WO2015044101A1 - Procédé de conversion de substrat contenant du pentose - Google Patents

Procédé de conversion de substrat contenant du pentose Download PDF

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WO2015044101A1
WO2015044101A1 PCT/EP2014/070177 EP2014070177W WO2015044101A1 WO 2015044101 A1 WO2015044101 A1 WO 2015044101A1 EP 2014070177 W EP2014070177 W EP 2014070177W WO 2015044101 A1 WO2015044101 A1 WO 2015044101A1
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broth
fermentation
microorganism
liquid
process according
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PCT/EP2014/070177
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English (en)
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Johannes Petrus Smits
Elisabeth Maria Gierveld
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Dsm Ip Assets B.V.
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    • 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
    • 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
    • C12P7/14—Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
    • 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 invention is directed to a process for conversion of pentose containing substrate.
  • the invention relates to propagation and/or fermentation process wherein the microorganism is grown or maintained using toxic pentose containing lignocellulosic hydrolysate, in particular toxic C5-liquid.
  • lignocellulosic material may for example be pretreated, then hydrolysed and subsequently the resulting hydrolysate that comprises hexose and/or pentose sugar may be converted by microorganism, such as yeasts, into fermentation product.
  • microorganism such as yeasts
  • These processes may take place in a large scale Integrated Bioprocess Facility (IBF).
  • IBF Integrated Bioprocess Facility
  • the microbial fermentation with yeast is usually conducted under anaerobic conditions in the fermentation part of the IBF.
  • microorganism is propagated either in the IBF or elsewhere and shipped to the IBF. Propagation is usually conducted under aerobic conditions.
  • German patent 300662 there is known a process for the aerobic propagation of yeast wherein the propagation is started with broth that is strongly diluted and then undiluted broth is added slowly.
  • the part of the process of slow addition of broth is herein called fed-batch phase of the process.
  • the overall process including a fed-batch phase is herein called fed-batch process.
  • the advantage of the known fed-batch process is that excessive formation of ethanol is avoided and that larger broth concentrations than in diluted batch process can be used.
  • Toxicity of a lignocellulose hydrolysate or a lignocellulose hydrolysate fraction such as C5 liquid i.e. the liquid obtained during and often separated after acid pretreatement of lignocellulosic biomass, is a problem.
  • a toxic substrate such as C5 liquid can inactivate or kill the microorganism, so that fermentation is not possible or severely inhibit the microorganism so that the fermentation is impaired, which leads to an uneconomical process. Also toxicity more mildly may result in the inability for the microorganism to use pentose, e.g. xylose that without the toxic compound(s) would be converted by the microorganism.
  • An object of the invention is to provide a conversion process e.g. a propagation and/or fermentation process wherein pentose containing substrate is converted into product, e.g. microorganism biomass, such as yeast, or a fermentation product such as ethanol.
  • An object of the invention is to provide propagation and/or fermentation process wherein a toxic ligno-cellulosic hydrolysate, in particular toxic C5 liquid, may be fermented.
  • An object is to provide a propagation and/or fermentation process that has reduced fresh water use.
  • An object is to provide propagation and/or fermentation process that gives high product (e.g. ethanol) titer.
  • An object is to provide a propagation and/or fermentation process with a low loss of valuable ingredients from ligno-cellulosic hydrolysate.
  • a further object is provide a propagation and/or fermentation process that may be operated in a stable fashion.
  • Another object is to provide propagation and/or fermentation process that may be performed in multiple cycles, wherein part of the propagation and/or fermentation mixture may be used for the next round of propagation and/or fermentation.
  • a further object is to provide a fermentation process that avoids excess production of microorganism (e.g. yeast).
  • microorganism e.g. yeast
  • the invention provides a process for the conversion of pentose containing substrate in a reactor into fermentation product using a microorganism that can convert pentose, wherein the substrate is toxic to the microorganism, comprising the following steps:
  • the invention further relates to a process for producing a fermentation product which uses the microorganism according to the above conversion process, in particular where the fermentation product is ethanol.
