Patent Application
20220364132
The present disclosure generally relates to a method and a system for forming a storage stable hydrolysate from a lignocellulosic material and to a hydrolysate formed by such a method. It also relates to the use of the hydrolysate to reduce and/or control microbial contamination during storage and/or fermentation. Additionally, the present disclosure relates to a method and a system for reducing and/or controlling microbial contamination in a separate hydrolysis and fermentation (SHF) process.
Biorefineries producing commodities from renewable resources offer an alternative to oil refineries based on dwindling supplies of petroleum and permit a move towards improved energy security. Lignocellulosic residues from forestry and agriculture are attractive as feedstocks, since they are abundant, relatively inexpensive, and are not used for food. Lignocellulose consists mainly of lignin and two classes of polysaccharides, cellulose and hemicellulose. The polysaccharides can be hydrolyzed to sugars and converted to various fermentation products, such as bioalcohols, in processes based on biocatalysts, such as the industrially important baker's yeast Saccharomyces cerevisiae.
The hydrolysis of cellulose is typically preceded by a pretreatment, in which the hemicellulose is degraded and the cellulose is made increasingly accessible to cellulolytic enzymes. During hydrolysis, the cellulose present is partly converted into reducing sugars. The hydrolysis is a relatively long process, and may last up to 150 hours.
In the subsequent fermentation step, remaining cellulosic material is converted into reducing sugars. The fermentation is finished when the cellulosic material has been converted into fermentable sugars, and such fermentable sugars may be converted into target biochemicals, such as bioalcohols or acids and carbon dioxide. The fermentation step may take up to several days before being completed.
Microbial contamination is a widespread problem both during hydrolysis and during fermentation. Various species of Lactobacillus sp. are generally the predominant bacterial contaminants. Lactobacillus bacteria are known to be well adjusted to survival under conditions that most other bacteria are not, such as high ethanol concentrations, low pH and low oxygen conditions, commonly employed during fermentations. Undesirable presence of this type of contaminants is commonly controlled with the use antibiotics, which can cause not only waste disposal issues but also risk of introducing antibiotics resistance. Other types of organisms are also known to infect fermentations, such as wild yeast and fungi, but these are less prevalent than Lactobacillus sp.
A process wherein hydrolysis and fermentation are performed in separate steps, potentially under different process conditions, such as at a different pH and temperature, may be referred to as a separate hydrolysis and fermentation (SHF) process.
In an SHF process, the process may be discontinued between the hydrolysis and the fermentation steps. Under certain circumstances, it may be desirable to store the hydrolysate during a period of time prior to fermentation. The hydrolysate may for example need to be transported to a different location for subsequent fermentation.
During storage of the hydrolysate, the risk of bacterial growth is increased, which may result in an infected sugar hydrolysate. The introduction of such infected hydrolysate into the fermentation vessel may significantly impair the efficiency of the fermentation. The yield of the target chemical may be reduced as the Lactobacilli can inhibit fermenting microorganisms such as Saccharomyces sp., Escherichia coli, or desirable strains of Lactobacillus sp. An infected hydrolysate may also result in that the fermentation process must be shut down or delayed, causing loss of revenue and increased production costs.
There is therefore a need for improved methods to overcome problems with infections during hydrolysis and/or during subsequent fermentation. Particularly, there is a need to provide a hydrolysate from a lignocellulosic material, which is free from bacterial contaminants and which is suitable for storage and transport prior to fermentation.
In view of the above mentioned problems, it is an object of the present disclosure to provide improvements with respect to reducing and/or controlling contamination in a hydrolysate formed from a lignocellulosic material, and in a separate hydrolysis and fermentation (SHF) process. Particularly, the hydrolysate should be suitable for storage and transportation before fermentation.
According to a first aspect of the present disclosure, there is provided a method for forming a storage stable hydrolysate from a lignocellulosic material comprising:
a) pretreating the lignocellulosic material to form a pretreated lignocellulosic composition comprising a liquid component and a solid component; the solid component comprising at least lignin and cellulose
b) removing at least 80%, preferably at least 90% of the liquid component from the pretreated lignocellulosic composition to form a separated lignocellulosic component,
c) diluting the lignocellulosic component with a dilution liquid to form an aqueous slurry
d) subjecting the aqueous slurry to hydrolysis in the presence of at least one saccharification enzyme to form a hydrolysate,
e) adding an antimicrobial compound to the hydrolysate in an amount sufficient to form a storage stable hydrolysate, wherein the antimicrobial compound comprises at least one sulfur oxyanion.
As used herein, the term “storage stable hydrolysate” means a hydrolysate that may be stored for at least three weeks, preferably at least five weeks, such as at least eight weeks at room temperature without getting infected. In other words, the storage stable hydrolysate is substantially free from bacterial contaminants, and may therefore be safely introduced in a subsequent fermentation unit. The hydrolysate of the present invention provides an unfavorable environment for bacterial growth and survival, and is capable of maintaining its storage stable properties even during long periods of storage and transportation. If the hydrolysate is stored at 4° C., the storage period may be significantly longer.
A “lignocellulosic material” refers to a material comprising solely cellulose, or combination of cellulose, lignin and possibly hemicellulose. The lignocellulosic material may for example be wood residues or forestry residues, such as wood chips, sawmill or paper mill discards, or agricultural residues, such as sugarcane bagasse.
“Pretreating the lignocellulosic material” refers to pretreating a lignocellulosic material in order to modify its properties such that the cellulose becomes more accessible during subsequent hydrolysis. The pretreatment may involve one or several pretreatment methods known to the skilled person. The pretreatment can be made by hydrothermal/chemical, physical and biological methods. For example, the pretreatment may involve acid pretreatment, alkali pretreatment, ammonia fiber explosion, acid catalyzed or non-catalyzed steam explosion, auto hydrolysis, organosolv, sulfite or Kraft pulping. Further, the pretreatment may include impregnation, which refers to absorption or mass transfer of an impregnation fluid or gas to the lignocellulosic material, followed by above exemplified processing and/or pre-treatment. The fluid may e.g. be solvents or solutions of acids such as sulphurous acid, sulphuric acid, ammonia, sulfite/bisulfite, sulfate hydroxides, and alcohols. The impregnation may also be performed using gas, such as a SO2-gas or CO2— gas, or with the combination of a gas and fluid of any given combination, concentration and proportion. The pretreatment may also comprise steaming. Steaming refers to a process used to drive air out from the cellulosic biomass to facilitate further hydrolysis of the cellulose. Steaming is a well-known method for pretreating e.g. lignocellulosic biomass.
