Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
  • C12M41/32—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of substances in solution
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
  • C12M21/12—Bioreactors or fermenters specially adapted for specific uses for producing fuels or solvents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/58—Reaction vessels connected in series or in parallel
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/36—Adaptation or attenuation of cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
  • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
  • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/85—Saccharomyces
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present disclosure generally relates to a method and system for control of a microbial fermentation process involving fermentation of sugars from lignocellulosic biomass to fermentation products.
• Lignocellulosic residues from forestry are attractive as feedstocks for the production of green chemicals and fuels, since they are abundant, relatively inexpensive, and not used for food.
• Lignocellulose consists mainly of lignin and two classes of polysaccharides, cellulose and hemicellulose.
• Cellulose is composed of polysaccharide chains of several hundred to over ten thousand linked glucose units
• hemicellulose is a polysaccharide composed of xylose, other pentose sugars and various hexose sugars, e.g. glucose and mannose.
• lignocellulose cellulose and hemicellulose are tightly associated to lignin, a polyphenolic compound that ties the cellulose and hemicellulose polymers together, thus providing the lignocellulose with rigidity and mechanical strength.
• various pretreatment and hydrolysis steps are typically used to separate cellulose, hemicellulose and lignin and subsequently to degrade the cellulose and hemicellulose polysaccharides to fermentable saccharides in a fermentation media, often referred to as “hydrolysate”.
• the fermentable saccharides are then converted to ethanol by means of fermenting microorganisms, such as yeast or bacteria, and the ethanol is recovered by means of distillation.
• the yeast Saccharomyces cerevisiae for example, metabolizes hexose sugars and is a microorganism suitable for industrial processes for cellulosic bioethanol production.
• Saccharomyces cerevisiae does not naturally ferment xylose that comprises a major part of many types of lignocellulosic material, why much attention has been put forth to engineering yeast to also acquire the ability to ferment xylose to ethanol and therefore to more fully utilize the available sugars from lignocellulose.
• xylose is taken up by the same transmembrane transporters as glucose, but that the transmembrane transporters have a lower affinity towards xylose than towards glucose. Additionally, synthesis of the transmembrane transporters is induced by glucose but not by xylose, meaning that the amount of transmembrane transporters gets much lower when glucose has been exhausted and xylose is the only remaining fermentable sugar.
• a method for control of a microbial fermentation process involving fermentation of sugars from lignocellulosic biomass to fermentation products comprising the steps of:
• - providing a fermentation media comprising at least xylose and glucose
• the fermentation of xylose relative the fermentation of glucose is increased.
• the relative fermentation rate of xylose to the fermentation rate of glucose is increased.
• the inventors have realized that the elevated ethanol concentration results in that the yeast ferment the xylose and glucose more equally compared to normal conditions, or conditions in which the ethanol concentration is not intentionally increased to the elevated ethanol concentration.
• the elevated ethanol concentration delays the glucose fermentation compared to that of xylose, so that more xylose can be fermented while glucose is still present to induce transmembrane transporters.
• transmembrane transporter affinity towards glucose is reduced.
• this is advantageous as instability issues related to the decrease in xylose fermentation of the engineered yeast related to the loss of xylose fermenting capability, in which the glucose-only fermenting yeasts outgrows glucose/xylose-fermenting yeasts, can be reduced or even avoided. This is further described later in the text.
• the elevated ethanol concentration is adapted to achieve a delay of the glucose fermentation compared to that of xylose.
• the elevated ethanol concentration is adapted such that the fermentation of xylose relative the fermentation of glucose is increased.
• the yeast ferments the xylose and glucose more equally compared to normal conditions (i.e. a condition in which the ethanol concentration has not been intentionally increased to the elevated ethanol concentration).
• the ethanol producing substance is a substance that will be converted to ethanol in situ.
• the ethanol producing substance is e.g. sugar, such as glucose or mannose.
• the elevated ethanol concentration is adapted such that the fermentation rate of xylose is above 20 % the fermentation rate of glucose, such as e.g. above 40 %, or above 60 % or above 80 % the fermentation rate of glucose.
• the elevated ethanol concentration may be adapted to correspond to the desired relation or ratio of the fermentation rate of xylose to the fermentation rate of glucose.
• the elevated ethanol concentration is adapted such that the fermentation rate of xylose is equal to, or substantial equal to, the fermentation rate of glucose.
• fermentation xylose and glucose can be carried out at an equal, or substantially equal fermentation rate.
• the elevated ethanol concentration makes it possible to keep glucose present longer and thus the transmembrane transporters in the yeast induced until xylose is exhausted (i.e. all xylose is fermented).
• the elevated ethanol concentration is adapted such that the fermentation rate of xylose is within 70 % - 100 % of the fermentation rate of glucose.
• the elevated ethanol concentration is adapted such that the xylose fermentation rate is unaffected, or almost unaffected, while the glucose fermentation rate is reduced compared to normal conditions (i.e. a condition in which the ethanol concentration has not been intentionally increased to the elevated ethanol concentration).
