. Preferably the sugar syrup is molasses, such as originating from sugar cane or sugar beet. In one embodiment, the sugar syrup has a Brix value of at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 Brix or even at least 95 Brix. In one embodiment, the sugar syrup has between 5-95 Brix, 10-95 Brix, 15-95 Brix, 20-95, 25-95 Brix, 30-95 Brix, 35-95 Brix, 40-95 Brix, 45-95 Brix, 50-95 Brix, 55-95 Brix, 60-95 Brix, 65-95 Brix, 70- 95 Brix, 75-95 Brix, 80-95 Brix or 90-50 Brix.

[0092] I.ii Pretreatment of the sugar syrup

[0093] In one important aspect of the invention, the sugar syrup is pretreated by applying a microbial and enzyme cocktail to the sugar syrup, said cocktail comprising (i) a culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants and (ii) a dextranase enzyme and / or a culture of one or more dextranase producing probiotic microorganisms.

[0094] Unwanted contaminants include microorganisms which are not considered safe for human consumption - e.g. pathogens, such as microorganisms which produce toxins. Further unwanted contaminants include microbial contaminants affecting ethanol fermentation process by inhibiting the yeast, the contaminants affecting final quality of the protein feed. For example mycotoxin producing molds, and common pathogens like E. coli constitute unwanted contaminants.

[0095] The presence of unwanted microbial contaminants may be detected by measuring the level of toxins (e.g. by HPLC or other analytical method) or by detection of the presence of the organisms by standard microbial detection methods - as recognized by a person skilled in the art.

[0096] A probiotic microorganism is applied to the sugar syrup (such as molasses) in order to enhance microbial stability and reduce unwanted contamination. The positive effect of such probiotic microbial pretreatment of the sugar syrup is demonstrated in Example 1.

[0097] Traditionally, the sugar syrup would be chemically and / or thermally pretreated or have its microbial load reduced by other means to ensure a reduction in unwanted contaminants. Meanwhile, for the present invention, such steps may be omitted or at least significantly reduced due to the probiotic microbes preventing the growth of other unwanted microbial contaminants. The potential problem of contamination mostly arises when the provision of the sugar syrup includes a 'storage step', such as the time required to transport the sugar syrup to the ethanol plant from its origin, or during other general 'storage' conditions prior to its use in the present process.

[0098] Microbial pretreatment is a solution towards enhancing shelf life of the sugar syrups, without using pretreatment methods mentioned in the state of the art. The sugar syrup is pretreated by applying a microbial and enzyme cocktail comprising (i) a culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants and (ii) a dextranase enzyme or a culture of one or more dextranase producing probiotic microorganisms.

[0099] In one embodiment, the microbial and enzyme cocktail for the pretreatment of the sugar syrup comprises one or more probiotic fungus or fungi- i.e. one or more probiotic fungal cells or probiotic fungal cell cultures - suitable for limiting growth of unwanted contaminants in the sugar syrup. In a preferred embodiment, the microbial and enzyme cocktail for the pretreatment of the sugar syrup comprises one or more filamentous fungus which does not produce mycotoxins. In one preferred embodiment, the microbial and enzyme cocktail for the pretreatment of the sugar syrup comprises one or more Fusarium species - specifically a Fusarium species which does not produce mycotoxin. In one preferred embodiment, the microbial and enzyme cocktail for the pretreatment of the sugar syrup comprises Fusarium species for limiting growth of unwanted contaminants. In one preferred embodiment, the Fusarium species is Fusarium venenatum (e.g. Fusarium venenatum ATCC 20334which has GRAS status and is approved for human consumption).

[0100] In one embodiment, the microbial and enzyme cocktail for the pretreatment of the sugar syrup comprises one or more probiotic bacteria - i.e. one or more probiotic bacterial cells or probiotic bacteria cell cultures - suitable for limiting growth of unwanted contaminants in the sugar syrup. In a preferred embodiment, the microbial and enzyme cocktail for the pretreatment of the sugar syrup comprises of one or more probiotic lactic acid bacteria, such as a probiotic species of Lactobacillus and / or Streptococcus, for limiting growth of contaminants. In a preferred embodiment, the microbial and enzyme cocktail for the pretreatment of the sugar syrup comprises of one or more Lactobacillus species for limiting growth of contaminants. In one embodiment, the lactobacillus is selected from Lactobacillus sake!, Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus amylovorus, Lactobacillus hammesii and Streptococcus thermophilus. In a most preferred embodiment, the microbial and enzyme cocktail for the pretreatment of the sugar syrup comprises Lactobacillus sakei. Specifically, Lactobacillus sakei is able to control unwanted growth of Leuconostoc mesenteroides.

[0101] In one preferred embodiment, the one or more probiotic microorganism(s) suitable for limiting growth of unwanted contaminants comprise a combination of the fungal and lactobacillus species disclosed in the above paragraphs.

[0102] In one preferred embodiment, the culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants comprises Lactobacillus sakei and / or Fusarium venenatum, preferably a combination of Lactobacillus sakei and Fusarium venenatum. Lactobacillus sake! helps avoid / control contaminant growth of unwanted microbes in the sugar syrup, especially Leuconostoc mesenteroides; while the Fusarium venenatum helps avoid contamination from potential airborne contaminants.

[0103] The microbial and enzyme cocktail further comprises a dextranase enzyme and / or a culture of one or more dextranase producing probiotic microorganisms. The dextranase enzyme helps hydrolyze any dextran polymer present in the sugar syrup.

[0104] In one embodiment, the dextranase enzyme in the microbial and enzyme cocktail is a purified or partly purified enzyme. The dextranase may derive from a dextranase producing microorganism, having been purified by standard procedures known by a person skilled in the art. Examples of dextranase producing microorganisms, from which dextranase may be derived, include Penicillium luteum, Penicillium funiculosum, Aspergillus carneus, Bacillus subtiis, Bacillus circulans, Lipomyces starkyi, Fusarium sp., Flavobacterium sp., Pseudomonas sp., Saccharomyces cerevisiae, Brevibacterium fuscum and Leconostoc dextranicus.

[0105] In one embodiment, the microbial and enzyme cocktail for the pretreatment of the sugar syrup comprises one or more dextranase producing probiotic microorganism. Most preferably, the dextranase is secreted by the dextranase producing probiotic microorganism. In one embodiment, the microbial and enzyme cocktail for the pretreatment of the sugar syrup comprises one or more dextranase secreting probiotic microorganisms. In one embodiment, the microbial and enzyme cocktail for the pretreatment of the sugar syrup comprises one or more dextranase producing probiotic bacteria - i.e. one or more dextranase producing probiotic bacterial cells or dextranase producing probiotic bacteria cell cultures. In a preferred embodiment, the dextranase producing probiotic bacteria comprise of one or more species selected from Lactobacillus bifidus (Bifidobacterium bifidum), Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus brevis, Streptococcus thermophilus, Streptococcus salivarius and Flavobacterim sp; most preferably Lactobacillus bifidus (Bifidobacterium bifidum). Dextranase activity in probiotic treated sugar syrup is demonstrated in Example 3.

[0106] In one preferred embodiment, the microbial and enzyme cocktail for the pretreatment of the sugar syrup comprises a culture comprising different probiotic species selected from species of Fusarium and Lactobacillus.

[0107] In one preferred embodiment, the microbial and enzyme cocktail for the pretreatment of the sugar syrup comprises of combination of (i) one or more probiotic strains of Fusarium fe.g. strain of Fusarium venenatum, such as strain ATCC 20334) and (ii) one or more probiotic strains of lactic acid bacteria, such as one or more probiotic strains of Lactobacillus and / or Streptococcus (e.g. strain of Lactobacillus bifidus (Bifidobacterium bifidum) and / or Lactobacillus sake!). In one most preferred embodiment, the microbial and enzyme cocktail for the pretreatment of the sugar syrup comprises a culture of Fusarium venenatum, Lactobacillus bifidus (Bifidobacterium bifidum), and Lactobacillus sake!.

[0108] In one embodiment, the microbial and enzyme cocktail for the pretreatment of the sugar syrup is performed by applying mixing the cocktail with the sugar syrup; in another embodiment, the cocktail is applied onto the surface of sugar syrups, such as by spraying it on the surface of the sugar syrup.

[0109] In one embodiment, the microbial and enzyme cocktail is applied to the sugar syrup immediately after production of the sugar syrup, for best avoidance of contaminants.

[0110] In one further embodiment, one or more chemicals suitable for limiting growth of unwanted contaminants is added to the sugar syrup. The one or more chemicals may be selected from ethanol, lactic acid and sodium propionate. In such embodiment when ethanol is added, it is preferably added to reach a concentration of 1-10 wt% in the sugar syrup, more preferably 4-9 wt%, most preferably 6-8 wt%. In such embodiment when lactic acid is added, it is preferably added to reach a concentration of 0.2-2 wt% in the sugar syrup, more preferably 0.3-1.5 wt%, most preferably 0.5-1.0 wt%. In such embodiment when sodium propionate is added, it is preferably added to reach a concentration of 0.05-1 wt% in the sugar syrup, more preferably 0.2-0.8 wt%, most preferably 0.3-0.6 wt%.

