WO2023148727A1 - Method for controlling prokaryotic contamination in yeast fermentation processes by biocides produced on-site - Google Patents

Abstract

The present disclosure provides a method for controlling contaminations of prokaryotes known to reside alongside yeast during fermentation processes, e.g., bacterial contaminations, by using a bromine/bromide-containing solution and/or a chloramine - containing solution. The method enables purifying yeast cultures prone to prokaryotic contamination, while maintaining the viability of the yeasts, thereby increasing the yield of ethanol production.

Description

METHOD FOR CONTROLLING PROKARYOTIC CONTAMINATION IN YEAST FERMENTATION PROCESSES BY BIOCIDES PRODUCED ON-

SITE

FIELD OF THE INVENTION

The invention provides a method for controlling contaminations of prokaryotes known to reside alongside yeasts during fermentation processes, e.g., bacterial contamination. Efficient control of bacterial contamination, while maintaining the viability of the yeasts, leads to an increase in the yield of end product production, e.g., ethanol.

BACKGROUND OF THE INVENTION

Ethanol-based biofuel (also termed “bioethanol”) is one of the sustainable fuels used in many countries. It can be used directly as pure ethanol or may be blended with gasoline. Bioethanol is less toxic, readily biodegradable and produces air-borne pollutants to a lesser extent than petroleum fuel.

Conventional production methods of ethanol use a variety of carbohydrate substrates, e.g., sugar (derived, for example, from sugar cane and sugar beet), starch (derived, for example, from com, wheat, or potatoes) or other polysaccharides. Monosaccharide sugars are converted into ethanol mostly by varieties of the yeast Saccharomyces cerevisiae during a fermentation process.

Generally, the fermentation process involves contacting a carbohydrate substrate-rich medium with yeasts and fermenting it until the substrate is consumed, or until the yeasts are inhibited by the increase in ethanol concentration. In many cases, the fermenting yeasts are discarded and fresh inoculi are used for each cycle of fermentation. In contrast, distilleries in Brazil have adopted a fermentation process during which the yeast cells are recycled (also referred to herein as the “Brazilian process of ethanol production”). At the end of each fermentation cycle of the Brazilian process for ethanol production, the fermented broth is processed to separate the yeast cells in the form of a concentrated cream from the liquid that undergoes further distillation. After being treated with water-diluted sulfuric acid (e.g., at a pH of 2.0- 2.5, for 1-2 hours), these yeast cells are returned to large-volume fermentation tanks (250-3000 L) for participating in a new fermentation cycle (Lopes et al. Ethanol production in Brazil: a bridge between science and industry. Brazilian Journal of Microbiology: 47S: 64-76. 2016). Overall, the Brazilian process of ethanol production results in a better ethanol yield, since less sugar is consumed for yeast cell multiplication. However, bacterial contaminants are also recycled into the fermentation process and may be troublesome, for example due to substrate competition, thereby reducing the overall ethanol yield during fermentation.

As known in the art, bacteria cause various additional problems during fermentation, generally leading to inhibition of yeast fermentation and reduction of industrial yields. These problems include, for example, production of organic acids and stimulating flocculation of the yeast cells. One of the particular challenges is the control of contamination by lactic and acetic acid bacteria growth, such as bacteria of the genus Lactobacillus sp and Acetobacter sp., which produce lactic acid or acetic acid instead of ethanol.

In order to reduce the level of bacterial contaminants, the Brazilian process of ethanol production uses dilute sulfuric acid to kill bacteria, as detailed above. Without this acidic treatment step, industrial fermentations are subjected to sugar losses and reduced ethanol yield due to the presence of bacterial contamination.

Currently, there are various methods employed to selectively eliminate bacterial contaminants without harming the yeasts during fermentation, among which is by using antibiotics. The most commonly used antibiotics are penicillin, virginiamycin, erythromycin, tylosin, tetracycline and monensin. However, there are several concerns involving the use of antibiotics, for example, the emergence of resistant bacteria. This concern arises in particular due to the presence of antibiotics residues in dried distillers’ grains (DDG), a nutrient-rich co-product of ethanol production derived from starting material such as corn, and sold as livestock feed. Various countries do not accept any antibiotic residues in inactive dried yeasts for animal feed, thus, it is desirable to avoid the use of antibiotics.

Various other methods are used to overcome the problems relating to bacteria contamination during ethanol production (fermentation). One of the most commonly used methods is the application of a biocide during the steps of the fermentation process. For example, the publication US 2012/0009641 (by Kulkarni et al.) suggests a method including a pretreatment in which the sugar cane is washed with water comprising a biocide. Additionally, biocides such as quaternary ammonium compounds, carbamates, glutaraldehyde, and halogenated non-oxidizing organic biocides are among the proposed possible solutions (disclosed in, inter alia, US 2011/0027846, and US 2010/0297719). In addition, chlorine dioxide and hop acids derivatives (alpha and beta fraction) are among the new antimicrobials used. Chlorine dioxide (CIO₂) is a commonly used oxidative biocide, which has been suggested for the fermentation processes, e.g., in US 9,926,576, however, it suffers from several disadvantages. First, being a strong oxidizing agent, CIO₂ lacks selectivity and while eliminating the bacteria it may also harm the yeasts and reduce its viability, hence leading to an overall reduction in ethanol yield. Furthermore, use of CIO₂ is associated with safety concerns, as production of CI2 gas may cause an explosion. A further proposed alternative for using antibiotics is a combination of a non-oxidizing biocide, a stabilized oxidizing biocide and an antibacterial peptide (WO 2011/116042).

Furthermore, the publication US 2003/0228373 relates to a composition for inhibiting microbial growth in industrial waters, including triamine and a biocide which is an oxidizing biocide (e.g., a brominated agent), a non-oxidizing biocide or a combination thereof. Additionally, the publication WO 2020/240559 discloses methods for microbial control with bromine-based compounds and cis-2-decenoic acid.

SUMMARY OF THE INVENTION

The inventors have found that halogen-based biocides produced on site by the action of chlorine or hypochlorite/hypochlorous acid on ammonia or some ammonium salts, can kill bacterial species associated with ethanol fermentation, with practically no harmful effect on the yeasts used in the fermentation process. Non-active compounds that are transformable on-site into efficient biocidal agents, by the action of Cl₂ or hypochlorite/hypochlorous acid, include ammonia and ammonium salts of the formula (NH4)mX^m- in which X is a counter anion derived from a strong mineral acid (e.g., H₂SO₄, HC1, HBr) and organic acids of the formula R2NCO₂H, e.g., carbamic acid H₂NCO₂H.

The non-active compounds set out above are applied in the same fashion in the ethanol fermentation process, for example, a solution of ammonia or the ammonium salt (NH4)mX^m- is mixed with e.g., sodium hypochlorite solution shortly before use, to generate the active species (which tend to degrade rapidly and therefore cannot be prepared beforehand), and the freshly prepared mix is added at a suitable stage into the ethanol fermentation process. However, one observation to make is that while ammonia and ammonium salts (X^Br) are only transformed into chloramines by the reaction with sodium hypochlorite, ammonium bromide is oxidized by sodium hypochlorite to produce in addition bromine-related active species. In any case, as far as their disinfection ability is concerned, either NH4Br, NH3 or (NH4)mX^m- can be used

according to the invention.

For example, experimental studies conducted by the inventors have shown that ammonium bromide and ammonium sulfate ((NH4)2SO₄), upon activation by sodium hypochlorite solution, turn into effective anti-bacterial agents against the Lactobacillus strain Lactobacillus fermentum. Under similar reaction conditions, these agents demonstrated negligible biocidal effect on the yeast Saccharomyces cerevisiae. These results demonstrate that the above biocides may be useful for controlling prokaryotic contamination in yeasts cultures.

