WO2010066743A2

WO2010066743A2 - Fermentation of isomaltulose

Info

Publication number: WO2010066743A2
Application number: PCT/EP2009/066643
Authority: WO
Inventors: Bianca Gielesen; Paul Klaassen; De Wilhelmus Theodorus Antonius Maria Laat
Original assignee: Dsm Ip Assets B.V.
Priority date: 2008-12-11
Filing date: 2009-12-08
Publication date: 2010-06-17
Also published as: WO2010066743A3

Abstract

The present invention relates to a process for the fermentation of isomaltulose. wherein isomaltulose obtained from plant material is contacted with cells of Saccharomyces. Preferably, the Saccharomyces is Saccharomyces cerevisiae. According to a preferred embodiment the specific growth rate on isomaltulose is 0.25 h^-1 or more under anaerobic conditions. Preferably the fermentation has a yield of ethanol of 0.3 g ethanol/g sugar or more.

Description

FERMENTATION OF ISOMALTULOSE

Field of the invention

The present invention relates to a process for the fermentation of isomaltulose, in particular as part of a process for the production of a fermentation product, such as bioethanol, from plant biomass.

Background of the invention

Biofuel ethanol production generally starts with plant biomass as starting material, herein designated as plant material. Depending on the type of plant material different conversion technology needs to be used.

In the short term, the production of bioethanol as a liquid transport fuel is largely dependent on starch and sugars from existing food crops. The sustainability of this industry would be enhanced by increases in the yield of starch/sugar per hectare without further inputs into the crops concerned.

There is an interest in the improvement of starch and sugar crops and also in increased biomass production in the starch and sugar crops.

Recent results from research on sugar cane suggest that total sugar content can be greatly increased by conversion of sucrose into a non-metabolizable isomer, isomaltulose. It was recently reported that vacuolar targeting of a highly efficient sucrose isomerase, which converts vacuolar sucrose to isomaltulose (also called palatinose), by enzymatic rearrangement of the glycosidic linkage from a (1 ,2)- fructoside in sucrose to a (1 ,6)-fructoside. This allowed accumulation of 0.5M isomaltulose in sugarcane stems without a reduction in sucrose concentration, resulting in a doubling of the total sugar concentration in juice from selected transgenic lines relative to their elite parent cultivar [1]. "The only reported enzymes the author could find that could degrade isomaltulose were mammalian, and they degraded isomaltulose slowly compared to sucrose. This suggests that either more appropriate enzymes/genes be found, or gene shuffling is required to increase the rate of metabolism. Such genes will have to be transformed into the organisms used to convert sugars to bioethanol or biobutanol" [2]. The degradation of isomaltulose by several yeasts has been disclosed in 1960 by Emeis and Windisch [4].

Summary of the invention Now therefore it is an object to provide a process for the conversion of isomaltulose. Another object is to provide such a process that has a high activity towards conversion of isomaltulose and a high yield of fermentation product. Another object is to provide simultaneously conversion of isomaltulose and production of useful products such as amino acids, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels, or other organic polymers, lactic acid, and alcohols such as ethanol, including fuel ethanol, 1-butanol, 2,-butanol, isobutanol and isoamylalcohol. Another object is to provide a process that allows conversion of isomaltulose as a sole carbohydrate source. Further object is to provide a process for the conversion of isomaltulose using a microorganism that is a non genetically modified (non-GMO).

One or more of these objects are attained according to the invention that provides a process for the fermentation of isomaltulose wherein isomaltulose obtained from plant material is contacted with cells of Saccharomyces.

Preferably, the Saccharomyces is Saccharomyces cerevisiae. According to a preferred embodiment the specific growth rate on isomaltulose is 0.25 h^"1 or more under anaerobic conditions. Preferably the fermentation has a yield of ethanol of 0.3 g ethanol/g sugar or more.

Brief description of the figures Figure 1 : Growth (Absorption (OD) at 600 nm) in time (h) of strains in medium according to example 1 : a) CEN PK 1 13-7D in sucrose medium, b) CEN PK 1 13-7D in sucrose/isomaltulose medium, c) CEN PK 1 13-7D in isomaltulose medium.

Figure 2: Growth (Absorption (OD) at 600 nm) in time (h) of strains in medium of sucrose/isomaltulose according to example 2. Strains BIE101 , CENPK1 13-7D, BIE105, and BIE119. Anaerobic fermentation (with waterlock). Figure 3: Growth (Absorption (OD) at 600 nm) in time (h) of strains in medium of isomaltulose according to example 2. Strains BIE101 , CENPK1 13-7D, BIE105, and BIE119.