[0111] I. Hi Fermentation

[0112] Yeast is used for fermenting sugars to ethanol. Specifically, Saccharomyces cerevisiae is used in the present invention for making ethanol. Specifically, in the first aspect of the invention, diluted sugar syrup is used as the sole or main substrate in the fermentation process (as illustrated in Figure 1A). In one preferred embodiment of the first aspect of the present invention, the pretreated sugar syrup is fermented anaerobically by yeast Saccharomyces cerevisiae. In another embodiment of the first aspect of the present invention, the pretreated sugar syrup is fermented aerobically by yeast Saccharomyces cerevisiae.

[0113] In one embodiment, fermentation is preferably performed at a dissolved solids content resulting in a Brix value of 30 Brix; no more than 40 Brix, no less than 10 Brix, i.e. between 10-40 Brix, preferably between 20-30 Brix, most preferably around 30 Brix. Hence, in a preferred embodiment, the pretreated sugar syrup is diluted to obtain such Brix value. The sugar syrup is diluted by an aqueous solution. Mixing is preferably performed during the fermentation process. In one embodiment, the fermentation process is performed at a temperature between 20-40 degree C, preferably a temperature between 28-36 degree C, more preferably between 30-32-degree C, to ensure optimal conditions for the yeast.

[0114] In one embodiment, the fermentation is performed at a pH between 4-7, preferably a pH between 4.5-6.5, more preferably between 5.0-6.0, most preferably between 5.0- 5.5; such as at a pH of 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5. Keeping the pH above 5.0 will ensure that any lactic acid and acetic acid present in the solution will not inhibit yeast. pKa (the acid dissociation constant) plays an important role in yeast growth. Acetic acid and lactic acid contamination is the most detrimental to yeast health. The volatile acids (VAs) are in protonated form below their pKa (pKa of acetic acid is 4.76; pKa of lactic acid is 3.86). At protonated state these VAs can pass through yeast cell membrane resulting in lowering cytoplasm pH. Yeast expels these VAs at the expense of ATP. However due to protonated form, these VAs keep on passing through yeast cell membrane barrier. The significant energy drain due to continuous expulsion of VAs results in arrest in fermentation (i.e. stuck / sluggish fermentation). An advantage of the present invention is that pH is maintained > pKa of these VAs as a most ideal way towards making the bioethanol fermentation process robust towards VA inhibition.

[0115] Fermentation may be performed in several steps, such as one, two, three or more steps in separate fermentation reactors to optimize the process, such as to allow for a continuous process rather than batch fermentation. In Figure 1A, a three-stage fermentation is shown as one embodiment of the present invention, but should not be considered as limiting for the invention.

[0116] In one embodiment, the fermentation is performed in a two stage fermentation in two separate fermenters, each stage / fermenter having a specific dilution rate to ensure a specific ethanol concentrations in each stage.

[0117] In one embodiment, the fermentation is performed in a three stage fermentation in three separate fermenters, each stage / fermenter having a specific dilution rate to ensure a specific ethanol concentration in each stage.

[0118] In one embodiment, the fermentation is performed in three stage continuous mode using pretreated and diluted sugar syrup having dissolved solids content resulting in a Brix value between 10-40 Brix, preferably between 20-30 Brix, most preferably around 30 Brix; where the ethanol concentration (w / w) in the first stage is maintained between 1% to 10%, preferably between 4% to 9%, more preferably between 5% to 7% most preferably between 5.5-6.0%; such as at 5.5%, 5.6, 5.7, 5.8, 5.0, or 6.0%. The steady state concentration of ethanol in stage 1 is achieved by continuous inflow of pretreated sugar syrup to the fermentation broth in the stage 1 fermenter and continuous outflow of fermented material from the stage 1 fermenter to the stage 2 fermenter, maintaining a dilution rate (D) in the range of 0.10 -0.55 IT1, preferable between 0.10 -0.30 IT1, more preferably between 0.10 -0.20h-1. An advantage of keeping the ethanol concentration in this desired range, especially between 5.5-6.0% in the first fermenter, is that potential contaminants, such as lactic acid bacteria (LAB), acetic acid bacteria (AAB) or others will be inhibited, while yeast is not inhibited. The residence time of the first fermenter is between 5 to 80 hours, preferably between 10 to 30 hours, more preferably between 12 to 15 hours.

[0119] The outflow from the first fermenter is transferred to a second fermenter where the ethanol concentration (w / w) is maintained between 6% to 18 %, preferably between 8 % to 16 %, more preferably between 9% to 12%; such as 9%, 9.5%, 10%, 10.50%, 11%, or 12%. The steady state concentration of ethanol is achieved by continuous inflow of fermented material from the stage 1 fermenter to the stage 2 fermenter and continuous outflow of fermented material from the stage 2 fermenter to the stage 3 fermenter, maintaining a dilution rate (D) in the range of 0.04-0.10 IT1, preferably between 0.04-0.08 IT1, more preferably between 0.04-0.06 IT1. The residence time of second fermenter is between 5 to 80 hours, between 5 to 60 hours, preferably between 10 to 40 hours, more preferably between 25 to 30 hours.

[0120] The outflow from the second fermenter is transferred to a third fermenter where the ethanol concentration (w / w) is maintained between 10 % to 15 %, preferably between 12% to 15%. The residence time of third fermenter is between 3 to 8 hours, preferably between 4 to 7 hours, more preferably between 5 to 6 hours. The constant ethanol concentration is achieved by continuous inflow of fermented material from the stage 2 fermenter to the stage 3 fermenter and continuous outflow of fermented material from the stage 3 fermenter to a distillation unit.

[0121] Preferably, the fermentation is a continuous fermentation, using diluted sugar syrup as feed into the fermentation, said fermentation comprising at least two fermenters, wherein the dilution rate of the first fermenter is between 0.10-0.55 IT1, resulting in a steady state ethanol concentration between 5-11 % (w / w) in the first fermenter, and wherein the dilution rate of the second fermenter is between 0.04-0.10 IT1, resulting in a steady state ethanol concentration between 10-13% (w / w) in the second fermenter.

[0122] In one embodiment, yeast is added to the pretreated and diluted sugar syrup having dissolved solids content resulting in a Brix value around 30 Brix; to obtain a fermentation broth. The fermentation both is fermented by continuous fermentation comprising at least two stages, preferably three stages. In one preferred embodiment, pretreated (diluted) sugar syrup is appropriately dosed to a stage 1 fermenter to maintain an ethanol concentration in the stage 1 fermenter between 5-11% (w / w); this ethanol concentration in stage 1 may be obtained by maintaining a dilution rate between 0.1 IT1to 0.55 h1in stage 1. The fermented mash from the stage 1 fermenter is then continuously dosed to a stage 2 fermenter to maintain an ethanol concentration in the stage 2 fermenter between 10-13% (w / w); this ethanol concentration in stage 2 may be obtained by maintaining a dilution rate between 0.04 h1to 0.1 IT1. In a further preferred embodiment, the fermented mash from the stage 2 fermenter is then dosed to a stage 3 fermenter, where ethanol concentration is maintained between 12-15% (w / w) by continuously dosing the fermented mash from stage 3 to a distillation unit.

[0123] In one embodiment, the fermentation is performed in three stage continuous mode using pretreated and diluted sugar syrup having dissolved solids content resulting in a Brix value between 10-40 Brix, preferably between 20-30 Brix, most preferably around 30 Brix; where the ethanol concentration (w / w) in the first stage is maintained between 5% to 11%, preferably between 5% to 9%, more preferably between 5% to 7% most preferably between 5.5-6.5%; such as at 5.5%, 5.6%, 5.7%, 5.8%, 5.0%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, or 6.5%, preferably around 6%. The steady state concentration of ethanol in stage 1 is achieved by continuous inflow of pretreated sugar syrup to the fermentation broth in the stage 1 fermenter and continuous outflow of fermented material from the stage 1 fermenter to the stage 2 fermenter, maintaining a dilution rate (D) in the range of 0.10 -0.55 IT1, preferable between 0.10 -0.30 IT1, more preferably between 0.10 -0.20b1. An advantage of keeping the ethanol concentration in this desired range, especially between 5.5-6.5% in the first fermenter, is that potential contaminants, such as lactic acid bacteria (LAB), acetic acid bacteria (AAB) or others will be inhibited, while yeast is not inhibited. The residence time of the first fermenter is between 5 to 80 hours, preferably between 10 to 30 hours, more preferably between 12 to 15 hours.

[0124] The outflow from the first fermenter is transferred to a second fermenter where the ethanol concentration (w / w) is maintained between 10% to 13%, preferably between 10% to 13%, more preferably between 11% to 12%; such as 11%, 11.5%, 12%, 12.5% or 13%, preferably around 12%. The steady state concentration of ethanol is achieved by continuous inflow of fermented material from the stage 1 fermenter to the stage 2 fermenter and continuous outflow of fermented material from the stage 2 fermenter to the stage 3 fermenter, maintaining a dilution rate (D) in the range of 0.04-0.10 IT1, preferably between 0.04-0.08 IT1, more preferably between 0.04-0.06 IT1. The residence time of second fermenter is between 5 to 80 hours, between 5 to 60 hours, preferably between 10 to 40 hours, more preferably between 25 to 30 hours.