• the elevated ethanol concentration corresponds to an ethanol content up to 8 wt% above that which would have been produced by fermentation of the fermentation media without intentionally increasing the ethanol concentration.
• the term "intentionally elevated ethanol concentration” is simply referred to as an “elevated ethanol concentration”.
• the intentionally elevated ethanol concentration is referring to an ethanol concentration, which at least during a portion of the fermentation process, that is higher than the ethanol concentration normally obtained during a corresponding portion of the fermentation process, under the same conditions and for the same input values, except for the means causing the elevated ethanol concentration.
• the elevated ethanol concentration may be reached by achieving a surplus ethanol concentration obtained by e.g. direct addition to an ongoing fermentation process, or to a feed to be subsequently used in a fermentation process, of ethanol, or of an ethanol containing fluid having a higher ethanol content than the broth in the fermentation system without such additions, causing the broth being processed to have a higher ethanol concentration during the remainder of the fermentation process than without this addition.
• the elevated ethanol concentration may alternatively be reached by direct addition to an ongoing fermentation process, or to a feed subsequently to be used in a fermentation process, of a compound that is converted to ethanol "in situ" causing the broth being processed in the fermentation system to for at least some of the remainder part of the fermentation process to contain a higher ethanol content, than the broth otherwise would have at the corresponding part of the fermentation without this addition.
• the step of fermenting comprises cofermenting the xylose and glucose.
• the elevated ethanol concentration balances the co-fermenting process of xylose and glucose, as the presence of glucose during the co-fermentation keep the transmembrane transporters induced until both xylose and glucose have been exhausted, effectively enabling a faster xylose fermentation at least through a part of the fermentation process.
• the step of fermenting comprises at least partly sequentially fermenting the xylose and glucose, for example by utilizing a plurality of fermentation units as will be described later in the text.
• at least a part of the fermenting comprises fermenting solely the xylose, and/or solely the glucose (possibly together with another hexose, such as mannose).
• the fermenting comprises the two sub-steps of fermenting solely the xylose and co-fermenting the xylose and glucose in a preferred order, and/or comprises the two sub-steps of fermenting solely the glucose and co- fermenting the xylose and glucose in a preferred order, wherein at least one of the sub-steps are performed at the elevated ethanol concentration.
• at least one the sub-steps may be performed at an untampered ethanol concentration, or at least at an ethanol concentration which is not intentionally increased to the elevated ethanol concentration.
• the method further comprises the steps of:
• the ethanol concentration is determined directly during the step of fermenting, e.g. based on an online measurement of the fermenting slurry comprising the fermenting media, the yeast, and any produced fermented product.
• the determination of the ethanol concentration during the step of fermenting is achieved by an ethanol sensor configured to measure the ethanol concentration.
• the ethanol sensor may e.g. be an inline mounted (to the fermentation media or a sub-stream thereof) infrared spectroscope configured and calibrated to measure the ethanol concentration.
• the delay between determining the ethanol concentration and adjusting the ethanol concentration in response to the determination is minimized.
• the ethanol concentration is determined in an off-gas from the fermenting slurry.
• the determination of the ethanol concentration during the step of fermenting is achieved by measuring the CO2 produced during the fermentation, and based on the measured CO2, calculating the produced ethanol amount (weight) to determine the ethanol concentration (g/L).
• the determination of the ethanol concentration during the step of fermenting is achieved by measuring ethanol gases in the off-gas.
• the elevated ethanol concentration is maintained during the step of fermenting, or at least the elevated ethanol concentration is maintained during the step of fermenting.
• the threshold limit is based on the relation or ratio of fermentation rates of glucose and xylose.
• Such threshold limit is beneficial to use, as at least one purpose of the elevated ethanol concentration is to reduce the difference in fermentation rates between glucose and xylose.
• the threshold limit of the elevated ethanol concentration is set based on that the fermentation rate of xylose is at least above 20 %, or above 40 %, or above 60 % or above 80 % the fermentation rate of glucose.
• the threshold limit may furthermore be based on fermentation parameters such as e.g. amount of fermentable sugars present in the media before being fermented or partially fermented.
• the step of fermenting is carried out in at least two separate fermentation units, wherein the concentration of ethanol is intentionally increased to the elevated ethanol concentration in at least one of the fermentation units.
• the step of fermenting can be divided between different fermentation units.
• the concentration of ethanol may be intentionally increased to the elevated ethanol concentration in at least two fermentation units.
• the step of fermenting is carried out in at least two separate fermentation units, wherein the concentration of ethanol is intentionally increased to the elevated ethanol concentration in at least one of the fermentation units, and wherein the concentration of ethanol is untampered, or at least not intentionally increased to the elevated ethanol concentration, in at least one of the fermentation units.
• the different fermentation units can be adapted based on the target sugar (e.g. xylose or glucose) to ferment.
• target sugar e.g. xylose or glucose
• the concentration of ethanol is untampered, or at least not intentionally increased to the elevated ethanol concentration (it may e.g. be reduced) as this is advantageous for glucose fermentation, while in another fermentation unit configured to co-ferment xylose and glucose, the concentration of ethanol increased to the elevated ethanol concentration.