As detailed above, bacterial contaminants are abundant in alcohol fermentation reactors (“alcohol” and “ethanol” are used herein interchangeably), affecting yeasts performance and alcohol yield. The laboratory models detailed herein seek to mimic the microorganism environment present during alcohol fermentation. Surprisingly, the experiments conducted demonstrate that each one of the activated biocidal agents are effective anti-bacterial agents against the tested bacterial species, as a single biocidal agent. Remarkably, at the tested biocidal dosage of 0.5 and 1 ppm of total chlorine equivalent (herein: “ppm TCE”, as elaborated below), there was a negligible effect on the viability of the tested yeasts at a variety of pH values. Furthermore, under certain conditions, even at a dosage higher than 1 ppm TCE, the biocides had a marginal effect on the yeasts tested, while demonstrating an anti-bacterial effect.

Therefore, microbial control is achieved using at least one biocide as described herein, leading to selective decontamination of prokaryotic growth during the fermentation process, e.g. bacterial growth, with limited effect on yeasts viability, thereby reducing or eliminating the need for application of antibiotics during fermentation procedures.

In other words, the present invention provides a method for selective bacterial decontamination and/or control during the fermentation process. The method comprises fermenting a fermentable substrate and yeasts in the presence of at least one biocide as defined herein, to produce ethanol and solids content, wherein the biocide may be present as the sole biocide and controls growth of bacteria without substantially affecting yeast population, and wherein the method further comprises distilling the fermented substrate to separate at least a portion of the ethanol from the solid content produced during fermentation. The method according to the invention provides a sustainable solution, by reducing or eliminating altogether the use of antibiotics during fermentation.

Therefore, by one of its aspects the present invention provides a method for producing ethanol by fermentation prone to prokaryotic contamination, said method comprising: a) providing a fermentable substrate; b) combining said fermentable substrate with yeasts in the presence of water to obtain a fermentation broth; and c) fermenting said fermentation broth to obtain a fermented broth; wherein the process further comprises contacting the yeasts with at least one biocide, wherein the biocide is produced on-site by the action of Ch, hypochlorite or hypochlorous acid on ammonia or an ammonium salt of the formula (NH4)mX^m- in which X^m- is a counter anion derived from a strong mineral acid and from carbamic acid of the formula H₂NCO₂H. For example, X^m- can be selected from Br-, SO₄ ^2- Cl- or H₂NCOO-.

In some embodiments, the method according to the present disclosure further comprises the steps of: d) separating said fermented broth into spent yeasts and ethanol-containing liquid; and optionally e) recovering said spent yeasts.

According to particular embodiments, the recovered yeasts are used for at least one additional fermentation cycle(s). In particular embodiments, the recovering step as herein defined comprises contacting said spent yeasts with the biocide and optionally with dilute sulfuric acid to obtain recovered yeasts.

In further embodiments, the biocide as herein defined is produced on site by the oxidation of NH4Br with sodium hypochlorite and in other embodiments, the biocide is chloramine produced on site by the oxidation of ammonia, (NH4)_mX^m- wherein X^m- is selected from SO₄ ^2- , Cl- or H₂NCOO , or a mixture thereof, with sodium hypochlorite. In further specific embodiments, the biocide as herein defined is chloramine produced on site by the oxidation of (NH4)2SO₄ with sodium hypochlorite.

In specific embodiments, the biocide is produced on-site by mixing a solution of sodium hypochlorite, supplied from a first tank, with a solution of ammonia or (NH4)mX^m-, supplied from a second tank, to form a biocide solution, which is combined with the yeasts (e.g., with the spent yeasts). In particular embodiments the biocide solution is a bromine/bromide-containing solution and/or a chloramine-containing solution.

In some embodiments, the at least one biocide is added at a dosage level of 0.2 to 150 ppm TCE, for example, at a dosage level of 0.2 to 25 ppm TCE. In further embodiments, the at least one biocide as herein defined is added in a continuous or an intermittent mode. In particular embodiments, the fermentable substrate as herein defined is derived from sugar-containing raw materials, preferably sugar beet, sugarcane, molasses, whey, sorghum or fruits, starch-containing feedstocks, preferably grain or root crops, such as corn, wheat, rice or cassava, or any combination thereof.

In further specific embodiments, the method according to the present disclosure is performed in the absence of antibiotics. In other embodiments the method according to the present disclosure further comprises adding an antibiotic in an amount of below 70% of the amount needed to control the contamination under identical conditions, but without the use of a biocide produced on-site by the action of Ch, hypochlorite or hypochlorous acid on ammonia or an ammonium salt of the formula (NH4)mX^m- in which X^m- is a counter anion derived from a strong mineral acid and from carbamic acid of the formula H₂NCO₂H.

The method according to the present disclosure is applicable to prokaryotic contaminants comprising lactic acid bacteria, preferably of the genera Lactobacillus (e.g., Lactobacillus fermentum or Lactobacillus plantarum), E. coli, or any combination thereof.

Yeasts suitable for fermentation according to the present disclosure are S. cerevisiae, preferably selected from the S. cerevisiae strains PE-2, S288c, baker’s yeasts, and CEN.PK113-7D.

The present disclosure further provides a method for purifying a yeast culture prone to prokaryotic contamination, said method comprising contacting said yeast culture with at least one biocide, wherein the biocide is produced on-site by the action of CI2, hypochlorite or hypochlorous acid on ammonia or ammonium salt of the formula (NITOmX^m- in which X^m- is a counter anion derived from a strong mineral acid and from carbamic.

In further embodiments of the method as herein defined, separating said fermented broth comprises centrifuging said fermented broth and collecting at least a portion of said spent yeasts. In the above and other embodiments, the method as herein defined further comprises separating ethanol from said fermented broth, for example, by distillation.

In other specific embodiments of the present disclosure, contacting the yeasts with the at least one biocide is performed during fermenting of said fermentation broth.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Figure 1 is a schematic representation of the Brazilian process for alcohol production using fermentation.

Figure 2 is a graph showing the effect of activated ammonium bromide (AmBr) at the indicated concentration range (ppm TCE) on the viability of the yeast Saccharomyces cerevisiae (upper graph) and of the bacterial strain Lactobacillus fermentum (9338, lower graph) at pH 3.5 by measuring the colony forming units per ml (CFU/ml) thereof. Abbreviations: CFU, colony forming units; Saccharomyces, Saccharomyces cerevisiae; Lactobacillus 9338, Lactobacillus fermentum', ppm, parts per million; TCE, total chlorine equivalents.

Figure 3 is a graph showing the effect of activated AmBr at the indicated concentration range (ppm TCE) on the viability of the yeast Saccharomyces cerevisiae and the bacterial strain Lactobacillus fermentum (9338) at pH 5.6 by measuring the CFU/ml thereof. Abbreviations: as detailed for Figure 2.

Figure 4 is a graph showing the effect of activated ammonium sulfate (AmSO₄) at the indicated concentration range (ppm TCE) on the viability of the yeast Saccharomyces cerevisiae (upper graph) and the bacterial strain Lactobacillus fermentum (9338, lower graph) at pH 3.5 by measuring the CFU/ml thereof. Abbreviations: as detailed for Figure 2.