[0125] The outflow from the second fermenter is transferred to a third fermenter where the ethanol concentration (w / w) is maintained between 12% to 15%, preferably between 13% to 15%. The constant ethanol concentration is achieved by continuous inflow of fermented material from the stage 2 fermenter to the stage 3 fermenter and continuous outflow of fermented material from the stage 3 fermenter to a distillation unit. The residence time of third fermenter is between 3 to 8 hours, preferably between 4 to 7 hours, more preferably between 5 to 6 hours.

[0126] II. Method for making ethanol from a feedstock

[0127] In a second aspect, the present invention concerns a fermentation process, using a pretreated sugar syrup (as disclosed in sections I.i and I.ii) as a sugar booster to an existing fermentation process.

[0128] In one embodiment, the present invention provides a method for making ethanol and optionally a protein feed or food product from a feedstock comprising glucose or glucosederived compound, wherein said method comprises fermenting said feedstock by continuous fermentation using yeast Saccharomyces cerevisiae, wherein said method further comprises providing a sugar syrup as disclosed in section I.i and pretreating the sugar syrup as disclosed section I.ii, and wherein said continuous fermentation is performed in at least two fermenters, wherein the first fermenter is continuously fed with a combination of the feedstock and pretreated sugar syrup, and wherein the dilution rate of the first fermenter is between 0.10-0.55 IT1, resulting in a steady state ethanol concentration between 5-11 % (w / w) in the first fermenter, and wherein the second fermenter is continuously fed with fermented material from fermenter 1, and wherein the dilution rate of the second fermenter is between 0.04-0.10 IT1, resulting in a steady state ethanol concentration between 10-13% (w / w) in the second fermenter, and optionally wherein pretreated sugar syrup is also continuously added to the second fermenter.

[0129] In one preferred embodiment, the present invention provides a fermentation method for making ethanol and optionally a protein feed or food product, wherein said fermentation is a continuous fermentation using yeast Saccharomyces cerevisiae, wherein said continuous fermentation is performed in at least two fermenters, wherein the first fermenter is continuously fed with a combination of a feedstock and a pretreated sugar syrup, maintaining a dilution rate of the first fermenter of between 0.10-0.55 IT1, resulting in a steady state ethanol concentration of between 5-11 % (w / w) in the first fermenter, and wherein the second fermenter is continuously fed with fermented material from fermenter 1, maintaining a dilution rate of the second fermenter of between 0.04-0.10 IT1, resulting in a steady state ethanol concentration between 10-13% (w / w) in the second fermenter, and optionally wherein pretreated sugar syrup is also continuously added to the second fermenter wherein said feedstock comprises glucose or a glucose-derived compound, and wherein said pretreated sugar syrup is a sugar syrup having been pretreated by applying a microbial and enzyme cocktail to the sugar syrup, said cocktail comprising (i) a first culture of one or more probiotic microorganism(s) suitable for limiting growth of unwanted contaminants in the sugar syrup and (ii) a dextranase enzyme and / or a second culture of one or more dextranase producing probiotic microorganism(s).

[0130] In one preferred embodiment, the present invention provides a method for making ethanol and a protein feed or food product, from a feedstock comprising starch and protein, said method comprising the steps of:

[0131] (a) providing the feedstock comprising starch and protein,

[0132] (b) adding an aqueous liquid to the feedstock, followed by mixing to obtain a feedstock slurry,

[0133] (c) optionally adjusting pH of the feedstock slurry to pH 5-7,

[0134] (d) adding one or more starch hydrolyzing enzyme(s) to the feedstock slurry, and incubating to obtain a feedstock hydrolysate,

[0135] (e) adding yeast Saccharomyces cerevisiae to the feedstock hydrolysate to obtain a fermentation broth,

[0136] (f) fermenting the fermentation broth by continuous fermentation in at least two connected fermenters, wherein the dilution rate of the first fermenter is between 0.10-0.55 IT1, resulting in a steady state ethanol concentration between 5-11 % (w / w) in the first fermenter, and wherein the dilution rate of the second fermenter is between 0.04-0.10 IT1, resulting in a steady state ethanol concentration between 10-13% (w / w) in the second fermenter, and

[0137] (g) separately recovering ethanol and protein, said method further comprising the steps of:

[0138] (h) providing a sugar syrup as disclosed in section I.i,

[0139] (i) pretreating the sugar syrup as disclosed in section I.ii,

[0140] (j) adding pretreated sugar syrup to one or more of the fermenters during the continuous fermentation. In a further preferred embodiment, a pretreated feedstock is used in the method of making ethanol, said pretreatment comprising applying to the feedstock a culture of one or more probiotic microorganism(s) suitable for limiting growth of unwanted contaminants in the feedstock, as further disclosed in section II. ii.

[0141] An advantage of the second aspect of the present invention is that it provides a method wherein the (pretreated) sugar syrup can be used 'as is' without diluting it with water, as a feeding medium to boost ethanol titers.

[0142] II. i Feedstock

[0143] The present invention provides a method for making ethanol and a protein feed or food product from a feedstock, wherein the pretreated sugar syrup (as disclosed in section I.ii) is added as a sugar boost to the fermentation.

[0144] The feedstock may be any substrate comprising sugars which can be directly fermented to ethanol by yeast Saccharomyces cerevisiae, or which may first be enzymatically hydrolyzed to more simple sugars which can then be fermented to ethanol by yeast. Specifically, the feedstock therefore comprises glucose or glucose-derived compound(s).

[0145] A glucose-derived compound refers to a compound comprising glucose, either as glucose -dimer, -oligomer, or -polymer, or as glucose in combination with other sugar molecules - all which may enzymatically can be hydrolyzed to obtain a monomer or dimer which Saccharomyces cerevisiae is capable of fermenting to ethanol, e.g. glucose or sucrose.

[0146] In one embodiment, the feedstock comprises starch. In such one embodiment, the present invention provides a fermentation method for making ethanol and a protein feed or food product from a feedstock comprising starch and protein, wherein the sugar syrup is added as a sugar boost to the fermentation process.

[0147] In one embodiment, the feedstock is grain and / or grain-derived products.

[0148] "Grains" are the edible seeds of plants. There are two main types of grain crops as cereals and legumes. Cereal grains are members of the Poaceae family and contain a high carbohydrate content. Wheat, rice, oats, barley, rye, barley, millet, corn, triticale, and sorghum are some examples of cereal grains. Grains also include pseudo-cereals like chia, quinoa, and buckwheat gains. Legumes or pulses are members of the pea family and they have a higher protein content than cereal grains. Chickpeas, mung beans, soybeans, common beans, lentils, and lima beans are some further examples of grains. "Grain-derived products" refers to products made using above mentioned grains and may comprise of high starch content. Flours made from above mentioned grains are examples of grain derived products. Bread, cakes, dough, chocolates, biscuits, breakfast cereals are further some examples of grain derived products. "Grain material" is used herein as a common term for grain and grain-derived products.

[0149] In one embodiment, the grain 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 grain material has a starch content between 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%.

[0150] In one embodiment, the grain material is selected from wheat, rice, oats, barley, rye, barley, millet, corn, triticale, and sorghum grain, and any product derived from these grains. In a preferred embodiment, the grain material is bread derived from wheat.

[0151] In another embodiment, the feedstock comprises cellulose. In such one embodiment, the present invention provides a fermentation method for making ethanol and a protein feed or food product from a feedstock comprising cellulose and protein, wherein the sugar syrup is added as a sugar boost to the fermentation process.

[0152] II. ii Pretreatment of the feedstock

[0153] In one embodiment, a probiotic microbial cell culture is applied to the feedstock material in order to enhance microbial stability and reduce unwanted microbial contamination in the feedstock material.

[0154] Traditionally, the feedstock would be dried or have its water content reduced by other means to ensure a less favorable environment for unwanted contaminants. Meanwhile, for the present invention, such drying step may be omitted or at least significantly reduced due to the probiotic microbes preventing the growth of other unwanted microbial contaminants. The potential problem of contamination mostly arises when the provision of the feedstock material includes a 'storage step', such as the time required to transport the feedstock material to the ethanol plant from its origin, or during other general 'storage' conditions prior to its use in the present process.

[0155] In one embodiment, the probiotic culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants in the feedstock is added to feedstock material having a moisture content (i.e. water content) greater than 5, 10, 15, 20, 25, 30, 35, 40 or even greater than 45%. In one embodiment, the probiotic culture is added to feedstock material having a water content between 10-50%, 15- 50%, 20-50%, 25-50%, 30-50%, 40-50%, or 45-50% (w / w). In one embodiment, the probiotic culture is added to feedstock material having a water content between 10- 15%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, or 10-45% (w / w). In one embodiment, probiotic culture is added to feedstock material having a water content between 10-40%, preferably between 15-40%, more preferably between 20-30% (w / w).

[0156] An advantage of the present invention is that it provides a method, wherein the feedstock material can be used 'as is', such as without decreasing the water content of the feedstock material. The microbial pretreatment is a solution towards enhancing shelf life of the feedstock, without decreasing its water activity as well as without decreasing nutritional properties. The microbial pretreatment will not only reduce and / or prevent bacterial and mold contamination in the initial feedstock material, but will also result in reducing contamination in the later ethanol fermentation.