The “solid component” of the pretreated lignocellulosic composition may comprise solid, unhydrolyzed or undissolved materials of the lignocellulosic material, such as lignin, cellulose, and/or hemicellulose.
The “liquid component” of the pretreated lignocellulosic composition may comprise water, one or more monosaccharides, oligosaccharides, and by-products of the pre-treatment process, such as hydrolysis and fermentation inhibitors, impregnation components and organic acids. Examples of hydrolysis and fermentation inhibitors include phenolic compounds, aliphatic acids, and furan aldehydes. These compounds may inhibit enzymes and yeast, during hydrolysis and fermentation, respectively.
The components comprised in the liquid component, particularly hydrolysis and fermentation inhibitors may have a negative effect on the efficiency of the hydrolysis and the fermentation process. The liquid component comprising such compounds is therefore removed from the pretreated lignocellulosic composition, as defined in step b) above. The removed liquid component may e.g. be utilized in or outside the process of the present disclosure, for example in ethanol fermentation (which typically does not require any separation or washing between the pretreatment and hydrolysis steps).
The step of removing the liquid component may include washing and/or centrifugation of the pretreated lignocellulosic composition. In embodiments, the method according to the present disclosure comprises an additional washing step after the liquid component has been removed from the pretreated lignocellulosic composition in step b.
The “separated lignocellulosic component” comprises the solid component of the pretreated lignocellulosic composition (i.e. unhydrolyzed or undissolved lignin, cellulose and/or hemicellulose), and small amounts of the liquid component. The lignocellulosic component comprises a ratio of solid component-to-liquid component of 80:20, e.g. 90:10, e.g. 95:5, or a ratio of solid component-to-liquid component of 80:20-95:5. The lignocellulosic component is substantially solid.
It should be noted that the separated lignocellulosic component is typically washed as previously mentioned, and the lignocellulosic component which is subsequently subjected to step c) of diluting, may comprise a ratio of solid-to-liquid of 40:60-70:30 (of which said liquid component of the pretreated lignocellulosic composition derived from step a) has been removed by at least 80%, preferably by at least 90%). In other words, the liquid component of the pretreated lignocellulosic composition from the pretreatment in step a) has been at least partly replaced with a washing liquid, typically water.
According to at least one example embodiment, the liquid component of the pretreated lignocellulosic composition may be referred to as inhibitory liquid component as it comprises hydrolysis and fermentation inhibitors. Thus, step b) may be stated differently: b) removing at least 80%, preferably at least 90% of the inhibitory liquid component from the pretreated lignocellulosic composition to form a separated lignocellulosic component,
Correspondingly, the lignocellulosic component may comprise a ratio of solid component-to-inhibitory-liquid component of 80:20, e.g. 90:10, e.g. 95:5, or a ratio of solid component-to-inhibitory-liquid component of 80:20-95:5. As stated above, the lignocellulosic component subsequently subjected to step c) of diluting may comprise between 30%-60% liquid.
The separated lignocellulosic component is subsequently diluted with a dilution liquid to form an aqueous slurry. The dilution liquid is typically water or a sufficiently clean process liquid. For example, process liquid (e.g. washing liquor) recirculated from an upstream or downstream point of the process, may be reused and utilized. Preferably, the dilution liquid is water.
The aqueous slurry formed in step c) is substantially free from hydrolysis and fermentation inhibitors, and other detrimental compounds. For example, the amount of hydrolysis and fermentation inhibitors is less than 10 mg/ml. According to at least one example embodiment, the hydrolysis and fermentation inhibitors less than 10 mg/ml are here referring to compounds reactive with sulphur oxyanions, such as e.g. sulfite or dithionite, for example hydroxymethyl furfural, HMF, furfural, phenolic and aromatic inhibiting compounds. According to at least one example embodiment, the hydrolysis and fermentation inhibitors less than 10 mg/ml are here excluding non-reactive compounds, i.e. which are not reactive with sulphur oxyanions, such as e.g. aliphatic acids. That is, according to such embodiment, inhibitors which do not react with sulphur oxyanions, e.g. sulfite or dithionite, are not relevant for aqueous slurry, and are not included in the limit of 10 mg/ml.
The aqueous slurry may thus be regarded as a “clean” or purified stream, wherein an appropriate amount of harmful by-products and hydrolysis and fermentation inhibitors have been removed. The aqueous slurry may comprise minor amounts of the liquid component of the pretreated cellulose composition formed in step a) of the method. Thus, the combined effect of step b) of removing at least 80%, preferably at least 90% of the liquid component (or inhibitory liquid component) from the pretreated lignocellulosic, and step c) of diluting the lignocellulosic component with a dilution liquid to form an aqueous slurry, results in a slurry being a purified stream, with no, or a minimum amount of harmful by-products and fermentation inhibitors.
The aqueous slurry comprises less than 20% by weight, preferably less than 10% by weight and more preferably less than 5% by weight of the liquid component of the pretreated lignocellulosic composition.
According to at least one example embodiment, the step c) of diluting comprises adding at least 0.1-6 parts, e.g. 1-5 parts, e.g. 2-4 parts, of dilution liquid, such as e.g. water. Thus, at least 10%, as compared to the lignocellulosic component (e.g. a lignocellulosic component having a ratio of solid-to-liquid of 40:60-70:30), of dilution liquid is added. Stated differently, a specific amount of dilution liquid is added to the lignocellulosic component, such that the suspended solids content in the resulting aqueous slurry is from 10% to 35% by weight, or 12% to 35% by weight, or from 15% to 35% by weight.
According to at least one example embodiment, the method comprising adjusting the pH of the lignocellulosic component or aqueous slurry.
The step of adjusting pH is typically separate from step c) of diluting.
In this regard, it should be noted that the addition of antimicrobial compounds comprising sulfur oxyanions to pre-treated lignocellulosic material that has not been subject to separation (i.e. step b)) would have a detoxifying effect; i.e. reducing the amount of fermentation inhibitors, such as phenolic compounds, aliphatic acids, and furan aldehydes and generally improving the conditions during a subsequent fermentation process. However, the addition of sulfur oxyanions to a non-separated pretreatment stream could in fact increase the growth of bacteria, as illustrated by Example 1 below. In order to yield an antimicrobial effect for such non-separated streams, a significant amount of sulfur oxyanions would be required. The introduction of too high amounts of sulfur oxyanions in this step is associated with unnecessary costs and material expending.
