Field of the invention

The invention is directed to a process for conversion of pentose containing substrate. In particular 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.

Background of the invention

There are nowadays processes proposed to use lignocellulosic material as a source for the production of fuel and of base chemicals. They are aimed at commercially-viable production of these products from lignocellulosic feedstocks.

In such processes 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. These processes may take place in a large scale Integrated Bioprocess Facility (IBF). The microbial fermentation with yeast is usually conducted under anaerobic conditions in the fermentation part of the IBF.

To be able to supply enough microorganism to the fermentation, microorganism is propagated either in the IBF or elsewhere and shipped to the IBF. Propagation is usually conducted under aerobic conditions.

From 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.

From Kollaras, A. et al, Ethanol Producer Magazine, August 2012, page 52- 54 there is known aerobic propagation of yeast (S. cerevisiae) on xylose containing stillage and it is described that: "Within a submerged aerobic propagator similar to that in which baker's yeast is grown, S. cerevisiae MBG 3248 converted acetic acid, lactic acid, ethanol, glycerol, residual six carbon sugars and xylose into yeast biomass at an observed yield of 0.35g of yeast per gram of total usable carbon". The xylose converted in feed yeast can better be used to produce fermentation product in the IBF. Disadvantage to all currently known conversion process on acidic lignocellulosic hydrolysate is that yeast growth is inhibited by acetic acid and/or sugar degradation products, such as furfural and HMF. Nevertheless such hydrolysate may be present and available from the IBF, and is cheaper than conventional propagation carbon sources, and thus would be a desirable carbon source.

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.

Various detoxification methods have been tried. Examples are such as water washing, over-liming, vaporization and ion exchange absorption. However, these methods resulted in many negative outcomes, including massive freshwater usage and wastewater generation, loss of the fine lignocellulose particles and fermentative sugars and incomplete removal of inhibitors. Among the most frequently used methods, water washing, considerable pretreated lignocellulose solids (sugars) were lost during the washing and liquid-solid separation step, thus leading to the loss of fermentation product (e.g. ethanol) of at least the same percentage. Furthermore, the considerable amount of water used led to high cost of the downstream wastewater treatment. Finally, the high water content in the pretreated feedstock led to the low ethanol titer in the consequent fermentation and then the high energy cost in distillation.

Summary of the invention

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). One or more of these objects are attained according to the invention.

Accordingly 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:

a) contacting the substrate with the microorganism wherein the substrate is diluted and a broth is formed;

b) removing or reducing one or more toxins from the broth by growing the microorganism until a detoxified broth is obtained;

c) keeping at least part of detoxified broth in the reactor; and

d) optionally repeating steps a), b) and c).

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.

Brief description of the drawings

Fig. 1 : Anaerobic fermentation of toxic C5 liquid (C5 pCS liquid)

(example 1 ) at dilution factor 54%, concentrations (g/L) glucose (x), xylose (A ) and ethanol (■) against fermentation time (h);

Fig 2: 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. Furfural concentration (g/L) at different dilution factors ( )10%, (■) 15%, (A ) 24%, (□) 34%, (o) 43% and (·)52% against fermentation time (h). 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 )

24%, (□) 34%, (o) 43% and (·)52% against fermentation time (h).

. 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

(♦)10%, (■) 15%, (A ) 24%, (□) 34%, (o) 43% and (·)52% against fermentation time (h). The dotted lines (24% and 52%) represent control experiments wherein no yeast was added.

. 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).

1 1 : Schematic overview of repeated batch fermentation for propagation (biomass formation) and fermentation (ethanol production).

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.