[0157] In one embodiment, the probiotic culture for limiting growth of unwanted contaminants in 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 comprise one or more lactic acid bacteria. In one embodiment, the probiotic bacteria comprise one or more homofermentative lactic acid bacteria (HoLAB). In one embodiment, the probiotic lactic acid bacteria is one or more strain of the genus Lactobacillus. In one embodiment, the lactic acid bacteria is selected from Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus plantarum (for example strain 1A7), Lactobacillus amylovorus, and Lactobacillus hammesii. In one embodiment, the probiotic bacteria is one or more strain of the genus Pediococcus. In one embodiment, the probiotic bacteria is selected from Pediococcus acidilactici (for example strain KTL / 05-7), Pediococcus pentosaceus (for example strain KTL / 05-8) and Pediococcus pentosaceus (for example srain KTU05-10).

[0158] In one embodiment, the probiotic microbe to be applied to the feedstock comprises one or more yeast. In one embodiment, Pichia anomala (for example strain SKM-T) is used in the pretreatment of the feedstock material.

[0159] In one specific embodiment, the combination of probiotic microbes Lactobacillus plantarum and Wickerhamomyces anomalus are used in the pretreatment of the feedstock material. Specifically strains Lactobacillus plantarum 1A7 and Wickerhamomyces anomalus LCF1695 may be used in the pretreatment of the feedstock material.

[0160] In one specific embodiment, one or more spore forming probiotic microbes selected from Bacillus licheniformis, Bacillus clausii, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus fusiformis and Bacillus megaterium (also called Priestia megaterium) may be used in the pretreatment of feedstock material.

[0161] In one preferred embodiment, the probiotic microbes applied in the method of the present invention comprise a combination of bacillus and lactobacillus species. In one preferred embodiment, a culture / suspension of one or more probiotic species of Lactobacillus and / or spore forming Bacillus is applied to the feedstock.

[0162] In one such preferred embodiment, the probiotic culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants in the feedstock comprises of combination of (i) one or more strains of lactic acid bacteria disclosed herein, such as selected from Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus plantarum (for example strain 1A7), Lactobacillus amylovorus, Lactobacillus hammesii, Pediococcus acidilactici (for example strain KTL / 05-7), Pediococcus pentosaceus (for example strain KTL / 05-8) and Pediococcus pentosaceus (for example srain KTU05-10), and (ii) one or more strains of Bacillus species as disclose herein, such as selected from Bacillus licheniformis, Bacillus clausii, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus fusiformis and Bacillus megaterium spore forming bacillus.

[0163] In one embodiment, the probiotic culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants in the feedstock comprises a species of Aspergillus, preferably a spore forming species of Aspergillus , such as a spore forming species of Aspergillus selected from Aspergillus luchuensis, Aspergillus sojae, Aspergillus awamori and Aspergillus oryzae.

[0164] In one important embodiment, the microbial pretreatment of the feedstock material is performed by applying the probiotic culture onto the surface of the feedstock material, such as on the surface of a feedstock material, e.g. by spraying them on the surface of the material, to avoid contamination by other unwanted microbial contaminants, preferably immediately after harvesting and / or immediately after disposal.

[0165] II. Hi Hydolysis

[0166] The feedstock material is hydrolyzed in an aqueous solution. Specifically, it is the pretreated feedstock (probiotic pretreatment as discussed in section II. ii), preferably pretreated starch feedstock, e.g. grain material, which is enzymatically hydrolyzed to obtain monomeric sugars for ethanol fermentation.

[0167] The feedstock may be chopped, milled or broken down I reduced in size by such known processes, before addition of the aqueous solution. In one preferred embodiment, the feedstock is milled to flour prior to the hydrolysis step.

[0168] Depending on the initial water content of the feedstock material, an aqueous solution is added to the feedstock material to obtain a feedstock slurry which has a desired dry matter percentage. In one embodiment, hydrolysis is performed at a dry matter content between 20-40%, preferably between 25-35%, more preferably between 28-33%. In one embodiment, one or more chemicals suitable for limiting growth of unwanted contaminants is added to the feedstock slurry. The one or more chemicals may be selected from ethanol, lactic acid and sodium propionate. In such embodiment when ethanol is added, it is preferably added to reach a concentration of 1-10 wt% in the slurry, more preferably 4-9 wt%, most preferably 6-8 wt%. In such embodiment when lactic acid is added, it is preferably added to reach a concentration of 0.2-2 wt% in the slurry, more preferably 0.3-1.5 wt%, most preferably 0.5-1.0 wt%. In such embodiment when sodium propionate is added, it is preferably added to reach a concentration of 0.05-1 wt% in the slurry, more preferably 0.2-0.8 wt%, most preferably 0.3-0.6 wt%.

[0169] The pH of the feedstock slurry is optionally adjusted to pH >5.5 before hydrolysis. In one embodiment, the pH of the feedstock is adjusted to pH 5-7, such as pH 5.5-7 before hydrolysis. In one such embodiment, the pH of the hydrolysis solution is adjusted by use of any alkali, such as an alkaline solution, e.g. potassium hydroxide, sodium hydroxide, or ammonia water.

[0170] The feedstock material is hydrolyzed at a pH between 4-7, preferably a pH between 5- 6.50, more preferably between 5.5-6.0. In some embodiments, pH adjustment is required at one or more time points during the hydrolysis to maintain the pH within these pH ranges.

[0171] In the present method, the feedstock material is hydrolyzed at a temperature between 30-65 degree C, preferably a temperature between 35-65 degree C, more preferably between 40-55 degree C, most preferably between 40-45 degree C, even more preferably between 50-55 degree C.

[0172] Mixing is preferably performed during the hydrolysis process.

[0173] Starch hydrolyzing enzymes are used for hydrolysis of feedstock comprising starch. One or more amylase enzymes are used for hydrolysis of the starch. In one embodiment, the amylase enzymes comprise alpha- and / or beta-amylases belonging to the E.C. 3.2.1 enzyme classification. In a further embodiment, a mixture of one or more of glucoamylase, acid amylase and cellulase are used in the hydrolysis step; these may also be used in combination with the alpha-amylase. In one specific embodiment, the commercially available enzymes Liquoflow® GO 2X and Saczyme® (from Novozymes A / S) are used either alone or in combination. Based on the feedstock for hydrolysis, dosing of the enzymes may be optimized as recognized by a person skilled in the art.

[0174] In one embodiment, an amylase enzyme is added to the feedstock slurry and the slurry is incubated to obtain a feedstock hydrolysate.

[0175] An advantage of the present invention is that it provides a method, wherein the conventional liquefaction for conversion of starch to sugars is instead performed by a cold mashing process, wherein the temperature is maintained below starch gelatinization temperatures and the enzymatic hydrolysis happens directly on raw (non-gelatinized) starch material.

[0176] Cellulose hydrolyzing enzymes are used for hydrolysis of feedstock comprising cellulose. One or more cellulase enzymes are used for hydrolysis of the cellulose. In one embodiment, the cellulose enzymes comprise beta-glucosidase belonging to the E.C. 3.2.1.21 enzyme classification, endocellulase belonging to the E.C. 3.2.1.4 enzyme classification and / or cellobiohydrolase belonging to the E.C. 3.2.1.91 enzyme classification. In a further embodiment, one or more lytic polysaccharide monooxygenases are further applied in the hydrolysis step; these may also be used in combination with the cellulose enzymes.

[0177] In one embodiment, a cellulose enzyme is added to the feedstock slurry and the slurry is incubated to obtain a feedstock hydrolysate.

[0178] Based on the feedstock for hydrolysis, dosing of the enzymes may be optimized as recognized by a person skilled in the art.

[0179] II. iv Fermentation

[0180] In one preferred embodiment of the second aspect of the present invention, a feedstock is fermented anaerobically by yeast Saccharomyces cerevisiae to produce ethanol, and the pretreated sugar syrup is used as a sugar booster in the fermentation process. In another embodiment of the second aspect of the present invention, a feedstock is fermented aerobically by yeast Saccharomyces cerevisiae to produce ethanol, and the pretreated sugar syrup is used as a sugar booster in the fermentation process.

[0181] The fermentation is performed in continuous mode using pretreated hydrolyzed feedstock (as disclosed in sections II. i, II. ii, and Il.iii herein) as primary substrate in the fermentation, and adding pretreated sugar syrup (as disclosed in sections I.i and i.ii) to one or more of the fermentation stages (as illustrated in Figure IB).

[0182] Fermentation may be performed in several steps, such as one, two, three or more steps in separate fermentation reactors to optimize the process, such as to allow for a continuous process rather than batch fermentation. In Figure IB, a three-stage fermentation is shown as one embodiment of the present invention, but should not be considered as limiting for the invention. The pretreated sugar syrup is added to one or more of the fermentation steps.

[0183] Mixing is preferably performed during the fermentation process. In one embodiment, fermentation a continuous fermentation performed at a dry matter content between 20-60%, preferably between 20-50%, more preferably 25-40%, most preferably between 28-32% in each fermenter.