Figure 5 is a graph showing the effect of activated AmSO₄ at the indicated concentration range (ppm TCE) on the viability of the yeast Saccharomyces cerevisiae (upper graph) and the bacterial strain Lactobacillus fermentum (9338, lower graph) at pH 5.6 by measuring the CFU/ml thereof. Abbreviations: as detailed for Figure 2. DETAILED DESCRIPTION OF THE INVENTION

By one of its aspects, the present disclosure provides a method for controlling a prokaryotic contamination in a yeast-based process for producing ethanol, also referred to herein as a fermentation process. It has been unexpectedly found by the inventors that by using a biocide as defined herein, namely, a biocide produced on-site by the action of Ch, hypochlorite or hypochlorous acid on ammonia or ammonium salt of the formula (NH4)mX^m- in which X^m- is a counter anion derived from a strong mineral acid and from carbamic acid of the formula H₂NCO₂H, it is possible to selectively eradicate prokaryotic growth without substantially affecting yeast viability. Thus, the method of the invention comprises contacting yeasts with a biocide as defined herein.

In other words, the present disclosure provides a method for producing ethanol by fermentation prone to prokaryotic contamination, said method comprising: a) providing a fermentable substrate; b) combining said fermentable substrate with yeasts in the presence of water to obtain a fermentation broth; and c) fermenting said fermentation broth to obtain a fermented broth; wherein the process further comprises contacting the yeasts with at least one biocide, wherein the biocide is produced on-site by the action of CI2, hypochlorite or hypochlorous acid on ammonia or ammonium salt of the formula (NH4)mX^m- in which X^m- is a counter anion derived from a strong mineral acid and from carbamic acid of the formula H₂NCO₂H.

The present disclosure further provides a method for purifying a yeast culture prone to prokaryotic contamination, said method comprising contacting said yeast culture with at least one biocide, wherein the biocide is produced on-site by the action of CI2, hypochlorite or hypochlorous acid on ammonia or ammonium salt of the formula (NH4)mX^m- in which X^m- is a counter anion derived from a strong mineral acid and from carbamic acid of the formula H₂NCO₂H.

The contacting step (namely the step involving bringing yeasts and the biocide as herein defined into physical contact) may be carried out by adding the biocide of the invention into a liquid containing yeasts or a liquid containing a yeast culture contaminated with a prokaryotic growth, using delivery systems currently employed in the treatment of industrial liquid reservoirs, either continuously or in a batch mode. This step may be performed at any time during the course of the process for producing ethanol, and at any stage thereof, by way of example, during the stage in which yeasts are recovered (recycled) after a fermentation step is completed (such yeasts are also referred to herein as “spent yeasts”) or during the pre-treatment step of the fermentable substrate, in order to decontaminate the substrate before the fermentation step commences. The contacting step may alternatively or additionally be performed during the fermenting step of the fermentation broth.

The contacting step of adding a biocide into a liquid containing yeasts is generally carried out with a dosage level of at least 0.2 ppm of total chlorine equivalent (TCE) of the biocide as herein defined, with the upper limit dictated by the process needs, as generally described below, but may be up to, e.g., 150 ppm of total chlorine equivalent. In other words, the biocide as herein defined is added in an application rate to furnish a concentration in the range of 0.2 ppm and 150 ppm in the yeast-containing liquid. The biocide as herein defined may be introduced under the dosage conditions described herein once, twice, three times or more during each cycle of fermentation.

Generally, as it has been demonstrated in the examples below, dosage levels of the biocide of between 0.5 and 2.0 ppm TCE are efficient in selectively eradicating prokaryotic contamination, when performed in an organic-free aqueous medium. It should be appreciated that in the presence of reducing sugars and other constituents of fermentation/fermented broth, the required dosage of the biocides may be higher. Thus, the dosage levels in an organic-free medium may serve as a standardized starting point for determining the required dosage levels of the biocide in the ethanol-production process. The process may thus also comprise pre-determining the maximum tolerable amount of the biocide for the specific strain of the yeast that is being used in the process (i.e., the dosage level that does not kill the yeasts while negatively affecting bacteria) and based on the results thereof, to select a dosage level that is lower than that maximum tolerable amount for the process, expecting that it will be sufficient to eradicate any reasonable prokaryotic contamination. Specifically, depending on the yeasts’ strain, said at least one biocide as defined herein may be used at a dosage level of 0.2-150 ppm TCE, e.g., of 0.2 - 125 ppm TCE, 0.2 - 100 ppm TCE, 0.2 - 75 ppm TCE, 0.2 - 50 ppm TCE, 0.2 - 25 ppm TCE, for example, 0.2 - 10 ppm TCE, or of 0.2 - 7 ppm TCE, or of 0.2 - 5 ppm TCE, e.g. of 0.5 - 5 ppm TCE. The actual concentration of the biocide required for the process may be determined via monitoring, continuously or intermittently, the TCE values of the biocide, and adjusting the dosage level of the biocide accordingly during the process, e.g., by adjusting the rate of introduction thereof, or by relying on pre-determined values, for example, described above. The required dosage levels are dependent on the specific composition of the medium into which the biocide is added, particularly by the organic content thereof. Particularly, in an organic-free medium, the biocide is added at a dosage level of 0.2 - 25 ppm TCE.

As may be appreciated by those skilled in the art, the term “purifying” means decontaminating or disinfecting a yeast culture at least partially, from prokaryotic contamination, such that the yeast culture or any medium comprising thereof (e.g., the fermentation broth) is substantially purified, namely at least 60% free, preferably at least 75% free, and more preferably at least 90% free from prokaryotic contamination.

Monitoring the presence of prokaryotic contamination may be performed during the fermentation process and at any step thereof. For example, the presence of prokaryotic contamination may be detected by periodically testing fractions of the fermentable substrate, fermentation broth, fermented broth and/or the recovered (spent) yeasts, e.g., by culturing such fractions under suitable conditions enabling prokaryotic growth and by using detection tools for identifying the type of prokaryotic contamination concerned (if any).

The fermentation process is usually carried out in an aqueous medium. Thus, all the components of the fermentation process, as described in greater detail below, may be added into, or contained in, an aqueous medium, for example distilled water.

Oxidizing the biocides as herein defined provide active bromine or active chlorine species in the media into which they are delivered, for example water as herein described. The oxidation is achieved with the aid of a chemical oxidant, e.g., chlorine, hypochlorite (derived for example from sodium hypochlorite) and hypochlorous acid. The dosage of the biocides described herein is usually expressed as parts per million of total chlorine equivalent (ppm TCE), i.e., the total oxidizing species present in the solution expressed as titrimetric equivalents of chlorine (Ch).

The total chlorine equivalent concentration may be determined, e.g., by an iodometric titration. The titration may be performed directly, e.g. using titrating burettes, or using an automated titrimeter, e.g., titroprocessor Titrino 848 plus. The oxidative species may be reacted with excess iodide solution to form iodine which is then titrated directly with a calibrated solution of thiosulfate, or with an excess of calibrated solution of thiosulfate, the remainder whereof is then quantitatively titrated with iodine. Additionally, total chlorine equivalent may be determined by the DPD (Diethyl-p- PhenyleneDiamine) reagent method using a spectrophotometer, e.g., Merck SQ-300.