13: Repeated batch anaerobic fermentation, dilution factor 24% C5 pCS liquid. Glucose (♦) (g/L supernatant) for cycles 1 -5 against total fermentation time (h)

14: Repeated batch anaerobic fermentation, dilution factor 24% C5 pCS liquid. Ethanol (♦) (g/L supernatant) for cycles 1 -5 against total fermentation time (h)

15: Repeated batch anaerobic fermentation, dilution factor 24% C5 pCS liquid. Furfural (♦) (g/L supernatant) for cycles 1 -5 against total fermentation time (h) Fig. 16: Repeated batch anaerobic fermentation, dilution factor 24% C5 pCS liquid. Xylose (♦) (g/L supernatant) for cycles 1 -5 against total fermentation time (h)

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)

18: Repeated batch anaerobic fermentation, dilution factor 24% C5 pCS liquid. HMF( ) (g/L supernatant) for cycles 1 -5 against total fermentation time (h) Detailed description of the invention

Throughout the present specification and the accompanying claims the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

According to the invention, 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;

b) removing or reducing one or more toxins from the broth by growing the microorganism until a detoxified broth is obtained;

c) keeping at least part of detoxified broth in the reactor; and

d) optionally repeating steps a), b) and c).

Advantages of the invention include:

• Efficient yeast biomass production on toxic C5 liquid: all suagars present in toxic C5 liquid are used for biomass production or ethanol production;

Yeast biomass adaptation to toxic C5 liquid during propagation fermentation;

• Omitting lag-phases during propagation and fermentation;

• Ethanol fermentation on toxic C5 liquid with yeast biomass already adapted to the toxic liquid during propagation or during previous fermentation result in absence of lag phase or adaptation phase;

• Short fermentation times on toxic C5 liquid;

• High ethanol conversion (High Yps) on toxic C5 liquid in ethanol fermentation

Reduced chance of contamination due to:

o Short fermentation times

Short periods of C6-sugar availability

Toxicity of broth

Possibility of low pH propagation and fermentation Herein reactor, 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. In an embodiment, the the pentose containing substrate is lignocellulosic hydrolysate, preferably a C5 liquid. In an embodiment, 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. In an embodiment, 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. The presence and level of inhibitory compounds in lignocellulose may vary widely with variation of feedstock, pretreatment method hydrolysis process. Examples of categories of inhibitors are carboxylic acids, furans and/or phenolic compounds. Examples of carboxylic acids are lactic acid, acetic acid or formic acid. Examples of 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. For furans: several hundreds of milligrams per liter up to several grams per liter, depending on the feedstock, the pretreatment and the hydrolysis conditions. For phenolics: several tens of milligrams per liter, up to a gram per liter, depending on the feedstock, the pretreatment and the hydrolysis conditions. In an embodiment, in the process according to the invention 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. In an embodiment, 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. In step a) any suitable pentose containing carbon source may be used. In an embodiment, in step a), 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.

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;

a2) dilution of pentose containing substrate with detoxified broth, optionally from an earlier cycle to form a broth; Advantegeous the process is repeated, i.e. conducted in cycles, e.g. 2-50 or more cycles. In such embodiment, 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. In an embodiment, 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. In an embodiment 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. Moreover, if 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,

Efficiency of distillation is discussed in Wei-Dong Huang et al, "Analysis of biofuels production from sugar based on three criteria: Thermodynamics, bioenergetics, and product separation", Energy Environ. Sci., 201 1 , 4, 784. One embodiment of a4) is illustrated in more detail now hereunder.

In case a C5 liquid is fermented, containing 45 g/kg xylose and 15 g/kg glucose, which is non-fermentable as such, one might expect up to 35 g/kg ethanol (= 3.5% w/w) as theoretical maximal ethanol yield. If dilution is required to 70% to lower the toxicity and to make the C5 fermentable, the theoretical maximal ethanol concentration will be 70% ^* 35.2 = 24.7 g/kg. (2.5% w/w) which increases distillation costs. By using ethanol containing fermentation broth for dilution, containing at least 3.5 % w/w ethanol, making maximal conversion of sugars in C5 liquid possible, the final theoretical maximal ethanol titer will be least 3.5%w/w.

Calculation: 70% ^* 3.5% + 30% ^* 3.5% = 3.5 %w/w. If ethanol containing fermentation broth of higher ethanol concentrations are used, the final ethanol concentration will increase accordingly, and hence the distillation costs will decrease.

According to Wei-Dong Huang et al, 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.