Accordingly, the separation, optional wash and subsequent dilution allows for the formation of a sugar rich aqueous slurry wherein less inhibitory compounds are present.
The aqueous slurry of step c) is then subjected to hydrolysis (step d) in the presence of at least one saccharification enzyme to form a hydrolysate.
The hydrolysis reaction is catalyzed by at least one saccharification enzyme. A “saccharification enzyme” refers to at least one enzyme that can convert or hydrolyze (ligno) cellulosic material into fermentable saccharides, such as monosaccharides and/or disaccharides. Such saccharification enzymes may be glycosidases, which hydrolyze polysaccharides. Examples of glycosidases include cellulose-hydrolyzing glycosidases, such as cellulases, endoglucanases, exoglucanases, cellobiohydrolases and β-glucosidases, hemicellulose hydrolysing glycosidases, such as xylanases, endoxylanases, exoxylanases, β-xylosidases, arabinoxylanases, mannanases, galactanases, pectinases and glucuronases, and starch hydrolysing glycosidases, such as amylases, cc-amylases, β-amylases, glucoamylases, cc-glucosidases and isoamylases, or any enzymes in the group of enzymes found in EC 3.2.1.x, such as EC 3.2.1.4, where EC is the Enzyme Commission number.
The hydrolysate formed in the process of the present disclosure may comprise sugars from lignocellulosic biomass, such as glucose, mannose, xylose, arabinose, galactose, sucrose and fructose.
In embodiments, the storage stable hydrolysate comprises at least glucose.
In embodiments, the storage stable hydrolysate comprises at least two different sugars.
The hydrolysis may be carried out at a pH in the range of 3 to 6, e.g. between 5 and 5.5. The temperature during hydrolysis may be in the range of from 40 to 70° C., e.g. between 50 and 60° C. These conditions allow the saccharification enzyme(s) to perform optimally. Furthermore, these conditions prevent the growth of undesirable microorganisms.
The method further comprises a step e) of adding an antimicrobial compound to the hydrolysate in an amount sufficient to form a storage stable hydrolysate, wherein the antimicrobial compound comprises at least one sulfur oxyanion.
Examples of antimicrobial compounds comprising at least one sulfur oxyanions are disulfite, bisulfite, dithionite, dithionate, sulfate, sulfite and thiosulfate.
The inventors have found that a method according to the present disclosure yields a stable and substantially “infection free” hydrolysate, wherein the presence of bacterial contaminants is eliminated, or substantially reduced. Such hydrolysate therefore allows for prolonged storage periods. A storage stable hydrolysate of the present disclosure may be stored up to three weeks, e.g. at least five weeks, such as at least eight weeks at room temperature without getting infected. If the hydrolysate is stored at 4° C., the storage period may be significantly longer. The hydrolysate may be used in a subsequent fermentation step, to provide a safe and contamination free fermentation. Furthermore, only small amounts of antimicrobial compound need to be added to yield a significant contamination reducing effect. The fact that the method involves a separation step prior to hydrolysis, and that only relatively pure sugar streams are utilized, allows the addition of antimicrobial compounds to “boost” the hydrolysate and preserve its infection free properties such that the hydrolysate becomes resistant to infections during storage and during subsequent fermentation.
According to at least one example embodiment, the step e) is performed prior any storage of the hydrolysate, at least prior to a storage of the hydrolysate of at least 1 day, such as at least 7 days, or at least 14 days or at least 21 days.
According to at least one example embodiment, the step e) is performed prior to any fermentation, or subsequent fermentation step, of the hydrolysate.
In embodiments, the aqueous slurry formed in step c) has a suspended solids content of from 10% to 35% by weight. Depending on the fermentation process and what conditions and target chemicals that are to be produced, the dilution and suspended solids content may be adjusted. If the suspended solid content is too high, a larger amount of enzymes would be required in the subsequent hydrolysis step. This would significantly increase production costs.
According to at least one example embodiment, the aqueous slurry formed in step c) has a suspended solids content of from 12% to 35% by weight, or from 15% to 35% by weight, or from 20% to 35% by weight.
A “target chemical” may be any chemical that can be prepared from pretreated and hydrolyzed cellulosic material in a process comprising fermentation. The target chemical may e.g. be alcohols or acids, alkanes, alkenes, aromatics, aldehydes, ketones, biopolymers, proteins, peptides, amino acids, vitamins, antibiotics and other pharmaceuticals. For example, the target chemical is lactic acid, butanol, succinic acid or isobutene. Ethanol is typically not the target chemical in the method of the present disclosure, since the production of ethanol does not require any separation between the pretreatment and hydrolysis steps.
In embodiments, the hydrolysis step b) is performed in the presence of oxygen. Preferably, the hydrolysis step is performed in the presence of air.
In other words, the hydrolysis is an aerobic process. This is beneficial as the hydrolysis of cellulose is improved. Enzymatic oxidation of cellulose reduces the forces holding the cellulose fibers together, which thereby increases the availability and accessibility for the hydrolytic enzymes to hydrolyze cellulose to sugars.
It is conceivable to add the antimicrobial compound prior to or during hydrolysis, however if the hydrolysis is performed in the presence of air (and temperatures of 40° C. or more), the sulfite oxyanions will disassociate and form gaseous sulfur dioxide (SO2) which will leave the hydrolysis with the air flow. In other words, the antimicrobial effect would diminish. Therefore, the addition of sulfur oxyanions is preferably made after hydrolysis.
In embodiments, the concentration of the antimicrobial compound is between 1 and 100 mM, e.g. between 5 and 50 mM, e.g. between 5 and 25 mM. Preferably, the concentration is in the lower range as a too high concentration may inhibit microbes utilized in a subsequent fermentation step.
As demonstrated by example 2 hereinafter, only small amounts of antimicrobial compounds are required to achieve an antimicrobial effect. This is mainly because the hydrolysate formed by the process of the present disclosure is substantially pure, and contains less inhibitors, and other compounds that may react and thus consume the oxyanions, which is the desired effect when such sulfur oxyanion compounds are used for conditioning or detoxification of lignocellulosic hydrolysates containing hydrolysis and fermentation inhibitors.