[0184] In one embodiment, fermentation is continuous fermentation performed at a sugar content having a Brix value between 10-40 Brix, preferably between 20-30 Brix, most preferably around 30 Brix in each fermenter.

[0185] In one embodiment, the fermentation process is performed at a temperature between 20-40 degree C, preferably a temperature between 28-36 degree C, more preferably between 30-32-degree C, to ensure optimal conditions for the yeast.

[0186] In one embodiment, the fermentation is performed at a pH between 4-7, preferably a pH between 4.5-6.5, more preferably between 5.0-6.0, most preferably between 5.0- 5.5; such as at a pH of 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5. Keeping the pH above 5.0 will ensure that any lactic acid and acetic acid present in the solution will not inhibit yeast, as disclosed in section I.iii.

[0187] In one embodiment, the fermentation is performed in a two stage fermentation in two separate fermenters, each stage / fermenter having a specific dilution rate to ensure a specific ethanol concentrations in each stage. In one such embodiment, pretreated sugar syrup is added to the first fermenter. In one such embodiment, pretreated sugar syrup is added to the first and the second fermenter.

[0188] In one embodiment, the fermentation is a continuous fermentation comprising at least two fermenters, wherein the first fermenter is continuously fed with a combination of the feedstock and pretreated sugar syrup, and wherein the dilution rate of the first fermenter is between 0.10-0.55 IT1, resulting in a steady state ethanol concentration between 5-11 % (w / w) in the first fermenter, and wherein the dilution rate of the second fermenter is between 0.04-0.10 IT1, resulting in a steady state ethanol concentration between 10-13% (w / w) in the second fermenter, and optionally wherein pretreated sugar syrup is also continuously added to the second fermenter.

[0189] In one embodiment, the fermentation is performed in a three stage fermentation in three separate fermenters, each stage / fermenter having a specific dilution rate to ensure a specific ethanol concentrations in each stage. In one such embodiment, pretreated sugar syrup is added to the first fermenter. In one such embodiment, pretreated sugar syrup is added to the first and the second fermenter. In one such embodiment, pretreated sugar syrup is added to the first, the second, and the third fermenter.

[0190] The sugar syrup can be added to any yeast Saccharomyces cerevisiae ethanol fermentation process running at an ethanol concentration of 5-8 % (w / w) (preferably around 6%) and a pH between 5.2 -6.5 (preferably pH 6.2) In one embodiment, the fermentation is performed in a three stage continuous mode. A feedstock as disclosed in sections II. i, II. ii, and Il.iii is used as primary substrate in the fermentation process, and sugar syrup as disclosed in sections I.i and I.ii is added as sugar booster to each stage:

[0191] The ethanol concentration (w / w) in the first stage is maintained between 5% to 11%, preferably with 5% to 9%, more preferably between 5% to 8%, more preferably between 5% to 7%, most preferably between 5.5-6.0%. The steady state concentration of ethanol in stage 1 is achieved by continuous inflow of pretreated hydrolyzed feedstock in combination with pretreated sugar syrup to the fermentation broth in the stage 1 fermenter and continuous outflow of fermented material from the stage 1 fermenter to the stage 2 fermenter, maintaining a dilution rate (D) in the range of 0.10 -0.55 IT1, preferable between 0.10 -0.30 h1, more preferably between 0.10 -0.20b1. An advantage of keeping the ethanol concentration in this desired range, especially between 5- 8% in the first fermenter, is that potential contaminants, such as lactic acid bacteria (LAB), acetic acid bacteria (AAB) or other will be inhibited, while yeast is not inhibited. The residence time of first fermenter is between 5 to 80 hours, preferably between 10 to 30 hours, more preferably between 12 to 15 hours.

[0192] The outflow from the first fermenter is transferred to a second fermenter where the ethanol concentration (w / w) is maintained between 6% to 15 %, preferably with 8 % to 15 %, more preferably between 10% to 13%; such as 10%, 10.50%, 11%, 11.5%, 12%, 12.5% or 13%. The steady state concentration of ethanol is achieved by continuous inflow of fermented material from the stage 1 fermenter in combination with pretreated sugar syrup to the stage 2 fermenter and continuous outflow of fermented material from the stage 2 fermenter to the stage 3 fermenter, maintaining a dilution rate (D) in the range of 0.04-0.10 IT1, preferably between 0.04-0.08 IT1, more preferably between 0.04-0.06 IT1. The residence time of second fermenter is between 5 to 80 hours, between 5 to 60 hours, preferably between 10 to 40 hours, more preferably between 25 to 30 hours.

[0193] The outflow from the second fermenter is transferred to a third fermenter where the ethanol concentration (w / w) is maintained between 10 % to 15 %, preferably between 12% to 15%. The constant ethanol concentration is achieved by continuous inflow of fermented material from the stage 2 fermenter in combination with pretreated sugar syrup to the stage 3 fermenter and continuous outflow of fermented material from the stage 3 fermenter to a distillation unit. The residence time of third fermenter is between 3 to 8 hours, preferably between 4 to 7 hours, more preferably between 5 to 6 hours. In one embodiment of the three stage fermentation stage disclosed above, yeast is added to the feedstock to obtain a fermentation broth. The fermentation broth is fermented to reach an ethanol concentration (% w / w) of between 5-8%. At this ethanol concentration, the addition of pretreated sugar syrup is initiated.

[0194] The dosing rate of the sugar syrup is preferably adjusted to compensate for the weight loss due during fermentation, e.g. due to exiting CO2 and some moisture. The typical evaporation loss is 4-6% in ethanol fermentation. The higher the ethanol production rate, the more the production of CO2 resulting in higher evaporation loss and thereby higher dosing rate of the sugar syrup to compensate the evaporation loss.

[0195] In one embodiment, the pretreated sugar syrup comprises between 1-20% of the total inflow volume to each fermenter in the continuous fermentation process. Preferably between 3-15%, more preferably between 5-10%.

[0196] In one embodiment, yeast is added to a feedstock to obtain a fermentation broth. The fermentation both is fermented by continuous fermentation comprising at least two stages, preferably three stages. In one preferred embodiment, pretreated sugar syrup is appropriately dosed to a stage 1 fermenter comprising the fermentation broth, said stage 1 fermenter being maintain at an ethanol concentration between 5- 8% (w / w); this ethanol concentration in stage 1 may be obtained by maintaining a dilution rate between 0.1 IT1’ to 0.55 h1in stage 1. In a further preferred embodiment, the fermented mash from the stage 1 fermenter is then continuously dosed to a stage 2 fermenter and pretreated sugar syrup is also appropriately dosed to the stage 2 fermenter, maintaining an ethanol concentration in the stage 2 fermenter between 10-13% (w / w); this ethanol concentration in stage 2 may be obtained by maintaining a dilution rate between 0.04 h1to 0.1 h’1. In a further preferred embodiment, the fermented mash from the stage 2 fermenter is then dosed to a stage 3 fermenter, and pretreated sugar syrup is also appropriately dosed to the stage 3 fermenter, maintaining an ethanol concentration between 12-15% (w / w) in the stage 3 fermenter, by continuously dosing the fermented mash from stage 3 to a distillation unit.

[0197] An advantage of the continuous mode is, there is no need to stop fermentation, hence a significant saving on Cleaning In Place (CIP) cycles, resulting in reduced usage of water, energy as well as chemicals. Moreover decreasing CIP also results in improving overall process downtime. In ethanol production , typically around 8-10 hours are lost in cleaning and restarting batch fermentation process (downtime). In traditional ethanol plants, due to the risk of contamination, such mode of operating the process in continuous mode is avoided. The current invention overcomes this potential limitation. The process of the present invention benefits from maintaining the pH around pH 5 (or higher) as this keeps the VAs in their unprotonated state whereby VA inhibition of the yeast is reduced, hence creating most favorable conditions for the yeast fermentation and ensuring less stress on the yeast, thereby avoiding potential arrest in fermentation. Further, as a result of the higher ethanol concentration obtained in the first stage of fermentation, potential contaminants are inhibited, thereby sending well adapted yeast to the second stage of fermentation, which overall lead to improved ethanol yields. Apart from this, continuous fermentation mode also results in significant reduction in downtime (time required to start up new fermentation), thus improving productivity of the entire process. in Method of making ethanol

[0198] In one aspect, the present invention provides a fermentation method for making ethanol and optionally a protein feed or food product, wherein said fermentation is a continuous fermentation using yeast Saccharomyces cerevisiae and is performed in at least two fermenters, wherein the first fermenter is continuously fed with a feed, wherein said feed comprises (i) a diluted pretreated sugar syrup or (ii) a combination of a feedstock and a pretreated sugar syrup, wherein said pretreated sugar syrup is a sugar syrup which has been pretreated by applying a microbial and enzyme cocktail to the sugar syrup, said cocktail comprising (i) a first culture of one or more probiotic microorganism(s) suitable for limiting growth of unwanted contaminants in the sugar syrup and (ii) a dextranase enzyme and / or a second culture of one or more dextranase producing probiotic microorganism(s), wherein said diluted pretreated sugar syrup is the pretreated sugar syrup which has been diluted using an aqueous solution to obtain a Brix value between 10-40 Brix, and wherein said feedstock comprises glucose or a glucose-derived compound.