  • Fig. 1 Anaerobic fermentation of toxic C5 liquid (C5 pCS liquid)
  • example 1 dilution factor 24% concentrations glucose (x), xylose (A ) and ethanol ( ⁇ ) against fermentation time (h);
  • Fig. 3 Determination of dilution factor. Anaerobic fermentations on diluted C5 pCS liquid (see example 1 ): Glucose consumption. Glucose concentration (g/L) at different dilution factors ( )10%, ( ⁇ ) 15%, (A ) 24%, ( ⁇ ) 34%, (o) 43% and ( ⁇ )52% against fermentation time (h).
  • Fig. 4 Determination of dilution factor. Anaerobic fermentations on diluted C5 pCS liquid (see example 1 ): Ethanol level. Ethanol concentration (g/L) at different dilution factors ( )10%, ( ⁇ ) 15%, (A ) 24%, ( ⁇ ) 34%, (o) 43% and ( ⁇ )52% against fermentation time (h).
  • Fig. 5 Determination of dilution factor.
  • Anaerobic fermentations on diluted C5 pCS liquid see example 1 ): Furfural consumption.
  • the dotted lines (24% and 52%) represent control experiments wherein no yeast was added.
  • Fig. 6 Determination of dilution factor.
  • Anaerobic fermentations on diluted C5 pCS liquid see example 1 ): Xylose consumption. Xylose concentration (g/L) at different dilution factors ( )10%, ( ⁇ ) 15%, (A )
  • . 7 Determination of dilution factor. Anaerobic fermentations on diluted C5 pCS liquid (see example 1 ): Acetic acid consumption. Acetic acid concentration (g/L) at different dilution factors ( )10%, ( ⁇ ) 15%, (A ) 24%, ( ⁇ ) 34%, (o) 43% and ( ⁇ )52% against fermentation time (h).
  • Fig. 8 Determination of dilution factor. Anaerobic fermentations on diluted C5 pCS liquid (see example 1 ): Hydroxymethylfurfural (HMF) consumption. HMF concentration (g/L) at different dilution factors
  • . 9 Biomass. Anaerobic fermentation on diluted C5 pCS liquid (see example 1 ): Biomass concentration (g/kg broth) at ( ⁇ ), start of fermentation ( ⁇ ) and yield (g/g sugar consumed) (A ) against dilution factor (C5 liquid % of total).
  • Fig. 10 Biomass. Aerobic fermentation on diluted C5 pCS liquid (see example 1 ): Biomass concentration (g/kg broth) at ( ⁇ ), start of fermentation ( ⁇ ) and yield (g/g sugar consumed) (A ) against dilution factor (C5 liquid % of total).
  • Fig. 12 Repeated batch aerobic propagation, dilution factor 24% C5 pCS liquid. Biomass concentration (g/kg broth) at end of fermentation ( ⁇ ), start of fermentation ( ⁇ ) and yield (g/g sugar consumed) (A ) against number of cycles.
  • Fig. 17 Repeated batch anaerobic fermentation, dilution factor 24% C5 pCS liquid. Acetic acid ( ⁇ ) (g/L supernatant) for cycles 1 -5 against total fermentation time (h)
  • the process for the conversion of pentose containing substrate in a reactor into product using a microorganism that can convert pentose, wherein the substrate is toxic to the microorganism comprising the following steps: a) contacting the substrate with the microorganism wherein the substrate is diluted and a broth is formed;
  • Efficient yeast biomass production on toxic C5 liquid all suagars present in toxic C5 liquid are used for biomass production or ethanol production;
  • propagator or fermentor may be used for the reactor in which growing of the organism may take place.
  • Propagation is herein aerobic or anaerobic fermentation with the aim to increase a microorganism population.
  • Fermentation is herein fermentation to produce a fermentation product.
  • the pentose containing substrate may be any pentose (e.g. xylose and/or L- arabinose) containing material that is toxic to the microorganism.
  • the pentose containing substrate may be lignocellulose or materials derived from lignocellulose.
  • the the pentose containing substrate is lignocellulosic hydrolysate, preferably a C5 liquid.
  • the pentose containing substrate is C5- liquid, such as C5 melasse.
  • the microorganism may be any microorganism that is able to convert pentose containing substrate to a useful product.
  • the microorganism may be a prokaryotic or eukaryotic organism.