In embodiments, the antimicrobial compound is selected from dithionite and sulfite. Such compounds are advantageous carriers of the sulfur oxyanion believed to primarily contribute to the antimicrobial effect.
Dithionite and sulfite are known to be used as reducing agents to improve the fermentability during fermentation, typically by means of alleviating the effect of fermentation inhibitors. This is for instance disclosed in WO2011/080129.
Sulfite (SO32−) is used in several large-scale industrial processes. Dithionite ([S2O4]2−) is an industrial chemical used in the pulp and paper industry for reductive bleaching and in the textile industry as a reducing agent in dyeing processes. Hence, both sulfite and dithionite may be available in large quantities. Further, it is to be understood the antimicrobial agent may comprise sulfite and/or dithionite in salt form, i.e. complexed with different cations. Examples include, but are not limited to, Na2SO3, NaHSO3, KHSO3, and Na2S2O4. According to at least one example embodiment, the antimicrobial compound is Na2SO3, NaHSO3, KHSO3, or Na2S2O4 or any mixtures thereof.
Dithionite, when dissolved in the hydrolysate, will form sulfite ions, and thus have a corresponding antimicrobial effect as adding sulfite. Correspondingly, a salt comprising sulfite or dithionite will, after being dissolved in the hydrolysate, form sulfite ions (either directly, or via the previously described dissolvement of dithionite.
Dithionite may be added in an amount of 1-50, e.g. from 4 to 25 mM. For example, by adding Na2S2O4 in an amount of 1-50, e.g. from 4 to 25 mM.
Sulfite may be added in an amount of 1-100, e.g. from 10 to 60 mM. For example, by adding Na2SO3, NaHSO3 or KHSO3 in respective amount of 1-100, e.g. from 10 to 60 mM.
It should be noted that another compound comprising sulfur oxyanion(s)
According to at least one example embodiment, the antimicrobial compound is a sulfur containing compound, which when dissolved in the hydrolysate forms free sulfur oxyanions. The sulfur containing compound is added in an amount resulting in 1-100, e.g. from 10 to 60 mM, of the sulfur oxyanion in the hydrolysate.
According to at least one example embodiment, the antimicrobial compound is a sulfite containing compound, e.g. sulfite, or a precursor or a derivate of a sulfite-containing compound, forming a sulfur oxyanion in the form of a sulfite ion when dissolved in the hydrolysate. The sulfite containing compound is added in an amount resulting in 1-100, e.g. from 10 to 60 mM, of the sulfite ion in the hydrolysate. It should be understood that another source of the sulfur oxyanion than sulfite can be used, and that the antimicrobial effect is related to the sulfur oxyanion in general, and not the sulfite in particular. However, according to at least one example embodiment, the antimicrobial effect of the sulfur oxyanion is related to when the sulfur oxyanion is sulfite, i.e. the antimicrobial effect of the sulfite.
In embodiments, the method of the present disclosure further comprises the step of subjecting the hydrolysate formed in step d) or step e) to separation to remove at least a portion of residual solid components formed during the hydrolysis.
Such solid components may e.g. include incomplete hydrolyzed cellulose and/or lignin. Preferably, the hydrolysate comprises less than 10% of such solid components.
It should be understood that the step of separation to remove at least a portion of residual solid components formed during the hydrolysis, may be referred to as a secondary separation (in relation to the separation described with reference to step b) above, which may be referred to as a primary separation). Thus, step e) may be performed prior or subsequent to such secondary separation, but always subsequent to the primary separation (which is performed prior to step d), i.e. the step of hydrolysis).
In embodiments, the method further comprises the step of controlling the amount of antimicrobial compound in the storage stable hydrolysate by:
In embodiments, the sulfur ion content is measured inline; i.e. the measurements are performed directly in the process line. Throughout the application text, the sulfur ion may be referred to as the sulfur oxyanion.
In alternative embodiments, the sulfur ion content is measured online; i.e. the measurements are performed on a sample diverted from the process line, such as in a bypass loop from the main process line.
Inline and online sulfur ion measurements are advantageous since time consuming manual sampling methods and subsequent laboratory analyses used for controlling process parameters can be avoided. In other words, inline and online sulfur ion measurements provide a swift and efficient approach for correcting an ongoing process.
The sulfur ion content may be measured by titration means, a sulfur probe or a sulfur sensor, which is configured to reflect the content of sulfur ions; i.e. sulfur oxyanions present in the hydrolysate.
The measured sulfur content is compared with a reference sulfur value, which may vary depending on the process conditions or the target chemical to be produced during subsequent fermentation. The reference sulfur value may be a predetermined threshold value. For example, the reference sulfur value is a reference sulfur ion content value, e.g. representing a predetermined threshold of the sulfur ion content.
If the sulfur ion content is lower than the reference sulfur value, additional sulfur oxyanions, e.g. more dithionite and/or sulfite may be added. For example, if less than 1 mM sulfur is present in the sample, more sulfite/dithionite may need to be added.
The measurement may be made directly after the introduction of the antimicrobial compound to ensure the quality and an efficient infection control of the sample.
The measurement(s) may also be performed during storage of the formed hydrolysate or directly before fermentation to make sure that the hydrolysate to be added to the fermentation vessel is free from contaminants.
According to a second aspect, there is provided a hydrolysate formed by the method as described hereinbefore.
After the hydrolysate has been formed, it may be stored before being fermented and processed to a target chemical. The hydrolysate of the present disclosure is adapted to remain stable and free from bacterial contaminants, and is therefore suitable for storage during relatively long periods of time, and for being transported to e.g. a different fermentation site.
According to at least one example embodiment, the hydrolysate is a substantially infection free hydrolysate. Thus, the presence of bacterial contaminants is eliminated, or substantially reduced. Such hydrolysate therefore allows for prolonged storage periods. According to at least one example embodiment, the level of bacterial contaminates is below a bacterial contaminates threshold. The bacterial contaminants threshold may e.g. be adapted such that the hydrolysate may be stored up to three weeks, e.g. at least five weeks, such as at least eight weeks at room temperature without getting infected. The hydrolysate may be referred to as a storage stable hydrolysate. If the hydrolysate is stored at 4° C., the storage period may be significantly longer. The hydrolysate may be used in a subsequent fermentation step, to provide a safe and contamination free fermentation. Furthermore, the hydrolysate may comprise only small amounts of antimicrobial compound to yield a significant contamination reducing effect. The fact that the hydrolysate is formed by at least a separation step prior to hydrolysis, and that only relatively pure sugar streams are utilized in the formation of the hydrolysate, allows the antimicrobial compounds in the hydrolysate to act as a “boost” and preserve its infection free properties such that the hydrolysate becomes resistant to infections during storage and during subsequent fermentation.