Figure 4: Isomaltulose left in medium (%) as function of time (h) in fermentation with strains in medium of sucrose/isomaltulose according to example 2. Strains BIE101 , CENPK1 13-7D, BIE105, and BIE119. Anaerobic fermentation (with waterlock).

Figure 5: Isomaltulose left in medium (%) as function of time (h) in fermentation with strains in medium of sucrose/isomaltulose according to example 2. Strains BIE101 , CENPK1 13-7D, BIE105, and BIE119. Anaerobic fermentation (with waterlock).

Figure 6: Ethanol production (g/l) as function of time (h) in fermentation with strains in medium of sucrose/isomaltulose according to example 2. Strains BIE101 , CENPK1 13-7D, BIE105, and BIE119. Anaerobic fermentation (with waterlock).

Figure 7: Ethanol production (g/l) as function of time (h) in fermentation with strains in medium of isomaltulose according to example 2. Strains BIE101 , CENPK1 13- 7D, BIE105, and BIE1 19. Anaerobic fermentation (with waterlock).

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.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

Isomaltulose

Isomaltulose (chemical name: 6-0-α-D-glucopyranosyl-D-fructose), also known by the trade names Palatinose™ by Suedzucker, Germany and NRGylose or Xtend™ -A-

Isomaltulose, is a disaccharide that is natural constituent of honey and sugar cane and has a very natural sweet taste. It has been used as a sugar in Japan since 1985. It is particularly suitable as a non-cariogenic sucrose replacement and is favorable in products for diabetics and prediabetic dispositions. In contrast to sucrose, isomaltulose is a sugar that does not cause tooth decay.

Plant material

The isomaltulose used according to the invention may be obtained from any suitable plant material comprising isomaltulose in conventional way, e.g. by extraction. In particular, isomaltulose may be derived from a plant which is capable of accumulating isomaltulose. The plant may be available naturally or be the result of plant breeding, or a genetically modified plant.

Such a plant may be capable of synthesizing and/or accumulating sucrose and capable of expressing an enzyme which catalyses the conversion of sucrose into isomaltulose. Such plants are described in detail in WO2004/099403 and

US2007240240. In such an approach, a plant may be genetically modified so as to appropriately express a sucrose isomerase which may allow the accumulation of an increased amount of total sugar as compared to a corresponding plant which has not been genetically modified in this way. In particular, the increase in total sugar content may be due to the accumulation of isomaltulose and/or trehalulose. The sucrose content may remain about the same as a corresponding plant which has not been genetically modified so as to express the sucrose isomerase.

Expression of the sucrose isomerase may be tailored for vacuolar compartmentalisation. A plant according to the invention may be any plant which can accumulate an increased amount of total sugar, a proportion of which is isomaltulose, when a sucrose isomerase is expressed in the plant. The plant may be a sugar cane plant or sugar palm or a beet plant, for example a sugar beet plant.

The plant material according to the invention is thus an isomaltulose containing material from vegetable origin, preferably a sugar and/or starch containing material. The carbohydrates include, but are not limited to starch, cellulose, beta-glucan, arabinoxylan, mannan, pectin, arabinan and xyloglucan. In one embodiment, the plant material is selected from the group consisting of sugar containing tubers, roots, leaf material, and sugar cane; and any combinations of the forgoing. In a most preferred embodiment, the plant material is, or is derived from, sugar cane or sugar beet. The plant material may e.g. be selected from the groups consisting of or any combination of the foregoing.

The plant material may also consist of or comprise a side stream from starch processing-e.g. C6 carbohydrate containing process streams that are not suited for production of syrups. In other embodiments, the plant material does not consist of or comprise a side stream from starch processing. As set out above, the isomaltulose is derived from a plant material. Isomaltulose, optionally together with other sugars, like sucrose, may be extracted from the plant material, for example, by crushing the plant material or by diffusion from the plant tissues into water or another suitable solvent. The whole or part of the soluble carbohydrates, including sucrose and/or isomaltulose may thus be recovered. Methods for fermentation using plant-derived carbohydrate feedstocks are well know to those skilled in the art with established processes for various fermentation products (see for example Vogel et al., 1996, Fermentation and Biochemical Engineering Handbook: Principles, Process Design and Equipment, Noyes Publications, Park Ridge NJ, USA and references cited therein). The processes described above are described in more detail below.