• a first fermentation unit in which the concentration of ethanol is intentionally increased to the elevated ethanol concentration is arranged downstream of a second fermentation unit in which the concentration of ethanol is untampered, or at least not intentionally increased to the elevated ethanol concentration (e.g. reduced).
• the fermentation unit or each fermentation unit, may comprise a vessel, i.e. a fermentation vessel.
• the initial amount of xylose relative the total amount of fermentable sugars in the fermentation media is at least 5 wt%.
• the fermentable sugars may e.g. be sugars which are fermentable by Saccharomyces cerevisiae or another variant of Saccharomyces.
• the initial amount of xylose relative the total amount of fermentable sugars in the fermentation media may be at least 10 wt%, or at least 15 wt%, or at least 20 wt%.
• the step of intentionally increasing the concentration of ethanol to an elevated ethanol concentration comprises at least one of the following: i) performing the step of fermenting in a continuous manner to in situ produce ethanol such that the elevated ethanol concentration is maintained during fermenting; ii) external addition of ethanol, e.g.
• the external addition of ethanol according to option ii) may e.g. be above 70 g/L.
• the external addition of sugars producing ethanol according to option iii) may be above 100 g/L, or above 140 g/L. Typically, 100 g sugars/L results in the formation of approximately 50 g ethanol/L.
• the intentionally increasing the concentration of sugars according to option iv) may be carried out by removing liquid, such as water, in a dewatering step.
• the fermentation media further comprises mannose
• the method further comprises the step of
• control of temperature of the fermentation by a set temperature T ⁇ 28 °C may be applied to at least one of the fermentation units.
• the yeast is a Saccharomyces sp. or an engineered variant thereof, e.g. a xylose fermenting engineered yeast.
• a xylose fermenting engineered yeast is a yeast which has be engineered to ferment xylose.
• the yeast may be Saccharomyces cerevisiae or uvarum.
• an engineered yeast, or a C5 yeast refers to a yeast configured to ferment pentoses (and hexoses), being any microorganism that is a member of the saccharomyces genus, and that has been engineered to be able to ferment one or more pentose sugars in addition to hexose sugars.
• a non-engineered yeast e.g. baker’s yeast or a C6 yeast refers to a yeast configured to ferment hexoses (but not pentoses), thus being any microorganism that is a member of the saccharomyces genus, and that is naturally limited in fermentation ability to ferment only hexose sugars.
• the C5 yeast is a yeast of the S. cerevisiae species that have been genetically engineered to be able to ferment one or more pentose sugars, such as xylose.
• xylose fermenting yeast, C5 yeast, xylose fermenting engineered yeast or simply engineered yeast are used interchangeably, which is to be understood to be different from a non-xylose fermenting yeast, or a C6 yeast (i.e. a nonengineered yeast).
• the fermentation microorganism of yeast may simply be referred to as yeast or xylose-engineered yeast.
• the sugars are derived from hardwood, softwood, agricultural waste or any mixture thereof.
• the fermentation process is a continuous fermentation process, or is a batch fermentation process in which the yeast is recirculated, wherein the yeast is subject to regeneration, and wherein the elevated ethanol concentration is adapted to prevent, or at least reduce, oppression of xylose fermenting yeast.
• the fermentation of xylose may be carried out for a longer time.
• a regeneration of the yeast will result in less advantage for a yeast cell that has shed the xylose fermentation ability compared to a yeast cell that has the xylose fermentation ability maintained.
• the elevated ethanol concentration corresponds to an ethanol content which is at least 2 wt% above that which would have been produced by fermentation of the fermentation media without intentionally increasing the ethanol concentration, or it is at least 50 % of the theoretical yield of ethanol obtainable by the used fermentation media.
• the ethanol concentration is intentionally increased compared to not intentionally increasing the ethanol concentration.
• the intentionally increased ethanol concentration e.g. of an increased ethanol content of at least 2 wt% is typically referring to the concentration (g/L) of ethanol above that which would have been produced by fermentation of the fermentation media without intentionally increasing the ethanol concentration.
• the amount in wt% of ethanol is typically as compared to the total amount (weight) of the fermentation media.
• the elevated ethanol concentration (g/L) is at least 25 % of the theoretical yield of ethanol obtainable by the used fermentation media.
• the ethanol concentration (g/L) is at least 25 % above the concentration obtainable by fermenting the used fermentation media at 90 % ethanol yield (90 % of theoretical yield of 0.51 g ethanol generated per gram sugar fermented).
• the elevated ethanol concentration corresponds to an ethanol content which is at least 2.5 wt%, or 3 wt%, or 4 wt%, or 5 wt% or 6 wt% or 7 wt% or 8 wt%, or at least 10 wt%, with an upper limit of e.g.
• the elevated ethanol concentration corresponds to an ethanol content which is between 6 wt% (or 8 wt% or 10 wt%) and 20 wt% above that which would have been produced by fermentation of the fermentation media without intentionally increasing the ethanol concentration.