  • the microorganism used in the process as may be (genetically engineered) microorganism. Genetic engineering is hereinafter described in more detail. Examples of suitable organisms are yeasts, for instance Saccharomyces, e.g. Saccharomyces cervisiae, Hansenula, Issatchenkia, e.g. Issatchenkia orientalis, Pichia, e.g.
  • Pichia stipitis or bacteria, for instance Lactobacillus, e.g., Lactobacillus lactis, Geobacillus, Zymomonas , e.g.Zymomonas mobilis, Clostridium, e.g. Clostridium, phytofermentans.
  • the microorganism is yeast.
  • Toxic herein means that the (undiluted) substrate can inactivate or kill the microorganism, so that fermentation is not possible or is severely inhibits the microorganism so that the fermentation is impaired, which leads to an uneconomical process. Also toxicity more mildly may result in the inability for the microorganism to use specific carbon sources, e.g. xylose, that without the toxic compound(s) would be converted by the microorganism.
  • Toxic compounds herein are also designated as inhibitory compounds or inhibitors. Inhibitors are common in pentose containing substrate. This may be caused by common origin of the pentose containing substrate: lignocellulose.
  • inhibitors in lignocellulose may vary widely with variation of feedstock, pretreatment method hydrolysis process.
  • categories of inhibitors are carboxylic acids, furans and/or phenolic compounds.
  • carboxylic acids are lactic acid, acetic acid or formic acid.
  • furans are furfural and hydroxy- methylfurfural.
  • Examples or phenolic compounds are vannilin, syringic acid, ferulic acid and coumaric acid.
  • the typical amounts of inhibitors are for carboxylic acids: several grams per liter, up to 20 grams per liter or more, depending on the feedstock, the pretreatment and the hydrolysis conditions.
  • the inhibitor the level of which is reduced is chosen from the group consisting of acetic acid, formic acid, furfural and hydroxymethyl furfural (HMF).ln an embodiment, one or more of following steps is conducted
  • Acidic lignocellulosic hydrolysate is common product from pretreatment wherein acid is used, which results in formation of acetic acid, HMF and furfural among others.
  • the lignocellulosic hydrolysate may be acidic.
  • the lignocellulosic hydrolysate comprises organic acid. Examples of organic acids possible in lignocellulosic hydrolysate are acetic acid and formic acid. In an embodiment the organic acid is acetic acid.
  • Steps a), b), c) and d) may be conducted in conventional way, though some parameters of these steps may be different then in the specific known conventional processes as described in more detail herein.
  • any suitable pentose containing carbon source may be used.
  • the substrate is diluted lignocellulosic hydrolysate, more specifically lignocellulosic hydrolysate that may be diluted in water or fermentation broth of a previous batch at an amount that does not exceed the predetermined maximum level.
  • step a) In step a) contacting the substrate with the microorganism wherein the substrate is diluted and a broth is formed; In an embodiment the diluting in step a) is conducted in the reactor.
  • the dilution may e.g. be accomplished according to the following embodiments:
  • a1 dilution of pentose containing substrate with water and putting mixture into reactor to form a broth. This is e.g. done by putting water in the reactor, adding the pentose containing substrate and then inoculating the microorganism on the resulting mixture in the reactor;
  • detoxified broth from an earlier cycle may be used to dilute the pentose containing substrate.
  • a3) dilution of the pentose containing substrate with liquid originating from a detoxified broth e.g. in fermentation of the pentose containing substrate, detoxified broth may be harvested and then separated into a liquid phase and a phase with solids. The liquid phase may be used in next cycle for dilution of the pentose containing substrate;
  • a4) dilution of pentose containing substrate with fermentation broth containing fermentation product e.g. ethanol
  • the addition of such broth may be either as a whole or as a liquid fraction of the broth,
  • the broth may optionally be diluted before addtion.
  • the broth is from a starch based ethanol production process.
  • Advantage of ethanol containing broth is that is has reduced toxicity and at the same time results in a higher erthanol concentration after fermentation, which makes recovery (e.g. distillation) of ethanol more efficient.
  • living yeast is present in the ethanol containing broth, that yeast will consume toxic compounds from the substrate (e.g. C5 liquid) and therefore additionally decrease the toxicity of the substrate,
  • starch and sucrose based spent fermentation broths may contain 4 - 12% ethanol. Any combinations of embodiments a1 )-a4) may be made and are included in the invention.