[0199] Characteristics of the sugar syrup and pretreated sugar syrup is as disclosed in sections I.i and I.ii; and characteristics of the feedstock is disclosed in sections II. i, II. ii, and Il.iii.

[0200] In a preferred embodiment, the first fermenter is continuously fed with the feed, maintaining a dilution rate of the first fermenter of between 0.10-0.55 IT1, preferably between 0.10 -0.30 IT1, more preferably between 0.10 -0.20b1, resulting in a steady state ethanol concentration in the first fermenter of between 5-11 % (w / w), preferably between 5-9%, more preferably between 5-7% most preferably between 5.5-6.0%; and wherein the second fermenter is continuously fed with fermented material from the first fermenter, maintaining a dilution rate of the second fermenter of between 0.04-0.10 h’1, preferably between 0.04-0.08 h’1, more preferably between 0.04-0.06 IT1, resulting in a steady state ethanol concentration in the second fermenter between 10-13% (w / w), preferably between 9-12%; more preferably 11-12%.

[0201] In a further preferred embodiment, the fermentation comprises a third fermentor, wherein the third fermenter is continuously fed with fermented material from the second fermenter, maintaining an ethanol concentration in the third fermenter between 12-15% (w / w), preferably between 13-15%.

[0202] Pretreated sugar syrup may optionally be continuously added to the second fermenter and / or third fermenter.

[0203] IV Recovery of ethanol

[0204] The first main product of the process of the present invention is ethanol. Ethanol may be recovered by distillation of the output from the fermentation tank. In one example, as recognized by a person skilled in the art, the fermented slurry may be heated to ~ 60°C, such as by using a heat exchanger, and then pumped into a distillation column where the temperature may be maintained at around ~ 80°C. Ethanol is evaporated off, while the generated whole stillage is sent to further separation and filtration for protein recovery.

[0205] Evaporated ethanol may further be rectified to achieve ~95% ethanol concentration. A molecular sieve may further be applied to dehydrate the ethanol product to a concentration of 99.99%. At the end, the dehydrated ethanol may be denatured by adding an appropriate denaturant.

[0206] V Recovery of protein

[0207] A second main product of the process of the present invention is a protein feed or food product. In one abonnement, the whole stillage from the distillation process is processed to produce a protein product, preferably a high protein product, such as a protein product comprising at least 40, 45, 50, 55, 60, 65, 70, 75%, or at least 80% protein in the final composition. Protein products having high protein content (w / w) are highly desirable.

[0208] To obtain such high protein product, the whole stillage must undergo separation and / or purification steps. In one embodiment, the whole stillage is separated into two fractions: thin stillage containing soluble fibers, fat, salts and proteins, and wet cake comprising of insoluble fiber and protein. The oil or fat content of thin stillage is further removed using separation technologies like disc stack centrifuge or decanter. The defatted or deoiled thin stillage may be boiled and may by recycled back. The wet cake is dried using conventional driers like tray, ring or DDGS (Distiller's dried grains with solubles) drier to obtain high protein.

[0209] In one embodiment, the oil or fat content in the wet cake is removed using organic solvents like supercritical CO2 or hexane or n-pantane or octanol.

[0210] VI. Products of the present invention

[0211] VI. i Ethanol

[0212] One of the products obtained from the present method is ethanol. As discussed above, the ethanol yields are improved compared to traditional ethanol processes.

[0213] In one embodiment, the ethanol purity is at least 99.3%; Density at 20 degrees Celsius is maximum 0.7915 kg / liter; Water content is maximum 0.5% (by mass); Methanol is maximum 0.5% (by mass), and total acids (as acetic acid) is maximum 0.007% (by mass)

[0214] VI. ii Protein feed or food product

[0215] One of the products obtained from the present method is protein.

[0216] The term "protein feed product" refers to a protein product which based on its purity can be used as feed, such as for animal consumption. The term "protein food product" refers to a protein product which based on its purity can be used as food, such as for human consumption.

[0217] In the present context, purity is measured as amount of toxins - such as deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV), and zearalenone (ZON). By pretreating the sugar syrup and feedstock material using only probiotic microorganisms, the protein product obtained by the method of the present invention is essentially pure in the sense that it is free of toxins, as no other potentially toxic microbial contaminations can arise in the process due to the presence of the probiotic microbes. The probiotic microbes themselves do not produce toxins.

[0218] Fungal contamination is the biggest challenge in ethanol fermentation due to its toxins. For a protein product, which goes into human food or animal feed, having fungal toxins present, such as aflatoxins, are highly unfavorable and also not allowed in many jurisdictions. However, the presence of probiotic lactic acid bacteria (dead cells, as everything will be killed on distillation column) is on the contrary seen as added value, especially as non-gmo probiotics.

[0219] In one embodiment, the protein product has Dry matter of 90%; Crude protein of > 60%; Crude fat of > 8%; and Essential amino acid (for example, Lysine) of > 2 %.

[0220] VII. Advantages and commercial application

[0221] As disclosed above, the method of the present invention provides many advantages compared to traditional ethanol fermentation and has direct commercial application. The following list summarizes some of the advantages of the present process:

[0222] • Reducing contamination in sugar syrup (e.g. molasses) resulting in improved microbial stability

[0223] • Reducing dextran formation in sugar syrup (e.g. molasses) resulting in improved sucrose yield, decreased viscosity, improved utilization of the sugars for yeast fermentation processes

[0224] • Reducing inhibitory effect of volatile acids like Butyric acid resulting in robust and faster fermentation.

[0225] • Improving productivity of ethanol fermentation by dosing sugar syrup.

[0226] • Improving the productivity of ethanol fermentation by increasing the ethanol production rate due to increased dilution rate, compared to a process not applying sugar syrup addition.

[0227] • Reducing contamination in starch-feedstock resulting in improved microbial stability

[0228] • Avoiding the need to use antimicrobial agents to control contamination resulting in better protein quality (protein without antimicrobial residues).

[0229] • Avoiding the need to thermal pretreatment resulting in energy and water savings and the cost associated with

[0230] • Overall reduction of contamination risk in the fermentation process

[0231] • Reducing water consumption in the ethanol process.

[0232] • Reducing CIP cycles (CIP= Cleaning in Place).

[0233] • Producing a pure high protein product which may be used as food or feed product.

[0234] • Reducing energy requirement

[0235] • Reducing GHG emissions on ethanol.

[0236] • Improving productivity of ethanol fermentation by eliminating or reducing downtime. VIII. Numbered embodiments

[0237] Numbered embodiment 1. A fermentation method for making ethanol and optionally a protein feed or food product, wherein said fermentation is a continuous fermentation using yeast Saccharomyces cerevisiae, wherein said continuous fermentation is performed in at least two fermenters, wherein the first fermenter is continuously fed with a combination of a feedstock and a pretreated sugar syrup, maintaining a dilution rate of the first fermenter of between 0.10-0.55 IT1, resulting in a steady state ethanol concentration of between 5-11 % (w / w) in the first fermenter, and wherein the second fermenter is continuously fed with fermented material from fermenter 1, maintaining a dilution rate of the second fermenter of between 0.04-0.10 IT1, resulting in a steady state ethanol concentration between 10-13% (w / w) in the second fermenter, and optionally wherein pretreated sugar syrup is also continuously added to the second fermenter, wherein said feedstock comprises glucose or a glucose-derived compound, and wherein said pretreated sugar syrup is a sugar syrup which has been pretreated by applying a microbial and enzyme cocktail to the sugar syrup, said cocktail comprising (i) a first culture of one or more probiotic microorganism(s) suitable for limiting growth of unwanted contaminants in the sugar syrup and (ii) a dextranase enzyme and / or a second culture of one or more dextranase producing probiotic microorganism(s).

[0238] Numbered embodiment 2. The method according to Numbered embodiment 1, wherein the sugar syrup is selected from molasses, fructose syrup, maltose syrup, glucose syrup, and sucrose syrup, preferably the sugar syrup is molasses, such as sugarcane molasses or sugar beet molasses.

[0239] Numbered embodiment 3. The method according to Numbered embodiment 1 or 2, wherein the first culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants in the sugar syrup comprises species of Lactobacillus and / or a species of Fusarium, preferably comprises a combination of Lactobacillus and Fusarium, more preferably comprises a combination of Lactobacillus sake! and Fusarium venenatum.

[0240] Numbered embodiment 4. The method according to any one of Numbered embodiments 1-3, wherein the second culture of one or more dextranase producing probiotic microorganisms comprises Lactobacillus bifibus. Numbered embodiment 5. The method according to any one of Numbered embodiments 1-4, wherein the microbial and enzyme cocktail comprises Lactobacillus sake!, Fusarium venenatum, and Lactobacillus bifibus.

[0241] Numbered embodiment 6. The method according to any one of Numbered embodiments 1-5, wherein the fermentation is initiated in the first fermenter by addition of yeast Saccharomyces cerevisiae to the feedstock, thereby obtaining a fermentation broth, and the fermentation broth in fermenter 1 is fermented to reach an ethanol concentration between 5-8% prior to the continuous feeding with the combination of the feedstock and the pretreated sugar syrup.