• the elevated ethanol concentration is at least 50 % - 80 % of the theoretical yield of ethanol obtainable by the used fermentation media.
• the ethanol concentration is denoted X g/L
• the amount of yeast is denoted Y g/L
• the yeast stress ratio is defined as X/Y, wherein X/Y is at least 10.
• the elevated ethanol concentration is reached by the external addition of ethanol, or ethanol producing substance corresponding to the same amount of ethanol, of between 10 g/L to 50 g/L, such as e.g. between 10 g/L to 20g/L or 30 g/L, or 40 g/L, or between 20 g/L to 30 g/L or 40 g/L or 50 g/L.
• the elevated ethanol concentration is 25 % higher compared to what would normally be produced by the fermentable sugars in the fermentation media.
• the elevated ethanol concentration is an ethanol concentration totaling above that typically achieved by fermenting the initially available fermentable sugars in the fermentation media at 25 wt% or higher sugar to ethanol conversion/yield.
• the elevated ethanol concentration is 25 % higher compared to what would normally be accomplished by the fermentation of the available sugars in the used fermentation media at 90 % ethanol yield (90 % of theoretical yield of 0.51 g ethanol generated per gram sugar fermented).
• the increased or elevated ethanol concentration is an ethanol concentration corresponding to an ethanol content totaling between 6 wt% and 9 wt% as compared to the total amount (weight) of the fermentation media.
• a system for control of a microbial fermentation process involving fermentation of sugars from lignocellulosic biomass to fermentation products comprises:
• a fermentation unit configured to ferment a fermentation media comprising at least xylose and glucose by means of fermentation microorganisms of yeast;
• the system further comprises an ethanol determination arrangement configured to determine the ethanol concentration in the fermentation unit, and control means for automatically adjusting the ethanol concentration in the fermentation unit in a predetermined manner in response of determining that the concentration of ethanol is below a threshold limit.
• the determination arrangement configured to determine the ethanol concentration in the fermentation unit comprises an ethanol sensor, or a sensor for measuring produced CO2 from the fermentation unit.
• the ethanol concentration (g/L) in the fermentation unit can be determined based on the measured CO2, and calculations of the produced ethanol amount (weight).
• the sensor if configured to measure ethanol gases in the off-gases from the fermentation.
• the ethanol sensor may e.g. be an inline mounted (to the fermentation media or a sub-stream thereof) infrared spectroscope configured and calibrated to measure the ethanol concentration.
• the control means may e.g. be a control arrangement.
• the control arrangement may comprise adequate equipment, such as e.g. a controllable valve connected to a supply unit comprising ethanol or an ethanol-producing substance, which controllable valve is arranged on a supply line to, or upstream of, the fermentation unit, and is configured to vary the supply of ethanol or ethanol-producing substance in response of determining that the concentration of ethanol is below a threshold limit by means of the ethanol determination arrangement.
• the means of intentionally increasing the concentration of ethanol comprises at least one of the following: a) means for operating the fermentation unit in a continuous manner to in situ produce ethanol such that the elevated ethanol concentration is maintained in the fermentation unit during the fermentation; b) means for providing external addition of ethanol to, and/or upstream of, the fermentation unit; c) means for providing external addition of sugars producing ethanol to, and/or upstream of the fermentation unit, such that the elevated ethanol concentration is maintained in the fermentation unit during the fermentation; d) means for intentionally increasing the concentration of sugars in the fermentation media in, and/or upstream of the fermentation unit.
• the means for operating the fermentation unit in a continuous manner may comprise an adequate fermentation unit, such as a continuous operable fermentation vessel.
• the means for providing external addition of ethanol may comprise a supply unit comprising the external ethanol, and a supply line for distributing the external ethanol to, and/or upstream of, the fermentation unit.
• the means for providing external addition of sugars may comprise a supply unit comprising the external sugars, and a supply line for distributing the external sugars to, and/or upstream of, the fermentation unit.
• the supply unit may be configured to supply both external sugars and external ethanol, for example in two separated sub-compartments of the supply unit, and a respective (or single) supply line.
• the means for intentionally increasing the concentration of sugars in the fermentation may comprises a liquid removal arrangement, or a dewatering unit.
• the control means or control arrangement may be configured to remove liquid in, or upstream of, the fermentation unit, e.g. for intentionally increasing the concentration of sugars in the fermentation media.
• system further comprises a temperature control arrangement configured to control the temperature of the fermentation unit during the fermentation by a set temperature T ⁇ 28 °C.
• the system and the fermentation unit is configured to carry out the fermentation as a continuous fermentation process, or as a batch fermentation process in which the yeast is recirculated, in which the yeast is subject to regeneration, and wherein the means of intentionally increasing the concentration of ethanol is adapted to prevent oppression of xylose fermenting yeast.