  • the diluting in step a) is conducted so that concentration of pentose containing substrate in the diluted substrate or broth is 54% or less. In an embodiment it is 43% or less, or 24% or less.
  • acetate e.g. 4 g/L as in the 24% diluted substrate may be converted.
  • WO201 1010923 an acetic acid converting C5 yeast strain is disclosed. With this strain the process may be made more robust in that more cycles of anaerobic fermentation may be possible.
  • step c) in step c): keeping at least part of detoxified broth in the reactor;
  • part of the fermentation broth may be used to isolate product from, and/or may be replaced by substrate.
  • product may be harvested from the detoxified broth, though part of the detoxified broth should be kept in the reactor to dilute pentose containing substrate in the next cycle. From the part taken from the reactor, product may be isolated.
  • step d) optionally repeating steps a), b) and c):
  • steps a), b) and c) conducting the repeating of steps a), b) and c) in 2-50 or more cycles.
  • the process is a propagation process and the product is microorganism biomass, which is the isolated product from the harvested broth in step c1 ).
  • the growing in step b) is advantageously conducted aerobically.Propagation processes are described in more detail herein below.
  • the proces is a fermentation process and the product is an aqueous mixture comprising fermentation product, which originates from separation of the harvested broth into a liquid fraction and a fraction comprising solids and the fermentation product is taken from the liquid fraction. Details of the fermentation process are described in more detail below.
  • the fermentation in step b) is conducted anaerobically.
  • a part of the anaerobic fermentation may be conducted aerobically, aimed at reducing the level of one or more inhibitors.
  • the fermentation process converts the pentose containing substrate into fermentation product.
  • the fermentation product is ethanol. Fermentation processes and fermentation products are described in more detail herein below.
  • Propagation is herein any process of microbial growth that leads to increase of an initial microorganism population. Fermentation is herein any process using microorganisms that leads to increase of product.
  • the process according to the invention may be executed in any known way. It may be conducted continuously, or in batch. In an embodiment, the process is conducted as a repeated batch process.
  • the method of the invention may be carried out in any suitable format. However, the method may conveniently be carried out using a repeated batch also called sequential batch reactor (SBR) protocol.
  • SBR sequential batch reactor
  • batch-wise grown cultures may be transferred sequentially to new batches.
  • cells may be cultivated in repeated batches by repeated, for example automated, replacement of the culture with fresh medium. Typically, at least about 50%, at least about 60%, at least about 70%, at least abaout 80% or at least about 90% of the culture is replaced with fresh medium.
  • the process is conducted in a single reactor. In the single reactor both propagation and fermentation can take place.
  • the concentration of toxic compounds in broth of step b) is reduced and the inhibitor is chosen from the list consisting of furfural, HMF and acetic acid. In an embodiment, the concentration of toxic compounds in broth of steps c) is decreased to substantially zero.
  • steps a) b) and c) are repeated until sufficient amount of microorganisms is obtained.
  • steps a) b) and c) are repeated until productivity of the microorganism declines and reaches levels which are unfavorable.
  • the harvested broths from step c) are collected and used for isolation of the microorganism. The isolated microorganism is than re-used in the propagation in steps a) b) and c) and/or fermentation step a), b) and c).
  • step b) the process time for growing the microorganism is designed to be enough for the microorganism to maximize multiplication, and to allow all or most of the sugars, acetic acid, HMF, furfural and/or other acidic inhibitors from the feed to be consumed, and at the same time short enough to prevent the production of excess ethanol.
  • propagation is conducted until at least five generations of growth of the microorganism population are realized. In an embodiment the propagation is conducted until growth of the microorganism population for 5 to 6 generations compared to the initial microorganism population. In an embodiment the batch phase of propagation is conducted until growth of the microorganism population for two generations and the fed batch phase for three or more generations.
  • a generation of growth herein means a doubling of microorganism biomass in weight
  • microorganisms may be isolated from the reactor as a whole broth, as part of the whole broth or as washed, concentrated and/or purified microbial biomass fed to a fermentation reactor.