According to at least one example embodiments, the level of bacterial contaminates in the hydrolysate, and the amount of antimicrobial compound, are adapted such that the hydrolysate may be stored three weeks, e.g. at least five weeks, such as at least eight weeks at room temperature without getting infected.
According to at least one example embodiment, the bacterial contaminates threshold is 3*108 bacteria cells per ml. That is, a hydrolysate which is a substantially infection free hydrolysate has a presence of bacterial contaminants below the bacterial contaminates threshold. According to at least one example embodiment, the step e) of adding an antimicrobial compound to the hydrolysate in an amount sufficient to form a storage stable hydrolysate, results in a hydrolysate which has a bacterial contaminants below the bacterial contaminates threshold during the storage (e.g. storage of at least 1 day, at least 7 days, or at least 14 days or at least 21 days).
According to at least one example embodiment, the concentration of the antimicrobial compound in the hydrolysate is between 1 and 100 mM, e.g. between 5 and 50 mM, e.g. between 5 and 25 mM. Preferably, the concentration is in the lower range as a too high concentration may inhibit microbes utilized in a subsequent fermentation step. According to at least one example embodiment, the antimicrobial compound is selected from dithionite and sulfite. Such compounds are advantageous carriers of the sulfur oxyanion believed to primarily contribute to the antimicrobial effect. According to at least one example embodiment, the antimicrobial compound is Na2SO3, NaHSO3, KHSO3, or Na2S2O4 or any mixtures thereof. In the case of dithionite, the hydrolysate may comprise an amount of 1-50, e.g. from 4 to 25 mM. For example, Na2S2O4 in an amount of 1-50, e.g. from 4 to 25 mM. In the case of sulfite, the hydrolysate may comprise an amount of 1-100, e.g. from 10 to 60 mM. For example, Na2SO3, NaHSO3 or KHSO3 in respective amount of 1-100, e.g. from 10 to 60 mM.
According to at least one example embodiment, the hydrolysate comprises glucose, a level of bacterial contaminates below a bacterial contaminates threshold, and at least 5 mM antimicrobial compound, such that the hydrolysate is resistant to an addition of a Lactobacillus sp. for at least 21 days. Thus, an addition of Lactobacillus sp. which with a level of bacterial contaminates above the bacterial contaminates threshold, or with less than 5 mM antimicrobial compound, would cause a significant reduction of glucose (i.e. glucose consumed due to infection) over the 21 days after the addition, is not causing an infection of the hydrolysate owing to the combination of a level of bacterial contaminates below a bacterial contaminates threshold, and at least 5 mM antimicrobial compound. In other words, the bacterial contaminates threshold is adapted such that said addition of Lactobacillus sp. does not cause an infection of the hydrolysate over at least 21 days for an hydrolysate comprising at least 5 mM antimicrobial compound. This is further exemplified in Example 4 below.
According to third aspect, there is provided the use of a hydrolysate formed by the method as described hereinbefore to reduce and/or control microbial contamination during storage and/or fermentation to a target chemical. Alternatively, use of a hydrolysate according to the second aspect of the invention is provided.
As used herein, “reduce and/or control microbial contamination” means that undesired microbial contaminants are kept at a minimum or a reduced level throughout the storage and fermentation periods. Bacterial infections commonly occurring during storage and fermentation are thus prevented. Thus, the control of microbial contamination is achieved by the level of bacterial contaminants and amount of antimicrobial compound, as explained with reference to the second aspect of the invention.
The technical effect of storing the hydrolysate is for example given in examples 2 and 4 below. From the examples, it is clear that the storing of a hydrolysate according to the invention does not cause any (significant) reduction in the sugar level (in example 4 measured as glucose concentration), as compared to a reference sample.
According to a fourth aspect, there is provided a method for reducing and/or controlling microbial contamination in a separate hydrolysis and fermentation (SHF) process comprising:
Fermentation of the hydrolysate may be performed by a fermenting organism; i.e. an organism that is capable of fermenting saccharides into a target chemical. The fermenting organism may be at least one eukaryotic or prokaryotic microorganism, such as bacteria and/or yeast. Examples of bacteria and yeasts which are capable of fermenting saccharides into other chemical compounds are known to the skilled person. Bacteria from Escherichia, Lactobacillus and Streptomyces may be used for the fermentation. Yeasts from Saccharomyces, Pichia and Candida may be used as the fermenting organism. The fermenting organism may for example be wild type, mutant or recombinant Saccharomyces cerevisiae. Using S. cerevisiae for producing a target chemical by means of fermentation is advantageous since S. cerevisiae is well established with regard to industrial fermentation and provides for a high product yield.
Again, the control of microbial contamination is achieved by the level of bacterial contaminants and amount of antimicrobial compound, as explained with reference to the second aspect of the invention. The method may further comprise the step of storing the storage stable hydrolysate prior to subjecting the storage stable hydrolysate to fermentation. The storage time may e.g. be at least 1 day, or at least 7 days, or at least 172 h, or at least 340 h, or at least 21 days, or at least 510 h. The technical effect of an improved fermentation is obvious from the examples 2 and 4 below. From the examples, it is clear that the storing of a hydrolysate according to the invention does not cause any (significant) reduction in the sugar level (in example 4 measured as glucose concentration, and in example 2 as a low concentration of lactic acid), as compared to a reference sample. Thus, as more sugars are available after storage, the fermentation is improved.
Preferably, the fermentation is a fed-batch or a continuous fermentation.
In other words, the fermentation is semi-continuous or continuous. Since the hydrolysate stream is added in a (semi) continuous manner during fermentation, it is highly important to secure the infection control in the hydrolysate stream. The continuous introduction of an infected hydrolysate may significantly impair the fermentation.
For example, a fed-batch fermentation process comprising at least one batch phase, feed phase and end phase, respectively, and having continuous addition of fermentation media during the at least one feed phase may be used.
In embodiments, the SHF process comprises steps for controlling the microbial fermentation in an automated or semi-automated fermenting process.