• the fermentation unit is a first fermentation unit and the system further comprises a second fermentation unit, the first and second fermentation units being configured to subsequently ferment the fermentation media, wherein the second fermentation unit is configured to ferment the fermentation media with an untampered ethanol concentration, or at least in the absence of intentionally increasing the ethanol concertation to an elevated ethanol concentration.
• the fermentation unit(s) may be fermentation vessel(s) as previously described, and may be arranged as described in relation to the first aspect of the invention. That is, for example, the second fermentation unit may be arranged upstream of the first fermentation unit, and each one of the first and second fermentation units may according to an alternative embodiment be configured to ferment the fermentation media with an intentionally increased ethanol concentration to an elevated ethanol concentration.
• the system may comprise another fermentation unit being configured to ferment the fermentation media with an untampered ethanol concentration, or at least in the absence of intentionally increasing the ethanol concertation to an elevated ethanol concentration (such as e.g. a decreased ethanol concentration).
• the sugar concentration of the fermentation media is at least 5 g/L.
• the fermentation media comprises galactose or alternatively galactose instead of glucose.
• the system further comprises a control unit with program code means comprising instructions to perform at least some steps of the method according to the first aspect of the invention.
• the program code means may comprise instructions for controlling parts of the system in order to perform at least some steps of the method according to the first aspect of the invention.
• the program code means may comprise instructions to open a valve to adapt the amount (weight) of the fermentation media fed to the fermentation unit and/or to open a valve to adapt the amount (weight) of ethanol or ethanol-producing substance fed to the fermentation unit.
• an arrangement for treatment of lignocellulosic biomass comprises a pretreatment arrangement for pretreating the lignocellulosic biomass with e.g. SO2, a hydrolysis unit arranged downstream of and in fluid communication with the pretreatment arrangement, and a system for control of a microbial fermentation process comprising at least one fermentation unit, such as a fermentation vessel, arranged downstream of and in fluid communication with the hydrolysis unit.
• the system for control of a microbial fermentation process is typically the same as that described with reference to the second aspect of the invention.
• the arrangement may comprise additional units and components known to those skilled in the art.
• a separation unit may be arranged between the pretreatment arrangement and the hydrolysis unit, and/or between the hydrolysis unit and the fermentation unit. Any ratio of sugars is based on weight (w/w) and may be referred to as a weight ratio, if nothing else is stated. Such ratio may e.g. be derived by taking a ratio of the sugar concentrations (g/L).
• the concentration is in the unit g/L if nothing else is stated, and the fermentation rate is in the unit g/h if nothing else is stated.
• the concentration of ethanol in the fermentation media may alternatively be expressed as wt%, as the skilled person knows how to translate the concentration in g/L to wt% using e.g. the density of the fermentation media.
• the fermenting of the fermentation media by means of fermentation microorganisms of yeast may be achieved by a substantial anaerobic fermentation process. It should be understood that when stating that the fermentation process may be substantial anaerobic, the fermentation of the fermentation media by means of fermentation microorganisms of yeast is carried out anaerobically, i.e. fermentation without the presence of oxygen, or at least in a process in which oxygen is not taking part in the fermentation. Stated differently, a substantial anaerobic fermentative process is a process without using oxygen as terminal electron acceptor. Throughout the application, the step of fermenting, (or co-fermenting or fermentation, or cofermentation) may refer to such substantial anaerobic fermentation process.
• Fermentation of a fermentation media may be defined as anaerobic metabolism of the sugars in the fermentation media.
• the fermentation microorganisms such as yeast
• fermentation of a fermentation media implies extraction of energy from carbohydrates in the absence of oxygen participation. The absence of oxygen participation is related to that the fermentation microorganism (or yeast) does not use the oxygen even if it is present as long as there is glucose available, oxygen is thus not participating in the process.
• the substantial anaerobic microbial fermentation process is carried out in an oxygen reduced environment.
• the oxygen reduced environment is oxygen deficient.
• oxygen reduced environment means that the amount of oxygen is reduced compared to the amount of oxygen present in air.
• the oxygen concentration in the oxygen reduced environment is reduced with at least 50%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% compared to the oxygen concentration in air (in vol %).
• about 1%, such as about 2%, such as about 5%, such as about 10%, or such as about 15% of oxygen is present in the oxygen reduced environment (oxygen concentration in vol %).
• Fig. 1 is a graph showing the progress of the co-fermentation of a fermentation media including three sugars (glucose, mannose and xylose) by a non-xylose fermenting yeast;
• Fig. 2 schematically illustrates an arrangement for treatment of lignocellulosic biomass including control of a microbial fermentation process in accordance with one example embodiment of the invention
• FIG. 3 schematically illustrates a system for control of a microbial fermentation process in accordance with one example embodiment of the invention
• Fig. 4 schematically illustrates the steps of a method for control of a microbial fermentation process in accordance with one example embodiment of the invention.
• Fig. 5 is a graph showing the results of four measurement trials in accordance with one example embodiment of the invention.
• Fig. 6 is a graph showing the results of four measurement trials in accordance with one example embodiment of the invention.
• Fig. 1 is a graph showing the progress of the fermentation in terms of weight loss (mg) over time (min).