[0242] Numbered embodiment 7. The method according to any one of Numbered embodiments 1-6, wherein the continuous fermentation comprises at least three fermenters, wherein the third fermenter is continuously fed with the fermentation material from fermenter 2, maintaining a dilution rate of the third fermenter of between 0.02-0.06 IT1, resulting in a steady state ethanol concentration between 12-15 % (w / w) in the third fermenter; and optionally wherein pretreated sugar syrup is also continuously added to the third fermenter.

[0243] Numbered embodiment 8. The method according to any one of Numbered embodiments 1-7, wherein the feedstock comprises starch and optionally protein, preferably wherein the feedstock is grain and / or one or more grain derived products, such as grain selected from wheat, rice, oats, barley, rye, millet, corn, triticale, and sorghum grain; and wherein said method comprises hydrolyzing the feedstock using one or more starch degrading enzyme(s), and wherein said hydrolyzed feedstock is fermented by the continuous fermentation using yeast Saccharomyces cerevisiae.

[0244] Numbered embodiment 9. The method according to any one of Numbered embodiments 1-8, wherein the method comprises pretreating the feedstock by applying to the feedstock a third culture of one or more probiotic microorganism(s) suitable for limiting growth of unwanted contaminants in the feedstock.

[0245] Numbered embodiment 10. The method according to any one of Numbered embodiments 1-9, wherein the third culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants in the feedstock comprises a species of Lactobacillus and / or a species of spore forming Bacillus, preferably comprises a combination of a species of Lactobacillus and a species of Bacillus, more preferably comprises a combination of (i) a species of Lactobacillus selected from Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus amylovorus, and Lactobacillus hammesii and (ii) a species of spore forming Bacillus selected from Bacillus licheniformis, Bacillus clausii, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus fusiformis and Bacillus megaterium. EXAMPLES

[0246] Example 1: Pretreatment of molasses and other sugar syrups with probiotic bacteria: Fusarium venenatum and Lactobacilus sake!

[0247] Lab scale microbial stability test on molasses and other sugar syrups was done using Fusarium venenatum strain ATCC 20334 and Lactobacilus sake!.

[0248] Fusarium venenatum was inoculate in the defined medium (VogellO) consisted of: 10 g glucose, 2.6 g Na3C6H5O7-2H2O, 2.52 g KNO3, 2.88 g (NH4)H2PO4, 1.6 g KH2PO4, 0.2 g MgSO4'7H2O, 0.1 g, CaCI2-2H2O, 2.5 mL of biotin solution and 5 mL of trace elements per liter. The trace elements solution consisted of 0.1 g citric acid, 0.1 g ZnSO4'7H2O, 0.02 g FeSO4-(NH4)2SO4-6H2O, 5 mg CuSO4-5H2O, 1 mg MnSO4-H2O, 1 mg H3BO3, 1 mg Na2MoO4-2H2O per 100 mL. The pH of the medium was adjusted to 5.8. Inoculum was prepared in 250 conical flasks containing 50 mL Vogel medium. Flasks were inoculated from slant and incubated on a rotary shaker (25 °C, 200 rpm, 48).

[0249] Lactobacillus sake! was propagated for 24 h at 30°C in MRS (non-selective medium for growth of lactic acid bacteria, Oxoid Laboratories, Hampshire, United Kingdom) with the addition of fresh yeast extract (5% [vol / vol]) and 28 mM maltose. pH is adjusted to 5.6.

[0250] Leuconostoc mesenteroides was propagated for 24 h at 30°C in MRS (non-selective medium for growth of lactic acid bacteria, Oxoid Laboratories, Hampshire, United Kingdom) with the addition of fresh yeast extract (5% [vol / vol]) and 28 mM maltose. pH is adjusted to 5.6.

[0251] The molasses was sterilized using autoclave (121 degree for 20 min). A sample of around 100 ml sterile molasses was transferred to sterile bottle ( 250 ml) * 8 sets. Table 1 gives an overview of experimental design.

[0252] The experiment was carried out for 10 days and analysis of molasses was done on the last day. A same procedure was repeated where molasses was replaced with glucose syrup, maltose syrup, and high fructose corn syrup.

[0253] Microbiological analysis: 1 ml of molasses sample was taken from each bottle and spread to nutrient agar and ROGOSA agar (selective for lactobacillus) respectively. Care was taken to take sample from the bottom of the bottle (to avoid fusarium) using sterile pipette tip. The plates were incubated at 28 degrees for 2 days. Table 2 summarizes the results.

[0254] Conclusions:

[0255] 1 No growth in set 1 confirms sterilization of molasses was done properly and no microorganism survived during autoclavation.

[0256] 2 Detection of microbial colonies in nutrient agar of set 2 indicates presence of various microorganisms in the air.

[0257] 3 No Detection of microbial colonies in set 3 indicates efficacy of Fusarium base pretreatment towards preventing airborne contamination (fusarium forms thick mat over surface and thus prevents airborne contamination).

[0258] 4 Detection of microbial colonies in set 4 indicates the inability of Lactobacillus sake! based pretreatment to prevent airborne contamination.

[0259] 5 Slime formation in set 5 means inability of Fusarium based pretreatment towards preventing or controlling Leuconostoc growth.

[0260] 6 No slime formation in set 6 indicates efficacy of Lactobacillus Sake! based pretreatment against controlling growth of Leuconostoc.

[0261] 7 No slime formation in set 7 indicates efficacy of Fusarium and Lactobacillus Sake! based pretreatment against controlling growth of Leuconostoc. Detection of microbial colonies on ROGOSA agar and not on nutrient agar indicates presence of Lactobacillus easel (lactobacilli grow very poorly on nutrient agar and very well on ROGOSA agar).

[0262] 8 No slime formation in set 8 indicates efficacy of Fusarium and Lactobacillus Sake! based pretreatment against controlling growth of Leuconostoc as well as preventing air born contamination. A combined effect was seen here, Lactobacillus easel can prevent growth of Leconostoc (based on results from set and Fusarium can prevent airborne contamination by forming thick mat (based on results from set 3). Example 2: Ethanol fermentation with probiotic pretreated molasses

[0263] A shake flask experiment was conducted to simulate ethanol fermentation process. The media comprises of a mixture of bread crump, Saczyme C mash (raw starch hydrolyzing enzyme), Saczyme yield (a mixture of cellulase and glucoamylase), Viscoferm (Xylanase) and urea. The liquefaction was carried out by increasing slurry temperature around 45°C for 2 hours. After adjusting pH to 6.20 using any base such as NaOH, KOH or ammonia water, the liquefact was added to six shake flasks (100ml in each flask).

[0264] Table 3 describes the experimental design. Innova Fit (yeast) was inoculated to all shake flasks at zero hours to kickstart fermentation. In shake flask 5 & 6, 10 ml of probiotic pretreated molasses was also added at zero hours, while in shake flasks 3 & 4 10 ml of probiotic pretreated molasses was added after 24 hours. No molasses was added to shake flasks 1 & 2.

[0265] The probiotic pretreated molasses was pretreated with Fusarium venenatum and Lactobacillus sake!.

[0266] All the flasks were incubated at 32 degree C for 72 hours. After 72 hours, fermentations were terminated, and 10 ml sample was collected from each shake flask. The samples were centrifuged, and supernatant was collected to do HPLC analysis for estimation of glucose, lactic acid, acetic acid and ethanol. Table 4 summarizes the results. As seen from the above table, Molasses addition at zero hour (shake flask 3 & 4) resulted in overgrowth of lactobacillus instead of yeast as seen from lactic and acetic acid concentrations. Average Ethanol concentration was 2.25% which was significantly lower than control where average ethanol concentration was 11.85% indicating inhibition of yeast by lactic acid. Glycerol to ethanol ratio, an indicator of yeast stress is 1.78 indicating severe stress (the ratio between 0.7 -1,0 is considered as normal). Ethanol fermentation was normal in 3rdset (shake flask 5 & 6) where molasses was addition at 24 hours. The slightly higher concentration (13.9) than control was due to extra sugar from molasses.

[0267] Conclusions:

[0268] Pretreated molasses contains lactic acid bacteria and fungi, the main purpose of those is to prevent growth of unwanted airborne microorganism. However, if added at the beginning of ethanol fermentation is not ideal. Here we showed a smart way to circumvent this challenge by dosing probiotic containing molasses after 24 hours of fermentation when ethanol concentration is > 6%. At this concentration, most of the microorganisms except yeast cannot grow and thus creating advantageous environment for the yeast. Initial pH adjustment > 5.20 ensures the lactic and acetic acid does not protonate and inhibit yeast, avoiding yeast stress.

[0269] Example 3: Effect of probiotic treatment on dextran

[0270] To validate production of dextranase by Lactobacillus bifidus in molasses, Lactobacillus bifidus was propagated for 24 h at 30°C in MRS (non-selective medium for growth of lactic acid bacteria, Oxoid Laboratories, Hampshire, United Kingdom) with the addition of fresh yeast extract (5% [vol / vol]) and 28 mM maltose. pH is adjusted to 5.6. ~ 100 ml. Molasses was autoclaved for 121°C for 25 minutes and inoculated with 1ml of Lactobacillus bifidus (Bifidobacterium bifidum) culture and incubated for one week at 30°C. A negative control was also prepared where 1 ml of sterile water was added in the molasses. After one week, both the samples were centrifuged, and supernatant was collected.