  • part of the broth is harvested and replaced by substrate in an amount according to the predetermined maximum level for product formation. In an embodiment this is done in the same reactor as the propagation.
  • the microorganism may be isolated from the harvested part of the broth and recycled to the propagator or fermentation reactor.
  • the biomass growth rate can be measured by various means:
  • the increase of biomass amount can be analyzed by determining the amount of cells per weight or volume unit of a culture using any of the following method or a suitable alternative method: ⁇ Turbidity
  • CFU/ml Colony Forming Unit
  • the microorganism is a pentose fermenting industrial yeast.
  • An industrial yeast cell may be defined as follows. The living environments of yeast cells in industrial processes are significantly different from that in the laboratory. Industrial yeast cells must be able to perform well under multiple environmental conditions which may vary during the process. Such variations include change in nutrient sources, pH, ethanol concentration, temperature, oxygen concentration, etc., which together have potential impact on the cellular growth and ethanol production of Saccharomyces cerevisiae. Under adverse industrial conditions, the environmental tolerant strains should allow robust growth and production. Industrial yeast strains are generally more robust towards these changes in environmental conditions which may occur in the applications they are used, such as in the baking industry, brewing industry, wine making and the ethanol industry. Examples of industrial yeast (S.
  • Ethanol Red® Fermiol®
  • DSM Fermiol®
  • Thermosacc® Longmand
  • the yeast is inhibitor tolerant.
  • Inhibitor tolerant yeast cells may be selected by screening strains for growth on inhibitors containing materials, such as illustrated in Kadar et al, Appl. Biochem. Biotechnol. (2007), Vol. 136-140, page 847-858, wherein an inhibitor tolerant S. cerevisiae strain ATCC 26602 was selected.
  • RN1016 is a xylose and glucose fermenting S. cerevisiae strain from DSM, Bergen op Zoom, the Netherlands.
  • the yeast is capable of converting hexose (C6) sugars and pentose (C5) sugars.
  • the yeast can an-aerobically ferment at least one C6 sugar and at least one C5 sugar.
  • the yeast is capable of using L-arabinose and xylose in addition to glucose an-aerobically.
  • the yeast is capable of converting L-arabinose into L-ribulose and/or xylulose 5-phosphate and/or into a desired fermentation product, for example into ethanol.
  • Organisms for example S.
  • able to produce ethanol from L-arabinose may be produced by modifying a host yeast introducing the araA (L-arabinose isomerase), araB (L- ribuloglyoxalate) and araD (L-ribulose-5-P4-epimerase) genes from a suitable source. Such genes may be introduced into a host cell in order that it is capable of using arabinose. Such an approach is given is described in WO2003/095627. araA, araB and araD genes from Lactobacillus plantarum may be used and are disclosed in WO2008/041840.
  • araA L-arabinose isomerase
  • araB L- ribuloglyoxalate
  • araD L-ribulose-5-P4-epimerase
  • araA gene from Bacillus subtilis and the araB and araD genes from Escherichia coli may be used and are disclosed in EP1499708.
  • araA, araB and araD genes may derived from of at least one of the genus Clavibacter, Arthrobacter and/or Gramella, in particular one of Clavibacter michiganensis, Arthrobacter aurescens, and/or Gramella forsetii, as disclosed in WO 200901 1591 .
  • the yeast may also comprise one or more copies of xylose isomerase gene and/or one or more copies of xylose reductase and/or xylitol dehydrogenase.
  • the yeast may comprise one or more genetic modifications to allow the yeast to ferment xylose.
  • genetic modifications are introduction of one or more xylA-gene, XYL1 gene and XYL2 gene and/or X S7-gene; deletion of the aldose reductase (GRE3) gene; overexpression of PPP-genes TAL1, TKL1, RPE1 and RKI1 to allow the increase of the flux through the pentose phosphate pathway in the cell.
  • GRE3 aldose reductase
  • PPP-genes TAL1, TKL1, RPE1 and RKI1 examples of genetically engineered yeast are described in EP1468093 and/or WO2006009434.
  • RN1016 a xylose fermenting strain is available strain by DSM, Netherlands. This strain is used in the examples.
  • the fermentation product herein may be any useful product.