One convenient way to produce the biocide on site and rapidly supply it to the fermentation process is by mixing an aqueous solution of NH3 or ammonium salt (NH₄)mX^m with an aqueous solution of the oxidant, e.g., hypochlorous acid or sodium hypochlorite, which are stored in separate tanks. The Cl₂ or hypochlorite/hypochlorous acid may be (for example) generated on-site electrochemically, by electrolysis of chloride salts. With the aid of metering pumps, two separate streams are supplied from the tanks and mixed shortly prior to use, such that the combined stream enters the intended site of use shortly after it has been formed (also referred herein “on-site” or “in situ”), or held in a mixing tank for a couple of hours before it is delivered to the fermentation process. The system is thus treated by the oxidation product(s) of NH₄Br with hypochlorite (which presumably consist of bromine/bromide -related species and chloramine in water) or by the oxidation product(s) of NH3 or (NH₄)_mX^m-

with HOCl/NaOCl. The latter are called chloramines, because one or more N-H bonds in the NH3/(NH4)_mX^m precursors is replaced with N-Cl bond. Monochloramine

is generally preferred and the reagents are proportioned accordingly, using, for example, ammonia or ammonium sulfate, as shown for example for ammonia and ammonium sulfate by the chemical equations below: NH₃ + NaOCl NH₂C1 + NaOH

(NH₄)₂SO₄ + 2NaOCl 2NH₂C1 + Na₂SO₄ + 2H₂O

Chloramine solution can also be prepared by the method described in US 6,222,071, reacting sodium hypochlorite with a mixture of ammonia and ammonium salt such as NH₄C1 at a very low temperature, or by the method described in US 7,045,659, reacting NH₄C1 with sodium hypochlorite at a slight molar excess of NH₄C1 (reaction with carbamic acid).

Therefore in some embodiments, the biocide according to the present disclosure is chloramine produced on site by the oxidation of ammonia, (NH₄)_mX^m- wherein X^m- is selected from SO₄ ^2- , Cl- or H₂NCOO-, or a mixture thereof, with sodium hypochlorite. In further specific embodiments, the biocide according to the present disclosure is chloramine produced on site by the oxidation of (NH₄)₂SO₄ with sodium hypochlorite.

In further embodiments, the biocide according to the present disclosure is produced on site by the oxidation of NH₄Br with sodium hypochlorite.

The oxidation product of ammonium bromide with hypochlorite (also called in the experimental section below "activated ammonium bromide" or "bromide activated chloramine" (BAC)) or the solution of chloramine (also called in the experimental section below "activated NH3/ (NH4)mX^m- may be supplied in a continuous

mode or at an intermittent dosing, optionally at pre-determined time intervals, depending on the requirements of the process.

Biocides as defined herein suitable for use in the present invention are available in the marketplace in different forms, i.e., solids (such as powders and compacted forms, e.g., granules and tablets) and liquids (e.g., aqueous concentrates or other flowable formulations that can be easily supplied to the fermentation process to be treated).

The contacting step may be conducted at any suitable pH value, yet it may be especially advantageous to select the pH value according to the requirements of the process. For example, between 2 and 6, e.g., between 2 and 4 for a contacting step performed during the recovery step of the yeasts, or between 4 and 6 for a contacting step performed during the fermenting step. As demonstrated in the Examples section below, selective eradication of bacteria over yeasts occurs both at pH 3.5 and 5.6, and therefore without being bound by a particular theory it is believed that this selectivity is not affected by the pH of the medium.

In some embodiments, the method according to the present disclosure further comprises the step of separating the fermented broth into spent yeasts and an ethanol-containing liquid; and optionally further recovering said spent yeasts.

In other words, the present disclosure further provides a method for producing ethanol by fermentation prone to prokaryotic contamination, said method comprising: a) providing a fermentable substrate; b) combining said fermentable substrate with yeasts in the presence of water to obtain a fermentation broth; and c) fermenting said fermentation broth to obtain a fermented broth; d) separating said fermented broth into spent yeasts and an ethanol-containing liquid; and e) recovering said spent yeasts, wherein the process further comprises contacting the yeasts (e.g., the recovered, spent yeasts) with at least one biocide, wherein the biocide is produced on-site by the action of Ch, hypochlorite or hypochlorous acid on ammonia or ammonium salt of the formula (NH4)mX^m- in which X^m- is a counter anion derived from a strong mineral acid and from carbamic acid of the formula H₂NCO₂H.

By way of example, the step of separating the fermented broth according to the present disclosure comprises centrifuging said fermented broth and collecting at least a portion of said spent yeasts. In various embodiments, the method according to the present disclosure further comprises separating ethanol from said fermented broth, for example, by distillation. Separating the fermented broth according to the present disclosure by centrifuging, collecting at least a portion of the spent yeasts and further distillation of the ethanol portion of the fermented broth are well known to a skilled artisan and are graphically depicted in Figure 1.

In various embodiments, the recovering step comprises contacting said spent yeasts with said biocide and optionally with dilute sulfuric acid to obtain recovered yeasts. In other words, the contacting step with the biocide as defined herein may be performed as a stand-alone decontamination step, e.g., as a method for purifying yeast culture from bacterial decontamination or as a step during yeast-based fermentation, in particular during a yeast recovering step, and optionally may be associated with an acidification step as known for the Brazilian process of ethanol production (e.g., contacting the spent yeasts with water-diluted sulfuric acid (H₂SO₄), at a pH of 2.0-2.5, for 1-2 hours). The contacting step with the biocide may be performed before or after the acidification step as defined above or concurrently therewith.

The recovered yeasts, namely spent yeasts that were subjected to the recovering step comprising contacting the spent yeasts with the biocide as herein defined and optionally with dilute sulfuric acid, are suitable to be used in at least one additional fermentation cycle(s).

Without being bound by a particular theory, it is believed that contacting potentially contaminated yeast culture with at least one biocide according to the invention during the fermentation process, e.g., the recovery step of the yeasts, may be sufficient to essentially eradicate prokaryotic contamination, thereby selectively inhibiting bacterial growth without significantly affecting the viability of the yeasts, if performed at a dosage level of the biocide as defined herein in the general range of between about 0.2 and about 150 ppm TCE, e.g., 0.2 - 125 ppm TCE, 0.2 - 100 ppm TCE, 0.2 - 75 ppm TCE, 0.2 - 50 ppm TCE, 0.2 - 25 ppm TCE, for example, 0.2 - 10 ppm TCE, or of 0.2 - 7 ppm TCE, or of 0.2 - 5 ppm TCE, e.g. of 0.5 - 5 ppm TCE, and/or at pH value of 2 to 4. The contacting time between the potentially contaminated yeasts culture and the biocide as herein defined is of e.g., between 30 and 120 minutes, or between 45 and 75 minutes. The contacting time suitable for the methods defined herein may be dependent on the nature of the prokaryotic contamination, on the particular strain(s) of the yeasts, on the dosage level of the biocide, and on many other factors. Ideally, the contacting time, as well as the effective concentration / dosage level for each particular process, may be predetermined in a separate experiment prior to the mass production process by ways known in the art, such as detailed herein.

Generally, the fermentable substrate is a mixture of nutritious sugars and/or oligo- and polysaccharides, consumed by the microorganisms utilized in the fermenting step. The raw materials for the process are pre-processed, mechanically, chemically, enzymatically, and/or by pre-fermentation with another microorganism, to make these nutrients accessible to the ethanol-fermenting microorganisms, as described below. Generally, there are three major steps in ethanol production by fermentation: (1) obtaining the fermentation broth, namely the solution that contains fermentable substrate, from raw materials, (2) converting sugars and/or other nutrients in the fermentable broth to ethanol by fermentation, thereby obtaining the fermented broth, and (3) separating ethanol and/or purifying it, by further processes, including, for example distillation.

The starting material for ethanol production, namely, the substance or material being fermented, also referred to herein as “fermentable substrate”, “substrate”, "fermentable mash", suitable for the present invention, may be derived, for example, from sugar- containing raw materials (e.g., sugar beet, sugarcane, molasses, whey, sorghum and fruits) or starch-containing feedstocks (e.g., grain or root corps, such as corn, wheat, rice, cassava, etc.). Preferably, the process may be used for producing so-called “first generation bioethanol”, namely ethanol directly related to a biomass that is more than often edible. In particular, the fermentable substrate is sugarcane juice, molasses or mixtures thereof.