In other words, the fermentation process may comprise the steps of:
The RSI set point is a target parameter; i.e. a target value or an interval corresponding to desirable or optimum process conditions. The RSI setpoint may be fixed and/or variable. The determination of the RSI setpoint may involve calculations and/or online following of the registered RSI signal during the fermentation process. Preferably, the RSI setpoint corresponds to a steady state condition in the fermentation vessel.
In a controlled fermentation method as defined above, a favorable steady state condition is reached in the fermentation. The steady state condition is associated with in situ propagation of microorganisms, which renders additional inoculum unnecessary. The risk for infections is significantly reduced by utilizing a stable storage hydrolysate according to the present disclosure.
PCT/EP2019/060706 discloses a method and a system for controlling the fermentation process, the entire content which is incorporated herein by reference.
With the method of the present disclosure, it is possible to store the hydrolysate for a prolonged period of time.
In embodiments, the method further comprises the step of storing the hydrolysate at least three weeks, preferably at least five weeks, such as at least eight weeks at room temperature prior to fermentation.
This is advantageous, and allows for the SHF process to be discontinued after hydrolysis. The hydrolysate may thus be removed, stored and potentially transported to a different fermentation system site.
It should be noted that if the hydrolysate is stored at 4° C., the storage period may be significantly longer.
The method may further comprise the step of recovering or concentrating the hydrolysate prior to fermentation. Depending on the target chemical to be produced and the fermentation process conditions, the hydrolysate may be concentrated to increase the final sugar concentration of the hydrolysate.
The sugar concentration may be in the range of 8 to 75%, e.g. from 20 to 70, preferably from 40 to 70% depending on the application.
In other words, the sugar content of the hydrolysate may be from 80 g/l to 750 g/l, e.g. from 200 to 700 g/l, e.g. from 400 to 700 g/l.
According to fifth aspect, there is provided a system for forming a storage stable hydrolysate from a lignocellulosic material comprising:
The means for removing at least 80%, preferably at least 90% of the liquid component from the pretreated lignocellulosic composition to form a separated lignocellulosic component, may e.g. be a separation unit. The separation unit may comprise at least one of the following separation devices: a screen, a filter, a decanter screw, a centrifuge. Hereby, a device configured to separate and remove the liquid component from the pretreated lignocellulosic composition is provided. The separation unit may comprise washing means, such as a supply of washing liquid, e.g. water.
The means for diluting the lignocellulosic component with a dilution liquid, e.g. water to form an aqueous slurry, may e.g. be a dilution unit. The dilution unit may comprise a dilution vessel or tank configured to provide a stream of dilution liquid (e.g. water) to the lignocellulosic component.
The means for adding an antimicrobial compound to the hydrolysate to form a storage stable hydrolysate, may e.g. comprise an antimicrobial compound source comprised in a container, and corresponding transfer means, e.g. piping or the like, to transfer the antimicrobial compound from the antimicrobial compound source to the desired location within the system.
In embodiments, the system further comprises means for separating the hydrolysate to remove at least a portion of residual solid components formed during hydrolysis. Such separation means are preferably arranged in fluid communication with and downstream of the hydrolysis unit.
The means for separating the hydrolysate to remove at least a portion of residual solid components formed during hydrolysis may be referred to as a second, or secondary, separation means (as compared to the means for removing at least 80%, preferably at least 90% of the liquid component from the pretreated lignocellulosic composition to form a separated lignocellulosic component, which may be referred to as a first, or primary, separation means). The second separation means may e.g. be a second separation unit. The second separation unit may comprise at least one of the following separation devices: a screen, a filter, a decanter screw, a centrifuge.
The hydrolysis unit may be configured to receive an air stream through an inlet of the hydrolysis unit, and to discharge the air stream by means of an outlet the hydrolysis unit.
Thereby, air is flushed through the hydrolysis unit and mixed with the aqueous slurry. This increases the efficiency of the enzymes in the hydrolysis unit, yielding a quicker and more efficient hydrolysis
In embodiments, the system of the present disclosure comprises means for controlling the amount of antimicrobial compound in the storage stable hydrolysate, comprising:
The means for measuring the sulfur ion content may be titration, a sulfur probe or a sulfur sensor arranged to measure the sulfur content after the antimicrobial compound has been added to the hydrolysate. The means for adding additional antimicrobial compound in case the sulfur ion content is lower than a reference sulfur value may e.g. comprise a valve arranged and configured to supply antimicrobial compound on demand (the valve is preferably comprised in the means for adding an antimicrobial compound to the hydrolysate to form a storage stable hydrolysate), and a control unit configured to determine whether or not the sulfur ion content is lower than a reference sulfur value, and in response to determining that the sulfur ion content is lower than the reference sulfur value, configured to control the valve to supply antimicrobial compound (preferably from an antimicrobial compound source).
According to a sixth aspect, there is provided a system for reducing and/or controlling microbial contamination in a separate hydrolysis and fermentation (SHF) process comprising:
The fermentation vessel may comprise agitation means to mix the hydrolysate; i.e. the fermentation media with fermenting microorganisms.
Furthermore, the fermentation vessel may comprise means for measuring a residual sugar indicator parameter, RSI, which directly or indirectly indicates the concentration of residual sugars in the fermentation vessel. The measuring means may include one or more sensors arranged in the fermentation vessel or in a loop outside the vessel. Sensors may e.g. be arranged in the inlet and/or outlet of the fermentation vessel to provide RSI signals.
The RSI may be a density indicator, for example obtained using optical measurements or refractive index (RI) measurements. The RSI could also be derived from measurements and calculations of carbon dioxide (CO2) generation, or be obtained by direct measurements of sugar concentration.
The fermentation vessel may further comprise means for determining and setting an RSI setpoint based on the rate of change of the measured residual sugar indicator parameter such that the RSI setpoint corresponds to a maximum rate of change, and control means for automatically adapting the amount of sugar added to the fermentation vessel in a predetermined manner in response to the measured residual sugar indicator parameter RSI and the RSI setpoint, so as to achieve and maintain the RSI setpoint, whereby efficient co-fermentation of sugars to fermentation products is obtained. Such means may comprise suitable control unit in communicative connection to the sensor (or other measuring means), and/or a valve configured to be operated based on the control unit.
The amount of sugar added to the fermentation vessel may be automatically adapted in a predetermined manner in response to the measured residual sugar indicator parameter RSI and the RSI setpoint, so as to achieve and maintain the RSI setpoint, whereby efficient co-fermentation of sugars to fermentation products is obtained.