  • it is a product selected from the group consisting of ethanol, n-butanol, isobutanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, fumaric acid, malic acid, itaconic acid, maleic acid, citric acid, adipic acid, an amino acid, such as lysine, methionine, tryptophan, threonine, and aspartic acid, 1 ,3- propane-diol, ethylene, glycerol, a ⁇ -lactam antibiotic and a cephalosporin, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels, including biofuels and biogas or organic polymers, and an industrial enzyme, such as a protease, a cellulase, an amylase, a glucanase, a lactase, a
  • n-butanol may be produced by cells as described in WO2008121701 or WO2008086124; lactic acid as described in US201 1053231 or US2010137551 ; 3-hydroxy-propionic acid as described in WO2010010291 ; acrylic acid as described in WO2009153047.
  • Example 1 Determination of the dilution factor for C5-liquid fermentation under anaerobic conditions
  • lignocellulosic hydrolysate liquid from acid pretreated corn stover, obtained after centrifugation was used for production of ethanol, using a pentose fermenting yeast RN1016.
  • the composition of the C5 liquid hydrolysate is given in table 1.
  • Table 1 Composition of the C5 liquid from lignocellulosic hydrolysate (HPLC (H-column) analysis). The data show that C5 liquid contains fermentable sugar, but is very toxic.
  • the C5-liquid was obtained from acid pretreated Corn Stover feedstock (30-40% dry matter) by solid-liquid separation.
  • the acid pretreated feedstock was centrifuged for 20 minutes at 18000g.
  • the C5-liquid part was separated and filtered over a 0.2 mm filter to remove all residual solid particles.
  • the liquid was stored at 4-6°C.
  • the low pH of the liquid (pH 1 -1 .5) prevented microbiological contamination.
  • the use of a filter was necessary for proper biomass determination during fermentation.
  • the C5-liquid was heated to 33 ⁇ 1 °C and pH was adjusted to 5.5 ⁇ 0.05 with KOH. This will further on be called as undiluted C5 liquid (also as C5 pCS liquid).
  • Cream yeast solution was produced for inoculation of propagation and fermentation in the following way:
  • a yeast stock suspension containing 50% glycerol and stored at -80°C
  • YEPD-medium containing 10 g/l yeast extract, 20 g/l pepton, 15 g/l glucose, and incubated for 20-24 h at 30°C at 200 rpm in a shaker incubator.
  • the broth was stored at 4-6°C for at least 15 h with a maximum of 72 h storage time.
  • the yeast culture was than centrifuged for 10 minutes at 1600 g and the pellet was suspended in a similar volume of cold water and centrifuged. The amount of wet pellet was measured and suspended in a same amount of cold tap water and used as inoculum of fermentation immediately. This method resulted in cream yeast with 10 ⁇ 1 % dry matter content.
  • Determination of the dilution factor for C5-liquid fermentation under anaerobic and aerobic conditions Determination of dilution factor is done to determine the minimal dilution of the toxic undiluted C5 liquid at which fermentation takes place within 24 h.
  • Undiluted C5-liquid was mixed with tapwater, to obtain dilution levels ranging from 20- 90% C5-liquid, and heated to 33 ⁇ 1 °C prior to fermentation.
  • Cream yeast was added to each diluted C5 mixtures at a level of 1 g yeast dry matter/kg diluted C5-mixture.
  • the fermentation was performed in the Alcohol Fermentation Monitor unit (Halotec) at 33 ⁇ 1 °C and 150 rpm, using 200 g broth in 250 ml Schott bottles.
  • Cream yeast was added to each diluted C5 mixtures at a level of 1 g yeast dry matter/kg diluted C5-mixture.
  • the fermentation was performed with 200 g broth in 2000 mL shake flask with foam caps in a shaker incubator at 33 ⁇ 1 °C and 200 rpm During fermentation, samples were taken daily for sugar, ethanol and organic acid analysis by flow NMR. At start and at the end of fermentation, biomass concentration was determined by weighting a known amount of fermentation broth centrifuging it for 10 minutes at 3200g. The obtained pellet was washed three-times with demi-water. Pellets were dried for at least 3 days at 105°C, cooled and weighted.
  • the C5 liquid dilution factor (the fraction of undiluted C5 liquid in diluted C5 liquid) was determined based on the minimal dilution at which all pentose sugars present in the fermentation broth were consumed.