In an embodiment, 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.

b) In step b): removing or reducing one or more toxins from the broth by growing the microorganism until a detoxified broth is obtained;

It is shown in the examples that when pentose containing substrate is diluted the microorganism (C5 yeast) is able to convert furfural and HMF during fermentation. By growing the yeast removed furfural and HMF from the broth giving a detoxified broth. However it is clear from the examples the acetic acid is not converted, This is disdavantageous when more than three cycles of repeated batch are conducted. To avoid the build up of acetic acid the following embodiments b1 ) and b2) may provide a solution, since it is possible to convert the acetic acid.

b1 ) using aerobic fermentation; In aerobic propagation and fermentation, low levels of acetate , e.g. 4 g/L as in the 24% diluted substrate may be converted.

b2) using as the microorganism a microorganism that is capable of converting acetic acid; In 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.

c) in step c): keeping at least part of detoxified broth in the reactor;

c1 ) harvesting part of the detoxified broth and isolating product therefrom; In a fermentation process, part of the fermentation broth may be used to isolate product from, and/or may be replaced by substrate. In this step 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.

In a propagation process that will be yeast biomass. In a fermentation process, fermentation product, e.g. ethanol. Advantegeously the broth is subjected to S/L separation, in an ethano process yeast may than be isolated as solid and ethanol may be distilled from the liquid phase. d) in step d) optionally repeating steps a), b) and c):

There are two advantageous embodiments here:

d1 ) conducting the steps a) b) and c) as a repeated batch reactor process; All steps a), b) and c) can advantageously be conducted in a batch reactor, this may even be the same batch reactor for propagation and fermentation. Such process is schematically illustrated in figure 1 1. Product concentration may be higher in repeated batch reactor process, In such a method, batch-wise grown cultures may be transferred sequentially to new batches.

d2) conducting the repeating of steps a), b) and c) in 2-50 or more cycles.

It is possible to repeat steps a) b) and c).

In an embodiment, the process is a propagation process and the product is microorganism biomass, which is the isolated product from the harvested broth in step c1 ). In that embodiment the growing in step b) is advantageously conducted aerobically.Propagation processes are described in more detail herein below.

In another embodiment, 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. In an embodiment, the fermentation in step b) is conducted anaerobically. Advantageously a part of the anaerobic fermentation may be conducted aerobically, aimed at reducing the level of one or more inhibitors. In an embodiment, the fermentation process converts the pentose containing substrate into fermentation product. In an embodiment, 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. In such a method, batch-wise grown cultures may be transferred sequentially to new batches. In such a method 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. In an embodiment the process is conducted in a single reactor. In the single reactor both propagation and fermentation can take place.

In an embodiment 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.

For propagation, in an embodiment steps a) b) and c) are repeated until sufficient amount of microorganisms is obtained. In fermentation in an embodiment, steps a) b) and c) are repeated until productivity of the microorganism declines and reaches levels which are unfavorable. In an embodiment 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).

In a propagation process, in 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.

In an embodiment, 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

(g)-

In step c) after sufficient propagation 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. In an embodiment 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.

Exponential growth in batch cultures The definition of a generation here is a doubling of microorganism biomass. The doubling of the amount of biomass can be described by Cx (biomass concentration) at given time to be given by the following equation:

Cx(t) = Cx(0) ^* e ^t) (eq. 1 )

The doubling time (Td in h) or generation time (Tg h) can be derived from the is equation by substituting Cx(t) = 2 ^* Cx(0). Td = Ι_Ν(2)/μ (hr) (eq. 2)

Where μ = specific growth rate in g biomass/g biomass/h or 1/h).

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

• Optical Density in the visible light spectrum (usual range: 600 nm to 700 nm) of a culture

• A pellet volume after centrifugation,

• The dry weight content after drying at constant weight at 105 °C · Cell count per volume (microscopically),

• Colony Forming Unit (CFU/ml) after plating on a solid agar medium and growing colonies on a plate from single cells

In an embodiment 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. cerevisiae) are Ethanol Red® (Fermentis) Fermiol® (DSM) and Thermosacc® (Lallemand). These commercial strains have to be genetically engineered for pentose fermentation as described herein below.