• the fermentation is here the simultaneous fermentation, or co-fermentation, of a fermentation media including three sugars (glucose, mannose and xylose) in a sugar mixture derived from the hemicellulose and cellulose of softwood, (Norway spruce).
• a non-xylose fermenting yeast bakeer’s yeast
• was used for the same sugar mixture in four trials the sugar mixtures being subject to various external addition of ethanol giving different elevated concentrations of ethanol (external addition of ethanol in grams per litre: 10 g/L, 30 g/L, 40 g/L and 50g/L).
• the present disclosure provides an arrangement 500 for treatment of lignocellulosic biomass comprising a pretreatment arrangement 501 for pretreating the lignocellulosic biomass, a hydrolysis unit 502 in which the pretreated biomass is subject to enzymatic hydrolysis by means of saccharification enzymes, the hydrolysis unit being arranged downstream of and in fluid communication with the pretreatment arrangement 501, and comprises a fermentation unit 503, such as a fermentation vessel 503, arranged downstream of and in fluid communication with the hydrolysis unit 502.
• the hydrolysate is fermented into a target chemical, e.g. ethanol, by means of a yeast.
• the fermentation vessel 503 is comprised in a system 1001 configured for control of microbial fermentation, which may comprise additional units and components, such as e.g. a supply unit 505 connected to the fermentation vessel 503.
• the arrangement 500 may also comprise a product recovery unit 504, such as distillation or ion exchange chromatography, arranged downstream of and in fluid communication with the fermentation vessel 503.
• a system for control of a microbial fermentation process, as the system 1001, will now be described further with reference to Fig. 3.
• Fig. 3 illustrates a system 1 for control of a microbial fermentation process involving fermentation of sugars from lignocellulosic biomass to fermentation products.
• the system 1 comprises a main fermentation unit 10 and a secondary fermentation unit 20.
• the secondary fermentation unit 20 is arranged upstream of the main fermentation unit 10.
• Each one of the main and secondary fermentation units may be a fermentation vessel 10, 20.
• the main and secondary fermentation units 10, 20 may constitute the fermentation unit 503 of Fig. 2.
• the main and secondary fermentation units 10, 20 are configured to ferment a fermentation media 3 comprising at least xylose and glucose by means of fermentation microorganisms of yeast.
• the fermentation media 3 may e.g. be fed to the system 1 by a hydrolysis unit as shown in Fig. 2.
• the main and secondary fermentation units 10, 20 comprises yeast and the fermentation media 3.
• the main fermentation unit 10 is arranged downstream of the secondary fermentation unit 20.
• the fermentation media 3 will first be at least partly fermented in the secondary fermentation unit 20, where after the remaining fermentation media together with any produced fermented product, will subsequently be fermented in the main fermentation unit 10.
• the main fermentation unit 10 may be referred to as a first fermentation unit 10, or first fermentation vessel 10
• the secondary fermentation unit 20 may be referred to as a second fermentation unit 20, or second fermentation vessel 20.
• the system 1 of Fig. 3 further comprises a moisture adjusting unit 5 in the form of a dewatering unit 5, configured to intentionally increase the concentration of sugars in the fermentation media 3 upstream of the main and secondary fermentation units 10, 20.
• the dewatering unit 5 may e.g. be arranged between the hydrolysis unit 503 and the main and secondary fermentation units 10, 20.
• the system 1 comprises a supply unit 30 configured to supply external ethanol and/or an external ethanol-producing substance, such as external sugars, e.g. glucose, and a supply line 32 connecting the supply unit 30 with the main fermentation unit 10.
• the supply unit 30 and supply line 32, as well as the dewatering unit 5, constitute different means of intentionally increasing the concentration of ethanol present in at least the main fermentation unit 10 during operation (i.e. during the fermentation) to an elevated ethanol concentration.
• the concentration of sugars is increased such that both the main and secondary fermentation units 10, 20 are subject to the increased concentration of sugars, and the resulting increased concentration of ethanol.
• the system 1 needs not to be provided with both the dewatering unit 5 and the supply unit 30 with the supply line 32, but any means (including those mentioned below) for intentionally increasing the concentration of ethanol to an elevated ethanol concentration is sufficient.
• the dewatering unit 5, and the supply unit 30 with the supple line 32 it is possible to reach the elevated ethanol concentration by the addition of ethanol or an ethanol-producing substance in at least the main fermentation unit 10.
• Fig. 3 embodied by a second supply unit 31 with corresponding second supply line 33 arranged between the main and secondary fermentation units 10, 20.
• a further option of intentionally increasing the ethanol concentration to the elevated ethanol concentration is by operating the main fermentation unit 10 in a continuous manner to in situ produce ethanol such that the elevated ethanol concentration is maintained in the main fermentation unit during the fermentation.
• the elevated ethanol concentration may be reached.
• the inventors have realized that the elevated ethanol concentration results in that the yeast ferment the xylose and glucose more equally compared to normal conditions, or conditions in which the ethanol concentration is not intentionally increased to the elevated ethanol concentration.