Process Steps

The main process steps of a plant material based production process according to the invention of which a fermentation of the present invention is part, may in one embodiment be described as separated into the following main process stages: Pretreatment (optional), Saccharification (optional), Fermentation and Distillation.

The individual process steps of a process according to the invention, for example an ethanol production process, may be performed batchwise, as a fed batch or as a continuous flow. For the invention, processes where all process steps are performed batch wise, or processes where all process steps are performed as a continuous flow, or processes where one or more process step(s) is(are) performed batch wise and one or more process step(s) is(are) performed as a continuous flow, are equally contemplated. The cascade process is an example of a process where one or more process step(s) is(are) performed as a continuous flow and as such contemplated for the invention. For further information on the cascade process and other ethanol processes consult: The Alcohol Textbook, Ethanol production by fermentation and distillation, Eds. T. P. Lyons, D. R. Kesall and J. E. Murtagh, Nottingham University Press 1995. The steps are illustrated below in more detail.

Pretreatment

For plant material that contains in addition to sugar also starch, such as for instance sweet sorghum or sweet corn, pretreatment and subsequent saccharification may be needed or beneficial, to release more sugar from the plant material. Thus, in such preferred embodiment of the process of the invention the process comprises as a first step a step of pretreatment of the plant material. Pretreatment may comprise a mechanical step, e.g. milling of the plant material, preferably dry milling, e.g. by hammer or roller mils. Grinding is also understood as milling.

The plant material may be milled in order to open up the structure and allowing for further processing. Two processes of milling are normally used in alcohol production: wet and dry milling. The term "dry milling" denotes milling of grains, like corn grains. In dry milling the whole kernel is milled and used in the remaining part of the process. Wet milling gives a good separation of germ and meal (starch granules and protein) and is with a few exceptions applied at locations where there is a parallel production of syrups.

As part of the pretreatment, there may be a hydrolysation or liquefaction process the plant material is broken down (hydrolyzed) into maltodextrins (dextrins). In a preferred embodiment, in the primary liquefaction process of the invention the plant material, preferably in the form of milled sugar cane plant material, is hydrolyzed to a DE (an abbreviation for dextrose equivalent) higher than 4. DE stands for "Dextrose equivalents" and is a measure of reducing ends on C6 carbohydrates. Pure glucose has DE of 100. Glucose (also called dextrose) is a reducing sugar.

Whenever an amylase hydrolyzes a glucose-glucose bond in starch, two new glucose end-groups are exposed. At least one of these can act as a reducing sugar. Therefore the degree of hydrolysis can be measured as an increase in reducing sugars. The value obtained is compared to a standard curve based on pure glucose-hence the term dextrose equivalent. The DE may, e.g., be measured using Fehlings liquid by forming a copper complex with the starch using pure glucose as a reference, which subsequently is quantified through iodometric titration. In other words: DE (dextrose equivalent is defined as the amount of reducing carbohydrate (measured as dextrose- equivalents) in a sample expressed as w/w% of the total amount of dissolved dry matter. It may also be measured by the neocuproine assay (Dygert, Li Floridana(1965) Anal. Biochem. No 368). The principle of the neocuproine assay is that CuSO4 is added to the sample, Cu<2+> is reduced by the reducing sugar and the formed neocuproine complex is measured at 450 nm.

The hydrolysis may be carried out by acid treatment or enzymatically. The liquefaction is preferably carried out by enzymatic treatment, preferably an alpha- amylase treatment. In one embodiment, the liquefaction is carried out by preparing a slurry comprising milled plant material, preferably milled sugar cane , and water, heating the slurry to between 60-95[deg.] C, preferably 80-85[deg.] C, and the enzyme(s) is (are) added to initiate liquefaction (thinning). This is also termed the "primary liquefaction", i.e. it occurs before the process step of jet-cooking. The liquefaction in the process of the invention is performed at any conditions (pH, temperature and time) found suitable for the liquefying enzyme used. Within the scope is a process of the invention, wherein a liquefaction step is performed at 60-95[deg.] C. for 10-120 min, preferably at 75-90[deg.] C. for 15-40 min. In one embodiment, the liquefaction step is performed at a pH in the range of about pH 4-7, preferably pH about 4.5-6.5. The pH of the slurry may be adjusted or not, depending on the properties of the enzyme(s) used.

Pretreatment as described above may not be needed for sugar containing plant material that does not contain appreciated amounts of starch, such as sugar beet, sugar cane and sugar palm. Sugar from the sugar palm may be tapped as a liquid solution from the stem of the tree, once a cut has been made in the stem.