In an embodiment 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. For example the yeast is capable of using L-arabinose and xylose in addition to glucose an-aerobically. In an embodiment, 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. cerevisiae strains, 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. The araA gene from Bacillus subtilis and the araB and araD genes from Escherichia coli may be used and are disclosed in EP1499708. In another embodiment, 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 . In an embodiment, 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. Examples of 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. 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. In one embodiment, 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 β-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 lipase, a lyase, an oxidoreductases, a transferase or a xylanase. 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.

For the recovery of the fermentation product in the Integrated Bioprocess Facility existing technologies are used. For different fermentation products different recovery processes are appropriate. Existing methods of recovering ethanol from aqueous mixtures commonly use fractionation and adsorption techniques. For example, a beer still can be used to process a fermented product, which contains ethanol in an aqueous mixture, to produce an enriched ethanol-containing mixture that is then subjected to fractionation (e.g., fractional distillation or other like techniques). Next, the fractions containing the highest concentrations of ethanol can be passed through an adsorber to remove most, if not all, of the remaining water from the ethanol.

The following Examples illustrate the invention. Examples

Example 1 : Determination of the dilution factor for C5-liquid fermentation under anaerobic conditions In example 1 , as 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.

Production of C5-liquid from acid pretreated Corn Stover

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. Prior to use, 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).

Production of Cream yeast Cream yeast solution was produced for inoculation of propagation and fermentation in the following way:

0.5 ml of a yeast stock suspension, containing 50% glycerol and stored at -80°C, was added to 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. Subsequently, 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.

0.1 g KH2P04/kg broth and 0.2 g (NH4)2S04/kg broth were added to each diluted C5-mixtures. The pH was checked and adjusted to 5.5±0.05 with KOH (if necessary). The pH was not controlled during fermentation.

Start of fermentation under anaerobic conditions;

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.

Start of fermentation under aerobic conditions;

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.

After 24 h, fermentations were stopped, and 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.

Results Figure 1 shows that in anaerobic fermentation at dilution factor X=54% (X is the ratio of C5 liquid in diluted C5 liquid in %) the pentose xylose is hardly consumed. The substrate at this concentration is toxic for the pentose fermenting yeast RN1016. However figure 2 shows that at X=24, xylose is converted within 24h. Dilution allows the anaerobic fermentation of the toxic substrate. To find the optimal ratio, the C5 liquid was diluted at several dilution factors (10, 15, 24, 34, 43 and 52% ration of C5 liquid in diluted C5 liquid). The results of the dilution test are given in figures 3 to 8.

It is clear from figure 3, that glucose is consumed in 24 h at X<52%. Figure 4 shows that ethanol level at maximum level in 24h at X<24%. It is clear from figure 3, that glucose is consumed in 24 h at X<52%. Figure 4 shows that ethanol level at maximum level in 24h at X<24%. It is clear from figure 5, that furfural is consumed in 24 h at X<52%. Figure 6 shows that xylose is consumed in 24h at X<24%. It is clear from figure 7, that there is no change in acetic acid concentration. Figure 8 shows that that HMF is consumed in 24h at X<43%. Figure 9 shows that in anaerobic fermentation the biomass increases in 48 h at X<34%. Figure 10 shows that in aerobic fermentation the biomass increases in 48 h at X<43%

The dilution of C5 liquid at which xylose is completely consumed in 24 h is chosen as the dilution factor of choice for this C5 liquid. At 24% C5 pCS liquid we observed:

Conversion of 4/L glucose within 10 h Conversion of 20 g/L xylose within 24 h

Complete consumption of toxic compounds HMF (0.12 g/L) and furfural (0.6 g/L) within 10 h

Biomass increase under anaerobic (+80 % biomass) and aerobic (+650 % biomass) conditions

Ethanol production yield is >90% for C6 and C5

But: High-dilutions of C5-liquid result in

Low ethanol concentrations end of fermentation (EoF) -> high distillation costs

Biomass concentration at end of propagation phase is limited by dilution of C5 liquid

Large capital expenditure (Capex) due to diluted streams

In examples 3 and 4 repeated batch experiments are shown, with repeated batch process according to the invention, these disadvantages may be overcome. A schematic overview of repeated batch fermentation for propagation (biomass formation) and fermentation (ethanol production) is given in figure 1 1.

Example 2: Propagation on 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".