• the fermentation of xylose relative the fermentation of glucose is increased.
• the relative fermentation rate of xylose to the fermentation rate of glucose is increased.
• the dewatering unit 5 is omitted, and the concentration of ethanol is untampered in the secondary fermentation unit 20, as described previously with reference to various sub-steps of fermenting.
• the secondary fermentation unit 20 may be configured to ferment glucose but not xylose, e.g. by using a non-engineered yeast such as baker’s yeast, while the main fermentation unit 10 is configured to co-ferment glucose and xylose in the presence of an ethanol concentration increased to the elevated ethanol concentration.
• the operation of the main and secondary fermentation units 10, 20 are typically controlled by a respective first and second control unit 41, 42.
• a general control unit is used to control the operation of both the main and secondary fermentation unit 10, 20.
• the second control unit 42 may be connected to a controllable valve 60 configured to control the flow of fermentation media 3 to the secondary fermentation unit 10 (or in embodiments in which the secondary fermentation unit 20 is omitted, to the main fermentation unit 10).
• the first control unit 41 may be configured to control the supply of external ethanol or external sugars from the supply unit 30 via the supply line 32 to the main fermentation unit 10.
• the system 1 may comprise a first sensor 45 arranged to measure parameters in the main fermentation unit 10, and a second sensor 46 arranged to measure parameters in the secondary fermentation unit 20.
• At least the first sensor 45 may be configured to determine the ethanol concentration in the main fermentation unit 10. Together with the first control unit 41 and the supply unit 30 with the supply line 32, the first sensor 45 may thus form an automatically ethanol adjusting arrangement configured to determine the ethanol concentration in the main fermentation unit 10 and automatically adjust the ethanol concentration in the main fermentation unit 10 by means of supply of external ethanol and/or external sugars. Additionally or alternatively, the first control unit 41 is connected to the dewatering unit 5 and may thus form an automatically ethanol adjusting arrangement together with the first sensor 45.
• the system 1 may furthermore comprise a temperature control arrangement 50, here comprising the first sensor 45 acting as a first temperature sensor arranged to measure the temperature in the main fermentation unit 10 and a first temperature control element 52 configured to control the temperature of the main fermentation unit 10 (e.g. by means of a heating or cooling element).
• the temperature control arrangement 50 comprises the second sensor 46 acting as a second temperature sensor arranged to measure the temperature in the secondary fermentation unit 20 and a second temperature control element 54 configured to control the temperature of the secondary fermentation unit 20 (e.g. by means of a heating or cooling element).
• the first and second temperature control elements 52, 54 may be controlled by the first and second control units 41, 42, respectively.
• the first and second control units 41, 42 may e.g. be configured to control the temperature in the respective main and secondary fermentation units 10, 20 by a set temperature T ⁇ 28 °C.
• the secondary fermentation unit 20 is optional and may be omitted, and that the fermentation unit may comprise a fermentation unit corresponding to the main fermentation unit 10 independent of the secondary fermentation unit 20.
• the invention will now be described with reference to the flow chart of Fig. 4 which e.g. includes the operation of the system 1 of Fig. 3.
• the flow chart of Fig. 4 discloses the steps of a method for control of a microbial fermentation process involving fermentation of sugars from lignocellulosic biomass to fermentation products,
• a fermentation media comprising at least glucose and xylose is provided.
• the fermentation media and its sugars may be derived from hardwood, softwood, agricultural waste or any mixture thereof.
• the fermentation media may e.g. be supplied from a hydrolysis unit as shown in Fig. 2.
• the initial amount of xylose relative the total amount of fermentable sugars in the fermentation media may be at least 5 wt%.
• the fermentation media is fermented by means of fermentation microorganisms of yeast.
• the step S10 may comprises co-fermenting the xylose and glucose and/or comprise fermenting the xylose or glucose separately.
• the step S10 is carried out in at least two separate fermentation units, wherein the concentration of ethanol is intentionally increased to the elevated ethanol concentration in at least one of the fermentation units, and the concentration of ethanol is untampered, or at least not intentionally increased to an elevated ethanol concentration, in at least one of the fermentation units.
• the yeast may e.g. be Saccharomyces or a variant thereof, e.g. a xylose fermenting engineered yeast.
• the concentration of ethanol present during the fermentation is intentionally increased to an elevated ethanol concentration by the addition of ethanol or an ethanol- producing substance.
• Such intentional increase in the ethanol concentration to the elevated ethanol concentration may be carried out prior to and/or during the step of fermenting by e.g. utilizing means as described with reference to Fig. 3.
• the elevated ethanol concentration may e.g. be adapted such that the fermentation rate of xylose is above 20 % the fermentation rate of glucose, such as e.g. above 40 %, or above 60 % or above 80 % the fermentation rate of glucose.
• the elevated ethanol concentration may e.g. correspond to an ethanol content of at least 6 wt% above that which would have been produced by fermentation of the fermentation media without intentionally increasing the ethanol concentration, or at least 50 % of the theoretical yield of ethanol obtainable by the used fermentation media.