Sugar cane may be processed to release sugar as follows: It may be washed, chopped, and shredded by revolving knives. The feedstock may be fed to and extracted by a set of mill combinations to collect a juice, called garapa in Brazil, that contain 10-15 percent sucrose, and bagasse, a fiber residue. The main objective of the milling process is to extract the largest possible amount of sugar from the cane, and a secondary but important objective is the production of bagasse with a low moisture content as boiler fuel, as bagasse may be burned for electricity generation, allowing the plant to be self-sufficient in energy and to generate electricity for the local power grid. The cane juice or garapa is then filtered and treated by chemicals and pasteurized. Before evaporation, the juice is filtered once again, producing vinasse, a fluid rich in organic compounds. The syrup resulting from evaporation is then precipitated by crystallization producing a mixture of clear crystals surrounded by molasses. A centrifuge may be used to separate the sugar from molasses, and the crystals are washed by addition of steam, after which the crystals are dried by an airflow. Upon cooling, sugar crystallizes out of the syrup. From this point, the sugar refining process continues to produce different types of sugar, and the molasses may be used in the process according to the invention e.g. to produce ethanol.

Sugar from sugar beet may be released in any known way. For example, this may be accomplished as follows. After reception at the processing plant, the beet roots are washed, mechanically sliced into thin strips called cossettes, and passed to a machine called a diffuser to extract the sugar content into a water solution. Diffusers are long vessels of many metres in which the beet slices go in one direction while hot water goes in the opposite direction. The movement may either be by a rotating screw or the whole unit rotates, and the water and cossettes move through internal chambers. There are three common designs of diffuser: the horizontal rotating 'RT' (Raffinerie Tirlemontoise, manufacturer), inclined screw 'DDS' (De Danske Sukkerfabrikker), or vertical screw "Tower". A less common design uses a moving belt of cossettes, with water pumped onto the top of the belt and poured through. In all cases the flow rates of cossettes and water are in the ratio one to two. Typically cossettes take about 90 minutes to pass through the diffuser, the water only 45 minutes. These are all countercurrent exchange methods that extract more sugar from the cossettes using less water than if they merely sat in a hot water tank. The liquid exiting the diffuser is called raw juice. The colour of raw juice varies from black to a dark red depending on the amount of oxidation, which is itself dependent on diffuser design. Raw juice or any material produced from that may be used in the fermentation according to the invention. Saccharifi cation

For starch containing plant materials, such as sweet sorghum, it may be necessary to release sugar from the starch in order to increase the useful product output of the process. This may be accomplished in by pretreating the plant material, for instance by liquefying the plant material. To produce low molecular sugars, DP1-2, which can be metabolized by yeast, the maltodextrin from the liquefaction is preferably further hydrolyzed; this is also termed "saccharification". The hydrolysis may be done enzymatically by the presence of a glucoamylase. An alpha-glucosidase and/or an acid alpha-amylase may also be present in addition to the glucoamylase.

The saccharification is preferably performed in the presence of an alpha- amylase, e.g., derived from a micro-organism or a plant. Preferred alpha-amylases are of fungal or bacterial origin. Bacillus alpha-amylases (often referred to as "Termamyl- like alpha-amylases"), variant and hybrids thereof, are specifically contemplated according to the invention. Well-known Termamyl-like alpha-amylases include alpha- amylase derived from a strain of B. licheniformis (commercially available as Termamyl(TM)), B. amyloliquefaciens, and B. stearothermophilus alpha-amylase. Contemplated alpha-amylase derived from a strain of Aspergillus includes Aspergillus oryzae and Aspergillus niger alpha-amylases. Commercial alpha-amylase products and products containing alpha-amylases include TERMAMYL(TM) SC, FUNGAMYL(TM), LIQUOZYME(TM) SC and SAN(TM) SUPER, (Novozymes A/S, Denmark) and DEX- LO(TM), SPEZYME(TM) AA, and SPEZYME(TM) DELTA AA (from Genencor Int.).

A full saccharification step may last up to 72 hours. However, the saccharification and fermentation may be combined in simultaneous saccharification and fermentation (SSF) step, and in some embodiments of the invention a pre- saccharification step of 1-4 hours may be included. Pre-saccharification is carried out at any suitable process conditions. In a preferred embodiment, the pre-saccharification is carried out at temperatures from 30-65°C, such as around 6O⁰C, and at, e.g., a pH in the range between pH 4-5, especially around pH 4.5. Thus in one embodiment, the process of the invention may further comprise a pre-saccharification step, as described herein, which is performed after the secondary liquefaction step and before the SSF step. For a plant material containing sugar and starch, a simultaneous saccharification and fermentation (SSF) process maybe employed as preferred embodiment. In such embodiment, there is no holding stage for the saccharification, meaning that yeast and saccharification enzyme (glucoamylase) is added essentially together in the fermentor.