The system may further comprise means for controlling the amount of antimicrobial compound in the fermentation vessel, comprising:
Such means may be arranged within the fermentation vessel for securing infection control during fermentation. The means for controlling the amount of antimicrobial compound in the fermentation vessel may be arranged and configured in a corresponding manner to the means for controlling the amount of antimicrobial compound in the storage stable hydrolysate of the fifth aspect of the invention.
In embodiments, the sulfur ion content is measured both prior to fermentation (i.e. to the storage stable hydrolysate) and during fermentation.
If the fermentation system also comprises means to control the microbial fermentation by means for measuring a residual sugar indicator parameter, RSI, the content of sulfur may be measured simultaneously with such RSI measurements.
If additional sulfur oxyanions are added in the fermentation vessel, this may also have a positive effect on the fermentation, e.g. it may increase the rate of the fermentation reaction or the total amount of target product produced in the fermentation reaction.
According to a seventh aspect, there is provided the use of dithionite or sulfite for decreasing microbial contamination of a hydrolysate formed from a lignocellulosic material.
As previously mentioned, dithionite, when dissolved in the hydrolysate, will form sulfite ions, and thus have a corresponding antimicrobial effect as adding sulfite. Correspondingly, a salt comprising sulfite or dithionite will, after being dissolved in the hydrolysate, form sulfite ions (either directly, or via the previously described dissolvement of dithionite. Thus, such compounds are advantageous carriers of the sulfur oxyanion believed to primarily contribute to the antimicrobial effect.
The various aspects of the present disclosure, including its particular features and advantages, will be readily understood from the following detailed description and the accompanying drawings, in which:
The present invention will now be described more fully hereinafter with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided for thoroughness and completeness, and fully convey the scope of the present invention to the skilled person.
The system comprises means 7 to remove at least 80%, preferably at least 90% of the liquid component from the pretreated lignocellulosic composition 6 to form a separated lignocellulosic component 8. As illustrated in
The liquid component is removed from the separation device, as illustrated by the liquid stream 9. The liquid component comprises water and byproducts formed during the pretreatment process. The liquid stream 9 may be utilized outside the process, e.g. for ethanol fermentation. The separated lignocellulosic component (comprising the solid component of the pretreated lignocellulosic composition) 8 is discharged from the separation unit 7 and is thereafter diluted.
Means 10 for diluting the lignocellulosic component 8 with a dilution liquid, preferably water, to form an aqueous slurry 11 may be performed in a separate dilution vessel or tank as illustrated in
The system 1 further comprises a hydrolysis unit 12 for hydrolyzing the aqueous slurry, which has both been pretreated and cleaned; i.e. separated and diluted with water.
The hydrolysis unit 12 comprises an inlet 13 for receiving the aqueous slurry 11, and an outlet 14 for discharge of the hydrolysate 15 after the hydrolysis has been completed.
A second separation unit 16 may be arranged to receive the hydrolysate 15 in order to remove residual solids formed during hydrolysis. As illustrated in
The system also comprises means 18 for adding antimicrobial compound to the hydrolysate 15 before separation in the second separation unit 16 or to the separated hydrolysate stream 19. It is also conceivable to add the antimicrobial compound directly to the hydrolysis unit 12 upon completion of the hydrolysis reaction. Antimicrobial compound may, if needed, also be added to the fermentation vessel 23. The dotted arrows in
The hydrolysis unit 12 may be configured to receive an air stream 20 through an inlet 21 and to discharge the air stream 20 through an outlet 22 of the hydrolysis unit 12.
In other words, the air stream 20 is mixed with the feedstock within the hydrolysis unit 12. This may be achieved by simultaneously stirring the feedstock in the hydrolysis unit 12. The air stream 20 allows for the enzymes present in the system to become more efficient, yielding a quicker and more efficient hydrolysis.
The air inlet 21 may be the same as the inlet 13 for receiving the aqueous slurry 11 and the air outlet 22 may be the same as the outlet 14 for discharging the hydrolysate stream 15. Alternatively, the air inlet 21 is arranged in a bottom portion of the hydrolysis unit 12 and the air outlet 22 is arranged in a top portion of the hydrolysis unit 12, as illustrated in
The system 1 may be connected to a fermentation vessel 23 arranged in fluid communication with and downstream of the hydrolysis unit 12.
A product recovery unit 24, such as distillation or ion exchange chromatography may be connected to the system downstream of the fermentation vessel.
The system may also comprise means 25 for controlling the amount of antimicrobial compound. Such means 25 comprises means for measuring the sulfur ion content of the hydrolysate (15 or 19) after a sufficient amount of antimicrobial has been added. The measuring means may be a sulfur probe or a sulfur sensor arranged or inserted into the process to measure the sulfur content after the antimicrobial compound has been added to the hydrolysate.
If the fermentation vessel 23 also comprises means to control the microbial fermentation by means for measuring a residual sugar indicator parameter, RSI, the content of sulfur may be measured simultaneously with such parameter.
After a storage stable hydrolysate has been formed, i.e. after the addition of antimicrobial to the hydrolysate stream 15, or optionally hydrolysate stream 19, the hydrolysate may be removed from the process and stored during a period of time, before being used for subsequent fermentation in the fermentation vessel 23.
Although
Terms, definitions and embodiments of the first aspect of the present disclosure apply mutatis mutandis to the other aspects of the present disclosure, and vice versa.
Non-detoxified spruce hydrolysate (with added 2 g/l yeast extract; 0.5% sodium phosphate; 0.5 g/l diammonium phosphate; 2 g/l peptone and diluted with water to 30 g/l glucose) was filter sterilized and used to represent feed media. An inoculum was prepared using MRS media and the same contaminated sample as used in previous experiments, and was incubated at 37° C. overnight (16 hours). Sodium sulfite was added at concentrations of 0 mM, 5 mM, 10 mM, 20 mM and 40 mM, respectively, to 25 ml serum bottles with 22.5 ml of the feed media. After inoculation, the bottles were placed on a multiple magnetic stirring plate at room temperature, with samples taken periodically and analyzed using HPLC.
The hydrolysate used may represent a feed stream where no separation of the pretreatment liquid and solid component has taken place. In other words, hydrolysis and fermentation inhibitors are still present in the feed stream.