• step S20 may be carried out by:
• Sub-step S22 by external addition of ethanol.
• the external ethanol may e.g. be added prior to, and/or during the step S 10 to a final concentration of 50 g/L.
• the external addition of ethanol is achieved by the first and second supply units 30, 31 with corresponding supply lines 32, 33.
• Sub-step S24 by external addition of sugars producing ethanol such that the elevated ethanol concentration is maintained during the step S10.
• the external addition of external sugars is achieved by the first and second supply units 30, 31 with corresponding supply lines 32, 33.
• Sub-step S26 the concentration of sugars in the fermentation media is intentionally increased to be above 100 g/L, or above 140 g/L or above 180 g/L, prior to, and/or during the step of fermenting. In Fig. 3, this is achieved by the dewatering unit 5 prior to the secondary fermentation unit 20.
• step S20 is combined with the step S 10 such that the step of fermenting, S10, is carried out in a continuous manner to in situ produce ethanol such that the elevated ethanol concentration is maintained, S20.
• the ethanol concentration is determined (typically during the step S10), and in a step S32, the ethanol concentration is adjusted to be above a threshold limit in response of determining that the concentration of ethanol is below the threshold limit.
• the threshold limit may e.g. be based on the relation of fermentation rates of glucose and xylose.
• the first sensor 45 is used together with the first control unit 41 to instruct the supply unit 30 to supply a predetermined amount of ethanol or ethanol - producing substance (e.g. external sugars) via the supply line 32 to the main fermentation unit 10.
• the temperature of the fermentation is controlled by a set temperature T ⁇ 28 °C.
• the fermentation media typically comprises mannose.
• the system 1 in Fig. 3 is configured to carry out the fermentation as a continuous fermentation process in the main and secondary fermentation units 10, 20, but it may as well be configured as a batch fermentation process in which the yeast is recirculated.
• the yeast is subject to regeneration, and the means of intentionally increasing the concentration of ethanol may be adapted to prevent oppression of xylose fermenting yeast.
• the method may comprise a step S50, e.g. comprised in the step S20, or performed instead of step S20, of adapting the elevated ethanol concentration to prevent oppression of xylose fermenting yeast.
• the method may, alternatively or in addition to the ethanol concertation and the temperature during the fermenting, comprise a step of collecting and monitoring various process parameters in a continuous or semi-continuous manner and a step of automatically adjusting the fermenting process in response to the monitored process parameters.
• Co-fermentation of a fermentation media including three sugars (glucose, mannose and xylose) in a sugar mixture derived from the hemicellulose and cellulose of softwood, (Norway spruce) was performed batch-wise with 25 ml cultures in 50 ml bottles, consisted of diluted spruce hydrolysate containing 51 g/L glucose, 29 g/L mannose and fortified with added xylose to a resultant concentration of 40 g/L xylose.
• Acetic acid was adjusted to 4.5 g/L to reflect the content in undiluted hydrolysate, pH was adjusted to 5.5 with ammonia, and 3 g/L urea was added to ensure sufficient nitrogen.
• Fig. 6 is a graph showing the results of trials 1 and 4 (i.e. external addition of ethanol with 10 g/L and 50 g/L, respectively) from Fig. 5, together with two trials carried out for the same sugar mixture and as described above with the only difference that the yeast is not capable to ferment xylose (i.e. a non-engineered yeast, e.g. baker’s yeast) for external addition of ethanol with 10 g/L and 50 g/L, respectively.
• Fig. 6 discloses a pairwise comparison of a xylose fermenting and a non-xylose fermenting yeasts, at the lowest and highest external ethanol additions.
• the xylose fermenting ability of the yeast also increases the ability to ferment the hexoses near the hexose limit, and that at an external addition of ethanol of 50 g/L there is no shift caused by different fermentation rates of hexose and xylose as the curve passes the hexose limit. This is believed to be because the xylose fermentation rate is unaffected by the ethanol addition, whereas the hexose fermentation rate is reduced to, or towards, the same level as the xylose fermentation rate.
• the xylose fermenting engineered yeast has an advantage in fermentation rate and thus growth rate also in the phase where hexoses still remains to be fermented. This is likely due to the fact that the fermentation rate of xylose in the presence of higher ethanol concentrations does not slow down as the fermentation approaches the hexose limit.
• a yeast which is incapable of fermenting xylose e.g. a yeast which has shed the xylose fermentation ability is at growth rate disadvantage already in the late hexose phase.
• the elevated ethanol concentration results in a higher stability of the xylose fermenting yeast, in for example a continuous fermentation process, even when running it continuously at conditions where hexose is still present.
• the ethanol concentration should preferably reach a certain threshold level or elevated ethanol concentration for the fermentation rates of xylose and glucose to be equal, or substantially equal (e.g. that the fermentation rate of xylose is within 70 % of the fermentation rate of glucose) and thus keeping the transmembrane transporters induced until xylose is exhausted.

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