Therefore, in various embodiments, the fermentable substrate is derived from sugar- containing raw materials, preferably sugar beet, sugarcane, molasses, whey, sorghum or fruits, starch-containing feedstocks, preferably grain or root crops, such as com, wheat, rice or cassava, or any combination thereof.

The fermentable substrate is therefore derived from the various suitable feedstocks, e.g., as referred to above, which have been (optionally) subjected to pretreatment processes suitable to the particular feedstock used, as known in the art. Methods used for pretreatment include physical pretreatment (e.g., mechanical milling to grind the substrate and facilitate downstream mashing), chemical pretreatment (e.g., acid hydrolysis, alkaline hydrolysis, ozonolysis, enzymatic hydrolysis), physicochemical pretreatment (e.g. ammonia fiber explosion or stream fiber explosion), and biological pretreatment (e.g., using different fungal species) procedures. Overall, pretreatment procedures as defined herein include any process necessary for converting the raw material feedstocks into fermentable sugar-containing substrate, namely sugars available for bioethanol production by yeast fermentation.

Various methods of pre-treating feedstocks for ethanol fermentation and numerous other aspects of the process have been reviewed, e.g., in Mohd-Azhar et al, Biochemistry and Biophysics Reports 10 (2017) 52-61 incorporated herein by reference.

For example, currently in Brazil, the fermentable substrate comprises molasses (which is the residue or by-product of sugar production) diluted with water, or a mix of sugar cane juice and molasses, and currently in the USA the fermentable substrate comprises a liquor of glucose resulting from the process of breakdown of starch extracted from corn.

The different starting materials vary in the extent to which they should be pre- processed (also termed herein “pre-treated”) prior to use thereof for fermentation by yeasts, as well known in the art. Thus, the process may comprise pre-treating the starting material to obtain a fermentable substrate or mash. For example, when sugar cane is used as the source material for the ethanol production process, the cane may be first washed. Preferably, the washing of the cane is carried out in alkaline water with a pH value of about 11, e.g., between 10 and 12. The washed cane is then milled to obtain a raw juice and solid residue. The cane solids can be used as a regular burning fuel (e.g., for heating of the liquids in the further steps of the process). The raw juice may then be pre-heated, e.g., to a temperature between 60 and 80°C, and then clarified, e.g., by addition of calcium oxide (quicklime) and/or passing sulfur dioxide through it. The clarified raw juice may then be phosphated to precipitate the residues of calcium, and heated in a decanting assembly to separate the inorganic component, e.g. at about 105 °C for about 2 hours. The decanted liquid may then be filtered off to remove the inorganic solids, which may be used as a fertilizer, whereas the liquid (called at this stage “clarified broth”) may be used for sugar manufacturing. The residues of the sugar manufacturing (molasses), or the clarified broth, may be diluted with water of clarified sugarcane juice, as described above, to produce wort, used as a fermentable mash.

By way of a further example, when the starch-containing feedstock corn is used as a source (raw) material, the pre-treatment thereof is a multistep process, as known in the art, and as briefly described below. The first step involves milling of the com, by dry or wet milling. When wet milling is employed, corn kernels are broken down into starch, fiber, com germ, and protein by heating in a sulfurous acid solution for prolonged time intervals, e.g., of 1-3 days, such as about 2 days. The starch is then separated and may be used as the starting material for producing ethanol (namely the fermentable substrate as herein defined), as well as corn symp, or food-grade starch. The first step of com milling by a dry milling process is grinding the com by using a suitable mill, e.g. hammer-mill or roller mill. Once the corn is broken down, it is mixed with heated water to form a mash or slurry. The slurry comprises at this stage, among other constituents, corn particles and cornstarch granules. Next, the com slurry undergoes gelatinization and liquefaction (also termed “cooking”) under conditions of temperature and acidity as detailed below, during which water interacts with the starch granules in the corn when the temperature is above 60°C and forms a viscous suspension. The liquefaction step is partial hydrolysis that lowers the viscosity, essentially breaking up long starch chains into shorter chains. In order to accomplish liquefaction, the reaction conditions are usually maintained at a pH in the range of 5.9 - 6.2, and ammonia and sulfuric acid are added to the tank to maintain the pH value. The enzyme alfa-amylase may also be added to the mash before jet cooking (for 2-7 minutes at 105-120°C) to improve the flowability of the mash. At this stage, shorter dextrin is produced but glucose is not yet formed. When alfa-amylase is utilized, there are several processes known in the art, involving enzymatic and heat hydrolysis in various order. The first process consists of adding alfa-amylase and incubating the material at 85-95°C. The second process consists of placing the mash in the jet cooker at 105-120°C for 2-7 minutes, then flowing thereof to a flash tank at 90°C and adding alfa-amylase three hours later. The third process consists of adding the alfa-amylase, heating in the jet cooker at 150°C, followed by flow to the flash tank at 90°C and adding more alfa-amylase. As known in the art, the enzyme alfa-amylase acts on the internal glycosidic bonds to yield dextrin and maltose, for liquefaction of the material. The next step of corn pre-treatment is saccharification, namely the process by which further hydrolysis to glucose monomers occurs, using the enzyme glucoamylase, which cleaves glycosidic bonds at dextrin ends to form glucose. The optimal reaction conditions required are a pH of 4.5 and a temperature of 55-65°C.

As known in the art, while pretreatment of raw feedstock material may be separated from the fermentation process, the fermentation process may commence along-side the hydrolysis of the raw material into a fermentable substrate, provided that the pretreatment conditions are suitable for fermentation.

In general, fermentation starts by the addition of a substrate (fermentable substrate), which may be composed, by way of a specific example, of sugar cane juice and/or molasses at any proportion, to an inoculum of a yeast culture contained in a bioreactor (fermenter vessel) in the presence of water. The substrate usually contains reducing sugars (e.g., sucrose, glucose, fructose, and their mixtures), usually present at concentrations of about 150-250 g/L. Fermenting may be performed as generally known in the art, e.g., in a batch mode (e.g., by a fed batch process), in a continuous mode, or in any combinations of these two.

The fermentable substrate is contacted with the yeast culture and water under desired conditions of, for example, inoculum (yeast culture) concentration, sugar concentration, temperature, oxygenation, pH, incubation time and mixing (agitation rate) as well known in the art. The mixture of yeasts, water and fermentable substrate (also referred to herein as “fermentation broth”) is then incubated for a certain time, to effect fermenting of the fermentable substrate. The typical temperature may be between 20°C and 37°C, preferably between 32°C and 34°C. The temperature may be controlled as generally known in the art, e.g. by using a double-jacketed vat and circulating a heat-exchange liquid, e.g. water, through the jacket. The temperature of the heat-exchange liquid may be controlled by a temperature-controlling unit, e.g., by a heating unit and/or a cooling unit. The temperature of the heat-exchange liquid may be controlled by a temperature measured in a fermenting vat, e.g. by an indwelling thermocouple. The optimum pH range for S. cerevisiae is 4.0-5.0.

The time necessary for the process, e.g., for full consumption of the starting material (i.e., until less than about 0.1% of fermentable sugars remain present) or other pre- determined end-point, is usually dictated by the consumption rate of the sugars and/or by the production of alcohol, either or both may be monitored continuously or at discrete time points, for process control purposes.