0.1 g KH2P04/kg broth and 0.2 g (NH4)2S04/kg broth were added to the diluted C5- mixture. The pH was checked and adjusted to 5.5±0.05 with KOH (if necessary). The pH was not controlled during fermentation.

Start of propagation under anaerobic 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.

Start of propagation under aerobic conditions; 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

During propagation samples were taken daily for sugar, ethanol and organic acid analysis by flow NMR. At start and end of propagation, biomass concentration was determined as described before.

After 24 h, undiluted C5 liquid was added, at a ratio according to the dilution factor previously determined. 0.1 g KH2P04/kg undiluted C5-liquid and 0.2 g (NH4)2S04/kg undiluted C5-liquid were added to the fermentation broth. Propagation continued for another 24 h under the same conditions as described above.

During fermentation, samples were taken for sugar, ethanol and organic acid analysis by flow NMR. At start and end of each fermentation step, biomass concentration was determined as described before.

The addition of undiluted C5 liquid was repeated until sufficient amount of yeast biomass was produced. Figure 12 shows results of the repeated batch propagation. It is concluded that in three cycles more than 12 g biomass per L may be obtained.

Example 3: Repeated batch fermentation without yeast recycling

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".

0.1 g KH2P04/kg broth and 0.2 g (NH4)2S04/kg broth were added to the diluted C5- mixture. The pH was checked and adjusted to 5.5±0.05 with KOH (if necessary). The pH was not controlled during fermentation.

Start of fermentation under anaerobic 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.

Start of fermentation under aerobic conditions;

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

During fermentation samples were taken daily for sugar, ethanol and organic acid analysis by flow NMR. At start and end of fermentation, biomass concentration was determined as described before. After 24 h, part of the anaerobic and part of the aerobic fermentation broth were removed and replaced by undiluted C5 liquid, according to the dilution factor previously determined.

0.1 g KH2P04/kg undiluted C5-liquid and 0.2 g (NH4)2S04/kg undiluted C5-liquid were added to the fermentation broth. Fermentations continued for another 24 h under the same conditions as described above. During fermentation, samples were taken daily for sugar, ethanol and organic acid analysis by flow NMR. At start and end of fermentation biomass concentration was determined as previously described.

After another 24 h, part of the fermentation broth was replaced by undiluted C5 liquid as described above and fermentation continued under the same conditions. This replacement was repeated every 24 h until sugar consumption and ethanol production decreased significantly.

The replaced broths and the final broth were collected for distillation of ethanol.

The results of the repeated batch fermentation without yeast recycling are given in figures 13 to 18.

It is clear from figure 13, that glucose is consumed in less than 4 cycles. Figure 14 shows that ethanol level is at maximum in cycle 3. It is clear from figure 15, that furfural is consumed in less than 4 cycles. Figure 16 shows that xylose is consumed in less than 3 cycles. It is clear from figure 17, that there the acetic acid concentration increases. Figure 18 shows that that HMF is consumed in less than 4 cycles.

Example 4: Repeated batch fermentation with yeast recycling

During fermentation with yeast recycling, similar conditions were used as during "Repeated batch fermentation without yeast recycling" described above, except that after each 24 h fermentation, broths were centrifuged for 15 minutes at 6000 g and the yeast biomass was separated from the supernatant and stored at room temperature for later use. Part of the supernatant was than replaced by undiluted C5 liquid of pH 5.5 according the dilution factor, and 0.1 g KH2P04/kg undiluted C5- liquid and 0.2 g (NH4)2S04/kg undiluted C5-liquid were added. Fermentations were continued under conditions described above by adding the biomass obtained by centrifugation.

This replacement was repeated every 24 h until sugar consumption and ethanol production decreased significantly.

Instead of separating all yeast biomass from the complete fermentation broth every 24 h, 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. During the first three cycles, 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). During the first two cycles, 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.

The graphs show that with the repeated batch fermentation method, ethanol levels after two cycles are much higher than after the first cycle which is a batch fermentation of diluted C5-liquid. So, the benefit of repeated batch is a cheaper distillation as a result of higher ethanol concentrations at the end of fermentation.

Also, by using the broth of a previous cycle as diluting medium, water usage is reduced.