As illustrated in
The aim of this experiment was to test the potential of sodium sulfite to control bacterial contamination in the storage tanks which contain sugar-rich hydrolysate streams before they are used in fermentations. The hydrolysate is representative of a hydrolysate according to the present disclosure.
To test the theory, synthetic media was made containing about 90 g/l glucose, 0.5% sodium phosphate/phosphoric acid buffer (pH 5.5), 0.5 g/l diammonium phosphate, 2 g/l yeast extract and 2 g/l peptone. This solution (45 ml) was added to 50 ml serum bottles, along with a magnetic stirrer bar, and autoclaved, where after sodium sulfite at concentrations of 5, 10, 20 and 40 mM was added, while some bottles had no sulfite added, which served as control (0 mM). In an Erlenmeyer flask, 150 ml MRS media was inoculated using a sample previously collected from a contaminated fermentation at SEKAB, previously showed to involve lactic acid bacteria. This culture was incubated at 37° C. for 36 hours and used to inoculate all the serum bottles (10% v/v). The serum bottles were then sealed with an airtight septum and a metal ring clamp and two needles were inserted through the septum to allow for sampling. The serum bottles were placed on a magnetic stirrer plate at room temperature (since storage tanks are typically kept at room temperature), with stirring at 100 rpm. Samples were taken periodically and analyzed for glucose and lactic acid using HPLC. Experiments were performed in triplicate. The experiments were continued for 11 days.
The only growth that took place in the experiment was in the control bottles, both based on the visual evaluation, which indicated no growth or turbidity in any other bottles, as well as the lactic acid data obtained with HPLC analysis. This seems to support the theory that sodium sulfite inhibits the growth of lactic acid bacteria under these conditions. As illustrated in
Experiments in this example were performed according to the description in example 2 with the difference that the sugar-rich hydrolysate contained 10% (v/v) non-detoxified spruce hydrolysate. This was done to simulate sugar-rich hydrolysate with some inhibitory impurities present that may arise from insufficient separation and/or washing.
In the “Experiment A” samples, serum bottles were inoculated with Lactobacillus sp. at time 0 hours and incubated at room temperature for 21 days. Samples were taken periodically and analyzed for glucose and lactic acid (not shown here) using HPLC.
In the “Experiment B” samples, serum bottles were inoculated twice—firstly on the first day and again two weeks later, to simulate a re-infection and test the longevity of the inhibitory effect of sodium sulfite.
In Experiment “C”, the third group of serum bottles were inoculated after two weeks of storage at room temperature and inoculated with Lactobacillus (after 14 days).
Table 1 illustrates the glucose consumption at different time points for experiments A-C.
Growth was observed in all three experiments for all samples except in those containing 40 mM sodium sulfite, indicated by the glucose consumption in these samples (see table 1). Similar to example 1, a detoxification effect was observed in the samples containing 5-20 mM sodium sulfite, which grew faster than the sample with no sulfite added (0 mM). Thus, in contrast to example 1, where addition of sodium sulfite resulted in a detoxification effect and facilitated growth of Lactobacillus sp., sodium sulfite levels of 40 mM instead introduced an antimicrobial effect in this example. While 10% (v/v) of lignocellulosic hydrolysate containing impurities may be considered a moderate to low level of impurities, this examples clearly shows the importance of creating sugar-rich hydrolysates with a minimum of inhibitory compounds. Presence of such compounds results in a reaction between the sodium sulfite and the inhibitory compounds, causing a detoxification of the hydrolysate and facilitating bacterial growth rather than introducing an antimicrobial effect. To displace the effect of detoxification caused by sodium sulfite addition (even at moderate levels of impurities) requires higher amounts of sodium sulfite, i.e. 40 mM in this experiment compared to 5 mM in examples 2 and 4.
Storage of sugar-rich hydrolysate with addition of 5-40 mM was performed in accordance with the description of example 2. In this example, the samples were prepared and handled as in example 2. In other words, the “Experiment A” samples in this example are identical to those of example 2, and these were used as a reference for the “Experiment B” and “Experiment C” samples, as well as for validating the results of example 2.
The “Experiment B” samples were inoculated with a Lactobacillus sp. at time 0 hours, and again after 336 hours. This was done to test the infection resistance of samples that were exposed to more than one culture of Lactobacillus sp; i.e. samples that were re-infected after two weeks.
The samples in “Experiment C” were stored at ambient room temperature for 2 weeks, after which Lactobacillus was added to the flasks, after 336 hours. Samples were incubated for another 7 days and a total of 21 days as for the other two experiments. Table 2 illustrates the glucose levels at various time points throughout the storage period.
The results of “Experiment A” verified the effect observed in example 2; i.e. that bacterial growth occurs in the absence of sodium sulfite. Flasks with added sodium sulfite showed no glucose consumption during three weeks, indicating that no bacteria were able to grow, and thus no consumption of sugar was evident.
The results of “Experiment B” are the same as “Experiment A”, but allows for a second effect of sodium sulfite addition to be demonstrated; i.e. that a first addition of Lactobacillus sp. does not decrease the effect of sodium sulfite significantly at any concentration above 5 mM to which a second addition of Lactobacillus sp. was made. No signs of bacterial growth was observed during 21 days for experiments containing 5 mM or more of sodium sulfite.
The results from “Experiment C” demonstrate that 14 days of storage of a sugar-rich hydrolysate at room temperature containing sodium sulfite concentrations above 5 mM does not decrease the antimicrobial effect of sodium sulfite. As seen in table 2, no growth was observed in such samples compared to the reference with no sodium sulfite addition. Hence, sodium sulfite at all concentrations tested (5, 10, 20 and 40 mM) inhibited the growth of Lactobacillus sp. over three weeks of incubation while samples containing no sulfite allowed for bacterial growth (see table 2). These results indicate that storage for at least three weeks can be achieved with additions of 5-40 mM sodium sulfite when no compounds (or substantially no compounds) that can react with the sodium sulfite are present (compared to e.g. example 3). Presence of such compounds rather results in detoxification effects demonstrated in example 1 and in example 3.
Even though the present disclosure has been described with reference to specific exemplifying embodiments thereof, many different alterations, modifications and the like will become apparent for those skilled in the art.
Variations to the disclosed embodiments can be understood and effected by the skilled addressee in practicing the present disclosure, from a study of the drawings, the disclosure, and the appended claims. Furthermore, in the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19205177.9 | Oct 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/079616 | 10/21/2020 | WO |