The fermenting step may comprise gradually feeding the fermentable substrate to be combined with the yeasts and water (namely, by fed batch mode). This gradual addition may be particularly advantageous, in order to reduce the production of toxic co- products, thereby causing less stress to the yeasts, and consequently increasing the yield of ethanol production. Alternatively, the fermentable substrate may be combined substantially completely with the yeasts and water to initiate the fermenting step. When gradual addition is employed, it can be carried out within about 4-6 hours, whereas fermentation is completed within 6-10 hours after the initiation of feeding, attaining 7- 10% (v/v) ethanol in the fermented broth (i.e. the mixture obtained from the fermenting step). During feeding and fermentation, the temperature inside the fermenter vessel is maintained at between 27°C and 37°C, preferably between 32°C and 34°C. Ethanol may be thus advantageously produced at an efficiency of at least 80%, preferably at least 90%, meaning that 90% of the sugar contained in the substrate has been converted into ethanol.

Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. Yeasts suitable for industrial ethanol production by fermentation are usually S. cerevisiae strains, however, other genera known in the art may also be used, e.g., Pichia Stipidis (for example, strain NRRL-Y-7124), or Kluyveromyces fagilis (for example, strain Kfl). The present invention is thus applicable to any yeasts suitable for industrial ethanol production by fermentation, in particular, at least one S. cerevisiae strain. Some non-limiting examples of S. cerevisiae strains are PE-2 strain, S288c, baker’s yeasts, and CEN.PK113-7D. The yeasts suitable for the process may be native, or genetically modified, e.g., to consume pentoses or to express genes encoding lytic enzymes effective against bacteria. Additionally, or alternatively, the yeasts may be a mixture of several genera and/or strains, to maximize the utilization of the nutrients and conversion to ethanol. Some additional yeast microorganisms, suitable for fermenting the fermentable substrate into ethanol are described, e.g. in Mohd Azhar 2017 (vide supra). Free yeasts or immobilized yeasts may be used.

Therefore, the yeast cells (interchangeably referred to herein as yeast culture) of the present invention are a yeast culture containing a heterogeneous collection of yeast cells at various stages of life cycle and may occasionally also include dead yeast cells. Yeast utilized in the process according to the invention may be free yeasts or immobilized yeasts.

In various embodiments, the yeasts suitable for use in connection with the present disclosure is S. cerevisiae. In particular embodiments, the yeasts suitable for use in connection with the present disclosure is S. cerevisiae selected from the S. cerevisiae strains PE-2, S288c, baker’s yeast, and CEN.PK113-7D.

The inoculum for fermentation may be prepared from fresh yeast cells (interchangeably referred to as “yeast culture”) or, as practiced in the framework of the Brazilian process for ethanol production (for example, as schematically presented in Figure 1), yeast cells or yeast culture collected and recovered at the end of a fermentation cycle (e.g., the previous cycle) are used again after being treated or recovered, for example as detailed herein). Such yeast culture may be recovered, inter alia, by contacting with the biocide of the invention (e.g., during the step of fermentation of sugars into ethanol or after this step is completed), as detailed herein.

As detailed above, after the fermentation step is completed, according to the acceptable Brazilian process for ethanol production, the yeasts are separated from the fermented broth and treated with an acid to remove contaminants, and thereby recycled or recovered, whereas according to other (non-Brazilian) processes the yeasts are processed into products generally non-related to further fermentation. The present disclosure provides, inter alia, recycling or recovering the yeasts participating in the fermentation process (also termed herein interchangeably “spent yeasts” or “spent yeast”), as detailed above.

Depending on the starting material, the fermented broth may contain coarse solids, e.g., remainders from the mashing process, as well as dispersed yeasts mass. The fermented broth is first subjected to separating the liquid fraction from the solids fraction. When the substrate contains insoluble solids and/or forms insoluble fermentation products, these can be separated from the fermented broth by decantation, filtration or any other method known in the art. Yeasts may be separated from the fermented broth (which does not contain any appreciable amount of other insoluble products), e.g., by centrifuging the fermented broth at a suitable g-force. The fermented broth may contain between 8% and 16% of yeasts, preferably between 10% and 14%; after centrifugation the obtained yeast cream (also termed herein “spent yeast”) may contain between 40% and 80% of yeasts. The liquid stream separated as supernatant during the centrifuging step (also termed “fermented wine”, i.e., liquid without the yeast cells) may be further processed to extract ethanol, e.g., distilled for ethanol recovery in a distillation unit.

The yeasts cream (or spent yeasts) may then be subjected to contacting with at least one biocide as detailed herein. Generally, the amount of yeasts undergoing recovery (recycling) is dictated by the needs of the process, with excess yeasts produced in the process being optionally removed for further processing or discarded. Briefly, the yeasts may be contacted consecutively with the biocide as defined herein, and acid to ensure decontamination. Alternatively, the yeasts may be first contacted with acid, to reduce the primary prokaryotic bioburden, and then with the biocide as defined herein. The pH of the medium may be adjusted to any suitable value according to the requirements of the particular yeast strain. Alternatively, the yeasts may be recovered using only the biocide, obviating the need to expose the fermenting organisms to an acid.

In other words, the yeasts may be contacted with the biocide solution as described herein, optionally concurrently with or preceded or succeeded by an acid treatment. Yeasts acid treatment may be performed in a designated yeasts treatment vessel, for example by diluting the spent yeasts with water (e.g., in a ratio 1:1) and reducing the pH to 1.8-2.5, e.g., with sulfuric acid (98%), and incubating for about 1 hour, at a temperature between 32 and 34°C.

Furthermore, the yeasts may be contacted with the biocide of the present invention in combination with natural products having antibacterial properties, such as, for example Hop compounds (extracted from Humulus Lupulus), Propolis and Chitosan as well as with bacteriophages, which naturally antagonize bacteria.

The recovered yeasts suspension may be at least partially transferred for a further (e.g., consecutive) fermenting step with a fresh substrate, e.g., to an empty fermentation vat (reactor). Even using the conventional Brazilian process for ethanol production, it may be possible to perform two fermentation cycles per day. It is readily evident that increasing the efficiency of yeasts recycling will improve the overall process outcomes.

The present invention is directed to provide microbial control over any prokaryotic contamination of yeasts, for example, bacterial species the presence of which is associated with yeasts-based ethanol fermentation, sometimes referred to as "natural contaminants" of yeasts. By a non-limiting example, the methods according to the invention are suitable for the control of lactic acid bacteria (LAB), for example of the genera (genus) Lactobacillus (e.g., Lactobacillus fermentum, Lactobacillus plantarum, L. vini, L. paracasei, L. delbrueckii, L. buchneri and L. brevis), Leuconostoc (e.g., Leuconostoc mesenteroides, L.citrovorum, L.dextranicum), Oenococcus, Pediococcus and Weissella, for the control of acetic acid bacteria (AAB), for example of the genera Acetobacter, Gluconobacter and Xanthomonas, for the control of E. coli or any combination thereof.

In specific embodiments, the prokaryotic contaminants in the context of the present disclosure comprise lactic acid bacteria. In various embodiments, the lactic acid bacteria according to the present disclosure are of the genus Lactobacillus. In further specific embodiments, the lactic acid bacteria of the genus Lactobacillus according to the present disclosure are Lactobacillus fermentum, Lactobacillus plantarum or a combination thereof. As detailed above, the fermented broth according to the present invention contains a liquid fraction and solid sediment, which may be separated. Separating the liquid fraction of the fermented broth from the solid fraction results inter alia in a solid fraction containing a minimal amount of biocide as described herein. The solid fraction according to the present invention may be further converted into a distillers dried grains (DDG) product, containing a minimal amount of the biocide and being essentially free of antibiotics.