[0271] Dextran from Leuconostoc mesenteroides (average mol wt 60000-80000) was purchased from Sigma-Aldrich (CAS no: 9004-54-0). Dextranase activity from probiotic treated molasses was measured by evaluating the amount of reducing sugars through 3.5-dinitrosalicylic acid (DNS) method (Miller, 1959). The enzyme assay was performed using 1ml supernatant from probiotic treated molasses, 370 pL of 100 mM citrate-phosphate buffer pH 6.0, 100 pL of 1% dextran solution. Same procedure was repeated for negative control. All the reagents were incubated at 50 °C for 10 min, and the reaction was stopped by adding 500 pL of the DNS reagent. Samples were heated for 5 min in a boiling water bath, and then 1 mL of water was added to each sample. Absorbance readings were performed at 540 nm. In order to obtain the amount of reducing sugars, a standard curve was constructed with varying glucose concentrations. One dextranase unit (U) was defined as the amount of the enzyme that catalyzes the release of 1 pmol of reducing sugar per minute under the assay conditions. All enzymatic activity measurements were performed in triplicate.

[0272] Results & conclusion:

[0273] The dextranase presence was confirmed and ~ 10 Dextranase unit (U) was detected in the molasses inoculated with Lactobacillus bifidus.

[0274] Example 4: Three stage continuous fermentation using pretreated molasses as substrate

[0275] Figure 1A illustrates an experimental set up of three stage fermentation system. A feed bottle with molasses pretreated with probiotics was prepared. The pretreated molasses was transferred to fermenter 1 (stage 1). After adjusting pH yeast was inoculated to kickstart fermentation. A minimum ethanol concentration of approx. 6 % was maintained in fermenter 1 to have bactericidal effect. Using cold water, the temperature in fermenter 1 & fermenter 2was maintained at 35°C and 30°C, respectively. Ethanol concentration was maintained ~ 10% ethanol (w / w) in reactor 2 by maintaining constant inflow of fermented medium (from reactor 1) and outflow from reactor 2. The fermented mash was continuously collected in final fermentation tank (also referred to as the beer well tank) where it was further fermented for another 6 -7 hours until reaching in final ethanol concentration of ~ 15% (w / w).

[0276] Example 5: Three stage continuous fermentation using pretreated molasses as sugar booster

[0277] Figure IB illustrates an experimental set up of three stage fermentation system. The liquefied substrate is transferred to fermenter 1 (stage 1). After adjusting pH yeast is inoculated to kickstart fermentation. Separately, a dosing bottle with molasses pretreated with probiotics is prepared.

[0278] A minimum ethanol concentration of approx. 6 % is maintained in fermenter 1 to have bactericidal effect. Using cold water, the temperature in fermenter 1 & fermenter 2 is maintained at 35°C and 30°C, respectively. Ethanol concentration is maintained ~ 10% ethanol (w / w) in reactor 2 by maintaining constant inflow of fermented medium (from reactor 1) and outflow from reactor 2.

[0279] After reaching steady state, the probiotic treated molasses dosing is started. The fermented mash is continuously collected in final fermentation tank (also referred to as the beer well tank) where it is further fermented for another 6 -7 hours until reaching in final ethanol concentration of ~ 15% (w / w).

[0280] REFERENCES Miller. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar.

[0281] Anal Chem., 426-428.

Claims

CLAIMS1. A fermentation method for making ethanol and optionally a protein feed or food product, wherein said fermentation is a continuous fermentation using yeast Saccharomyces cerevisiae and is performed in at least two fermenters, wherein the first fermenter is continuously fed with a feed, maintaining a dilution rate of the first fermenter of between 0.10-0.55 IT1, resulting in a steady state ethanol concentration in the first fermenter of between 5-11 % (w / w); and wherein the second fermenter is continuously fed with fermented material from the first fermenter, maintaining a dilution rate of the second fermenter of between 0.04-0.10 IT1, resulting in a steady state ethanol concentration in the second fermenter between 10-13% (w / w), wherein said feed comprises (i) a diluted pretreated sugar syrup or (ii) a combination of a feedstock and a pretreated sugar syrup, wherein said pretreated sugar syrup is a sugar syrup which has been pretreated by applying a microbial and enzyme cocktail to the sugar syrup, said cocktail comprising (i) a first culture of one or more probiotic microorganism(s) suitable for limiting growth of unwanted contaminants in the sugar syrup and (ii) a dextranase enzyme and / or a second culture of one or more dextranase producing probiotic microorganism(s), wherein said diluted pretreated sugar syrup is pretreated sugar syrup which has been diluted using an aqueous solution to obtain a Brix value between 10-40 Brix, and wherein said feedstock comprises glucose or a glucose-derived compound.

2. The method according to claim 1, further comprising a third fermenter, wherein the third fermenter is continuously fed with fermented material from the second fermenter, maintaining an ethanol concentration in the third fermenter between 12- 15% (w / w).

3. The method according to claim 1 or 2, wherein the feed comprises a combination of the feedstock and the pretreated sugar syrup, and wherein additional pretreated sugar syrup is continuously added to the second fermenter and / or third fermenter.

4. The method according to any one of claims 1-3, wherein the sugar syrup is selected from molasses, fructose syrup, maltose syrup, glucose syrup, and sucrose syrup, preferably the sugar syrup is molasses, such as sugarcane molasses or sugar beet molasses.

5. The method according to any one of claims 1-4, wherein the first culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants in the sugar syrup comprises species of Lactobacillus and / or a species of Fusarium, preferably comprises a combination of species of Lactobacillus and Fusarium, more preferably comprises a combination of Lactobacillus sake! and Fusarium venenatum.

6. The method according to any one of claims 1-5, wherein the second culture of one or more dextranase producing probiotic microorganisms comprises Lactobacillus bifibus.

7. The method according to any one of claims 1-6, wherein the microbial and enzyme cocktail comprises Lactobacillus sake!, Fusarium venenatum, and Lactobacillus bifibus.

8. The method according to any one of claims 1-7, wherein the dextranase enzyme is a purified or partly purified enzyme.

9. The method according to any one of claims 1-8, wherein the dextranase producing probiotic microorganisms is a probiotic microorganism, preferably Lactobacillus bifidus.

10. The method according to any one of claims 1-9, wherein the fermentation is initiated in the first fermenter by addition of yeast Saccharomyces cerevisiae to the feedstock, thereby obtaining a fermentation broth, and the fermentation broth in fermenter 1 is fermented to reach an ethanol concentration between 5-8% prior to the continuous feeding with the feed.

11. The method according to any one of claims 2-10, wherein the third fermenter is continuously fed with the fermentation material from the second fermenter and has a steady state ethanol concentration between 12-15 % (w / w).

12. The method according to any one of claims 2-11, wherein the third fermenter is continuously fed with the fermentation material from the second fermenter, maintaining a dilution rate of the third fermenter of between 0.02-0.06 IT1, resulting in a steady state ethanol concentration between 12-15 % (w / w) in the third fermenter.

13. The method according to any one of claims 1-12, wherein the feedstock comprises starch and optionally protein, preferably wherein the feedstock is grain and / or one or more grain derived products, such as grain selected from wheat, rice, oats, barley, rye, millet, corn, triticale, and sorghum grain; and wherein said method comprises hydrolyzing the feedstock using one or more starch degrading enzyme(s), and wherein said hydrolyzed feedstock is fermented by the continuous fermentation using yeast Saccharomyces cerevisiae.

14. The method according to any one of claims 1-13, wherein the method comprises pretreating the feedstock by applying to the feedstock a third culture of one or more probiotic microorganism(s) suitable for limiting growth of unwanted contaminants in the feedstock.

15. The method according to claim 14, wherein the third culture of one or more probiotic microorganism suitable for limiting growth of unwanted contaminants in the feedstock comprises a species of Lactobacillus and / or a species of spore forming Bacillus, preferably comprises a combination of a species of Lactobacillus and a species of Bacillus, more preferably comprises a combination of (i) a species of Lactobacillus selected from Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus amylovorus, and Lactobacillus hammesii and (ii) a species of spore forming Bacillus selected from Bacillus licheniformis, Bacillus clausii, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus fusiformis and Bacillus megaterium.

16. The method according to any one of claims 1-15, wherein the feedstock is a feedstock slurry obtained by adding an aqueous solution to the feedstock, and wherein said aqueous solution further comprises one or more chemicals suitable for limiting growth of unwanted contaminants in the feedstock slurry.

17. The method according to claim 16, wherein the one or more chemicals suitable for limiting growth of unwanted contaminants in the feedstock slurry is selected from ethanol, lactic acid and sodium propionate.

18. The method according to claim 16 or 17, wherein the concentration of ethanol in the feedstock slurry is 1-10 wt%, and / or the concentration of lactic acid in the feedstock slurry is 0.2-2 wt%, and / or the concentration of sodium propionate in the feedstock slurry is 0.05-1 wt%.

19. The method according to any one of claims 1-19, wherein the method further produces a protein feed or food product, by providing a feed comprising protein.

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