  • Ethanol production yield is >90% for C6 and C5
  • Biomass concentration at end of propagation phase is limited by dilution of C5 liquid
  • Propagation was done on diluted C5 liquid to produce yeast biomass.
  • Undiluted C5-liquid was mixed with tap water to obtain the target dilution level, resulting from "Determination of the dilution factor for C5-liquid fermentation under anaerobic and aerobic conditions".
  • Cream yeast was added to the diluted C5 mixture at a level of 1 g yeast dry matter/kg diluted C5-mixture.
  • the fermentation was performed in the Alcohol Fermentation Monitor unit (Halotec) at 33 ⁇ 1 °C and 150 rpm, using 200 g broth in 250 ml Schott bottles.
  • Cream yeast was added to the diluted C5 mixtures at a level of 1 g yeast dry matter/kg diluted C5-mixture.
  • the fermentation was performed with 200 g broth in 2000 mL shake flask with foam caps in a shaker incubator at 33 ⁇ 1 °C and 200 rpm
  • Fermentations are done to produce ethanol.
  • the undiluted C5-liquid was heated to 33 ⁇ 1 °C and pH was adjusted prior to start fermentation to 5.5 ⁇ 0.05 with KOH.
  • the undiluted C5-liquid was mixed with tapwater to obtain the target dilution level, resulting from "Determination of the dilution factor for C5-liquid fermentation under anaerobic and aerobic conditions".
  • Cream yeast was added to the diluted C5 mixture at a level of 1 g yeast dry matter/kg diluted C5-mixture.
  • the fermentation was performed in the Alcohol Fermentation Monitor unit (Halotec) at 33 ⁇ 1 °C and 150 rpm, using 200 g broth in 250 ml Schott bottles.
  • Cream yeast was added to the diluted C5 mixtures at a level of 1 g yeast dry matter/kg diluted C5-mixture.
  • the fermentation was performed with 200 g broth in 2000 mL shake flask with foam caps in a shaker incubator at 33 ⁇ 1 °C and 200 rpm
  • the replaced broths and the final broth were collected for distillation of ethanol.
  • yeast can be separated from only the part of the broth that is harvested after each 24h. In this case, not all biomass is recycled but only that part present in the harvested broth (Partial Recycling of Yeast).
  • the withdrawn broths and the final broth were collected using cycles of repeated batch fermentation on 25% diluted C5-liquid (containing 25% undiluted C5 liquid in the dilution), glucose is consumed within a couple of hours after start of the cycle until the fourth cycle [A].
  • Xylose is consumed, but the rate of consumption decreases with each cycle. Xylose consumption is zero in the fourth cycle [B].
  • Furfural [C] and HMF [D] are consumed completely until the fourth cycle.
  • ethanol reaches a concentration of 19 g/L [E], which is 168% of the ethanol level that is reached in batch fermentation on diluted C5 liquid (ethanol value end of the first cycle).
  • biomass increases [F] and assures the presence of sufficient amount of biomass even though part of the biomass is withdrawn at the end of each cycle.

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Abstract

La présente invention concerne un procédé pour convertir un substrat contenant du pentose en produit de fermentation dans un réacteur, au moyen d'un micro-organisme pouvant convertir le pentose, le substrat étant toxique pour le micro-organisme. Le procédé comprend les étapes consistant à : a) mettre le substrat en contact avec le micro-organisme, ce qui dilue le substrat et produit un bouillon ; b) éliminer ou réduire une ou plusieurs toxines du bouillon en faisant croître le micro-organisme jusqu'à obtenir un bouillon détoxifié ; c) laisser une partie au moins du bouillon détoxifié dans le réacteur ; et d) répéter éventuellement les étapes a), b) et c).
PCT/EP2014/070177 2013-09-24 2014-09-23 Procédé de conversion de substrat contenant du pentose WO2015044101A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
US10844363B2 (en) 2015-08-05 2020-11-24 Cargill, Incorporated Xylose isomerase-modified yeast strains and methods for bioproduct production

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10844363B2 (en) 2015-08-05 2020-11-24 Cargill, Incorporated Xylose isomerase-modified yeast strains and methods for bioproduct production

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