Preferably, the method according to the present invention obviates the use of antibiotics altogether, therefore in some particularly preferred embodiments the method according to the present disclosure is performed in the absence of antibiotics, namely, is essentially antibiotic -free. That is, the biocide as described herein is usually sufficient to maintain the yeast culture free from contamination. Nevertheless, it is envisaged that some contaminants may be particularly hard to control even at the highest applied dosage levels of the biocide. In such cases the process may comprise providing an antibiotic during the process, e.g., to the fermentable substrate, to the fermentable broth, and/or to the yeasts. In other words, the biocide as described herein may be applied in combination with antibiotics that is, before, after, or concurrently with antibiotics. Any antibiotic agent may be used in the framework of the present invention, preferably an antibiotic agent that eliminates bacterial contaminants without harming the yeasts during fermentation. By way of example, the antibiotic agent may be monensin, penicillin, virginiamycin, erythromycin, tylosin and tetracycline, to name but a few. The antibiotic agent may be applied at a dosage of about 0.2 to about 5 ppm. The antibiotic agent may preferably be applied during the yeast recycling step. It is noted that when an antibiotic is used, it is used in a concentration that is lower than the concentration needed to control the contamination under identical conditions, but without the use of biocide as herein defined. For example, when an antibiotic is used, it may be used in an amount of below 70% of the amount needed to control the contamination under identical conditions, but without the use of the biocide as defined herein, preferably, below 50%, more preferably below 30%.

In specific embodiments, the method according to any one of the preceding claims, further comprising adding an antibiotic in an amount of below 70% of the amount needed to control the contamination under identical conditions, but without the use of a biocide produced on-site by the action of Cl₂, hypochlorite or hypochlorous acid on ammonia or an ammonium salt of the formula (NH4)mX^m- in which X^m- is a counter anion derived from a strong mineral acid and from carbamic acid of the formula H₂NCO₂H.

EXAMPLES

Materials

Culture media, reagents and solutions used are as detailed in Table 1 below.

Table 1 List of materials

Microorganisms

The tests were conducted using the following bacteria and yeasts species, all obtained from the American Type Culture Collection (ATCC):

Lactobacillus fermentum, also referred to as L. fermentum (ATCC 9338); and Saccharomyces cerevisiae, also referred to as S. cerevisiae (ATCC 9763).

Methods

Preparation of microbial suspensions

The yeasts Saccharomyces cerevisiae and the Lactobacillus bacteria were sub-cultured from stock culture by streaking them onto culture plates containing Sabouraud Dextrose and MRS agar, respectively. The plates were incubated for 18-24 hours at 30 ± 2°C for Saccharomyces cerevisiae and at 37 ± 2°C for the Lactobacillus. Then, a second subculture was prepared from the first subculture, in the same manner, and the cultures were incubated as detailed above, namely, 18-24 hours at 30 ± 2°C for Saccharomyces cerevisiae and at 37 ± 2°C for Lactobacillus.

In order to prepare a microbial test suspension, sterile phosphate buffer solution (10 ml) was placed in a 100 ml flask with 5 gr of glass beads. A loop full of cells from the over- night grown culture was transferred into the phosphate buffer. The flask was shaken for 3 minutes, using a vortex. The suspension was aspirated from the glass beads and transferred to another tube. The number of cells in the suspension was adjusted, by means of a calibration curve, to 1.0xl0⁶-1.0xl0⁷ CFU/ml (colony forming units per ml), using a phosphate buffer solution.

Efficacy test procedure

The efficacy (namely, the anti-microbial activity) of activated ammonium bromide (AmBr) and activated ammonium sulfate (AmSO₄), at concentrations of between 0.5 to 5 ppm as Ch, were tested against the yeasts Saccharomyces cerevisiae and against a lacto-bacteria commonly found in fermentative processes, Lactobacillus fermentum (each having specified strain number and source as detailed above) under different pH conditions (at pH=5.6 and pH=3.5) according to a modification of the European standard EN 1040: 2005: "Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of basic bactericidal activity of chemical disinfectants and antiseptics - Tests method and requirements (phase 1)", as generally described below.

Briefly, tests were performed as follows. Phosphate buffer solution (100 ± 2 ml) was transferred into sterile flasks. Duplicate flasks were prepared for each tested biocide concentration. In addition, duplicate controls with no biocides were prepared. Each test vessel was brought to 30 ± 2°C and inoculated with pure culture to achieve a microbial count of at least 10⁶ CFU/ml at time zero, after which the biocide stock solution was added to the duplicate flasks in a manner such that the volume of biocide stock added does not exceed 1% of the total volume of the flask. Flasks were placed in a shaker to provide mixing, under a constant temperature of 30 ± 2°C during the contact periods.

Following 1 hour ± 5 minutes of contact time, 1 ml of the tested mixture was pipetted into a tube containing 9.0 ml neutralizer (Sodium thiosulfate neutralizer for conditions of pH=5.6 and universal neutralizer for conditions of pH=3.5, both neutralizers prepared as described above). After 5 min ± 10 sec, a sample of 1 ml was taken (in duplicate) and after a serial dilution, inoculated by the pour plate method on a Petri dish containing Sabouraud agar for S. cerevisiae and MRS agar for lactobacillus (L. fermentum). The plates were incubated at 37 ± 2°C for 48 hours for lactobacillus and at 30 ± 2°C for 48 hours for S. cerevisiae. After incubation, visible growth was observed and the colonies forming units (CFU) were counted and recorded.

Preparation 1

Preparing activated AmBr solution (BAC)

Activated ammonium bromide (also named herein “BAC” and “Fuzzicide®”) was prepared by the formation of a mixture of a 1.2:2 molar ratio of (NH4)Br:NaOCl according to equation (1):

NH₄Br+ NaOCl -> BAC + NaCl (1)

Based on equation (1), the concentration of activated ammonium bromide (BAC) is based on the concentration of sodium hypochlorite (NaOCl). Equal volumes of the reactants were mixed in order to obtain activated ammonium bromide at a concentration of 50% of the concentration of the reactant NaOCl.

In particular, activated ammonium bromide was prepared as follows:

1. A 100 ml solution of NaOCl was prepared (aim to 1000 ppm). The Cl₂ concentration was determined by the DPD Method (Diethyl-p-PhenyleneDiamine) reagent method using a SQ-300 spectrophotometer: Merck SQ-300 or by iodometric titration, using a titroprocessor: Titrino 848 plus.

2. The result obtained in (1) was divided by the molecular weight of Cl₂ (71 gr/mol).

3. The result obtained in (2) was multiplied by the molecular weight of NH₄Br (98 gr/mol) and by 1.2 (the factor of excess the molar ratio).

4. The result in (3) is the required mass of NH₄Br. A 100 ml solution of NH₄Br was prepared using this amount of NH₄Br.

5. The equal volumes obtained in (1) and (4) were mixed as follows: 25 ml NaOCl solution was added in one stroke to a stirred solution (using a magnetic stirrer) of the 25 ml NH₄Br solution at ambient temperature.

6. The concentration of the activated ammonium bromide as Cl₂ concentration obtained is expected to be half of that obtained in (1) in view of the two-fold volume dilution. Preparation 2

Preparing activated AmSO4 solution (Chloramine)

Activated ammonium sulfate (namely, chloramine solution) was prepared by the formation of a mixture of a 1.2:2 molar ratio of (NH4)₂SO₄:NaOCl according to equation (2):

According to equation (2), the concentration of the product (monochloramine) is based on the concentration of the sodium hypochlorite (NaOCl). Equal volumes of the reactants were mixed in order to obtain chloramine at a concentration of 50% of the concentration of the reactant NaOCl.

In particular, the chloramine solution was prepared as follows:

1. A 100 ml solution of NaOCl was prepared (aim to 1000 ppm). The Cl₂ concentration was determined by the DPD Method or iodometric titration, as detailed above.

2. The result obtained in (1) was divided by the molecular weight of Cl₂ (71 gr/mol).

3. The result obtained in (2) was multiplied by the molecular weight of (NH₄)₂SO₄ (132.14 gr/mol) and by 0.6 (the factor of the molar ratio).

4. The result in (3) is the required mass of (NH₄)₂SO₄. A 100 ml solution of (NH₄)₂SO₄ was prepared using this amount of (NH₄)₂SO₄.

5. The equal volumes obtained in (1) and (4) were mixed as follows: 25 ml NaOCl solution was added in one stroke to a stirred solution (using a magnetic stirrer) of the 25 ml (NH₄)₂SO₄ solution at ambient temperature.

6. The concentration of the chloramine solution formed in (5) is expected to be half of that obtained in (1) in view of the two-fold volume dilution.

EXAMPLE 1

The anti-bacterial effect of activated ammonium bromide (AmBr)

The effect of activated AmBr was tested on the bacterial strain L. fermentum and on the yeasts Saccharomyces cerevisiae, as detailed above and the results obtained under a pH of 3.5 and of 5.6, are graphically shown in Figure 2 and Figure 3, respectively. Figures 2 and 3 show that activated AmBr is an effective anti-bacterial agent against the tested bacteria, at all of the tested concentrations thereof and under both tested pH conditions, after a contact time of 1 hour with the bacteria. Remarkably, at the biocidal concentrations of 0.5 and 1 ppm, there was either none or a negligible effect on yeasts viability. Furthermore, even at a concentration of 2.5 ppm, at the tested pH of 3.5 (Figure 2), activated AmBr had a marginal effect on the yeasts tested, while demonstrating an excellent bactericidal effect on the Lactobacillus bacteria.

EXAMPLE 2

The anti-bacterial effect of activated ammonium sulfate (AmSO4)

Next, the effect of activated ammonium sulfate solution (0.5 to 5 ppm TCE) was tested on the bacterial strain L. fermentum (ATCC 9338) and on the yeasts Saccharomyces cerevisiae (ATCC 9763), as detailed above, under a pH of 3.5 and of 5.6 and the results of these experiments are graphically shown in Figure 4 and in Figure 5, respectively.

As shown in Figure 4 and in Figure 5, activated ammonium sulfate is an effective anti- bacterial agent against the tested bacteria, at all of the tested concentrations thereof and under both tested pH conditions, after a contact time of 1 hour with the bacteria. Remarkably, at the biocidal concentrations of 0.5 and 1 ppm, there was either none or a negligible effect on yeasts viability. Furthermore, even at a concentration of 2.5 ppm (at a pH of 5.6, Figure 5), activated ammonium sulfate had a marginal effect on the yeasts tested, while demonstrating a bactericidal effect on the Lactobacillus bacteria.

Claims

CLAIMS:

1. A method for producing ethanol by fermentation prone to prokaryotic contamination, said method comprising: a) providing a fermentable substrate; b) combining said fermentable substrate with yeasts in the presence of water to obtain a fermentation broth; and c) fermenting said fermentation broth to obtain a fermented broth; wherein the process further comprises contacting the yeasts with at least one biocide, wherein the biocide is produced on-site by the action of Ch, hypochlorite or hypochlorous acid on ammonia or ammonium salt of the formula (NH4)mX^m- in which X^m- is a counter anion derived from a strong mineral acid and from carbamic acid of the formula H₂NCO₂H.

2. The method according to claim 1, further comprising d) separating said fermented broth into spent yeasts and an ethanol-containing liquid; and optionally e) recovering said spent yeasts.

3. The method according to claim 2, wherein the recovering step comprises contacting said spent yeasts with said biocide and optionally with dilute sulfuric acid to obtain recovered yeasts.

4. The method according to claim 2 or claim 3, wherein said recovered yeasts are used for at least one additional fermentation cycle(s).

5. The method according to any one of claims 1 to 4, wherein the biocide is chloramine produced on site by the oxidation of ammonia, (NH4)mX^m- wherein X^m- is selected from SO₄ ^2- , Cl- or H₂NCOO , or a mixture thereof, with sodium hypochlorite.

6. The method according to any one of claims 1 to 5, wherein the biocide is chloramine produced on site by the oxidation of (NH4)₂SO₄ with sodium hypochlorite.

7. The method according to any one of claims 1 to 4, wherein the biocide is produced on site by the oxidation of NEUBr with sodium hypochlorite.

8. The method according to any one of the preceding claims, wherein said at least one biocide is added at a dosage level of 0.2 to 150 ppm TCE.

9. The method according to any one of the preceding claims, wherein said at least one biocide is added at a dosage level of 0.2 to 25 ppm TCE.

10. The method according to any one of the preceding claims, wherein said prokaryotic contaminants comprise lactic acid bacteria, E. coli, or any combination thereof.

11. The method according to claim 10, wherein said lactic acid bacteria are of the genus Lactobacillus.

12. The method according to claim 11 , wherein said lactic acid bacteria of the genus Lactobacillus are Lactobacillus fermentum, Lactobacillus plantarum or a combination thereof.

13. The method according to any one of the preceding claims, wherein the at least one biocide is added in a continuous or an intermittent mode.

14. The method according to any one of the preceding claims, wherein said yeast is S. cerevisiae.

15. The method according to any one of the preceding claims, wherein said yeast is S. cerevisiae selected from the S. cerevisiae strains PE-2, S288c, baker’s yeast, and CEN.PK113-7D.

16. The method according to any one of the preceding claims, wherein the biocide is produced on-site by mixing a solution of sodium hypochlorite, supplied from a first tank, with a solution of ammonia or (NH4)mX^m-, supplied from a second tank, to form a biocide solution, which is combined with the yeasts.

17. The method according to claim 16, wherein the biocide solution is a bromine/bromide-containing solution and/or a chloramine-containing solution.

18. The method according to any one of claims 2 to 17, wherein separating said fermented broth comprises centrifuging said fermented broth and collecting at least a portion of said spent yeasts.

19. The method according to any one of the preceding claims, further comprising separating ethanol from said fermented broth.

20. The method according to claim 19, wherein said separation is performed by distillation.

21. The method according to any one of the preceding claims, wherein said fermentable substrate is derived from sugar-containing raw materials, preferably sugar beet, sugarcane, molasses, whey, sorghum or fruits, starch-containing feedstocks, preferably grain or root crops, such as corn, wheat, rice or cassava, or any combination thereof.

22. The method according to any one of the preceding claims, wherein said contacting the yeasts with at least one biocide is performed during fermenting of said fermentation broth.

23. The method according to any one of the preceding claims, performed in the absence of antibiotics.

24. The method according to any one of the preceding claims, further comprising adding an antibiotic in an amount of below 70% of the amount needed to control the contamination under identical conditions, but without the use of a biocide produced on- site by the action of Ch, hypochlorite or hypochlorous acid on ammonia or an ammonium salt of the formula (NH4)mX^m- in which X^m- is a counter anion derived from a strong mineral acid and from carbamic acid of the formula H₂NCO₂H.

25. A method for purifying a yeast culture prone to prokaryotic contamination, said method comprising contacting said yeast culture with at least one biocide, wherein the biocide is produced on-site by the action of CI2, hypochlorite or hypochlorous acid on ammonia or ammonium salt of the formula (NH4)mX^m- in which X^m- is a counter anion derived from a strong mineral acid and from carbamic acid of the formula H₂NCO₂H.