Fermentation

The strain used in the fermentation process according to the invention is of the genus Saccharomyces. Preferably the strain (also cell or cells) used is Saccharomyces cerevisiae . Preferably, the Saccharomyces cerevisiae cells are capable of growth on isomaltulose as sole hydrocarbon source. More preferably the Saccharomyces is a baker's yeast. In a particular preferred embodiment, the Saccharomyces cerevisiae is Ethanol Red®, commercially available from Fermentis, USA, Product No. 42138.

The strain may be wild type, developed by selective cultivation or a genetically modified strain. Preferably the strain is non-GMO. In one embodiment the fermentation is conducted under anaerobic conditions.

An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than 5, 2.5 or 1 mmol/L/h.

In another embodiment the fermentation is conducted under oxygen-limited conditions.

An oxygen-limited fermentation process is a process in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid. The degree of oxygen limitation is determined by the amount and composition of the ingoing gasflow as well as the actual mixing/mass transfer properties of the fermentation equipment used. Preferably, in a process under oxygen-limited conditions, the rate of oxygen consumption is at least 5.5, more preferably at least 6 and even more preferably at least 7 mmol/L/h.

Preferably the specific growth rate on isomaltulose μ_MAx is 0.05 h^"1or more, 0.1 h^"1 or more, 0.15 h^"1 or more, 0.2 h^"1 or more, 0.25 h^"1or more. More preferably, the specific growth rate μ_MAx is 0.30 h^"1 or more or 0.35 h^"1 or more.

Preferably, wherein the specific isomaltulose uptake of the cells QS_MAX is 0.25 g isomaltulose/(g biomass^*h) or more, 0.50 g isomaltulose/(g biomass^*h) or more, more preferably 1.00 g isomaltulose/(g biomass^*h) or more, most preferably 2.00 g isomaltulose/(g biomass^*h) or more, 2.50 isomaltulose/(g biomass^*h) or more, 3.00 g isomaltulose/(g biomass^*h) or more.

In a bioethanol production process conducted according to the invention, preferably the Yield of ethanol based on sugar is 0.3 g ethanol/g sugar or more, 0.4 g ethanol/g sugar or more, more preferably 0.45 g ethanol/g sugar or more, most preferably 0.51 g ethanol/g sugar or more. The theoretical maximum yield for isomaltulose is 0.54 gr ethanol/g isomaltulose.

Preferably, the fermentation is a co-fermentation of isomaltulose with one or more other sugars. The one or more other sugars is preferably chosen from the group: fructose, glucose, sucrose, threhalulose. Most preferred other sugar is sucrose.

The Saccharomyces used for the fermentation is added to the fermentation medium, usually in the form of a mash, and the fermentation is continued until the desired amount of useful fermentation product such as ethanol is produced; this may, e.g., be for 24-96 hours, such as for instance 35-60 hours. Next to the sugars, the fermentation medium may comprise other constituents, common in fermentation, such as solvents, usually water, nutrients etc. The temperature and pH during fermentation is at a temperature and pH suitable for the microorganism in question, such as, e.g., in the range about 32-45⁰C, e.g. about 34⁰C, above 34⁰C, at least 34.5[deg.] C, or even at least 35⁰C, and at a pH e.g. in the range about pH 2-6, preferably pH is 2,5-4,5, e.g. about pH 3.5. A low pH is advantageous to avoid contamination of unwanted microorganisms, such as for instance lactic acid bacteria.

The fermentation reactor may be a continuously stirred tank reactor, with and without cell recycling, reactors in series, reactors under vacuum, immobilized cell reactors and tower fermentors with flocculating yeast or a centrifuge to separate the yeast from the ethanol.

In a preferred embodiment, the fermentation is conducted in high OD (as defined herein in the examples) fermentation. The OD is preferably 150 or more, or 100, 200, 300, 400, 500 or more. Preferably the fermentation is conducted with a cell recycle.

Alternative to fermentation a yeast extract from the yeast according to the invention may be used as an enzyme mix to produce glucose and fructose from the isomaltulose. The resulting glucose and fructose may than be fermented in a conventional way to useful products, such as ethanol.

Fermentation products Fermentation products which may be produced according to the invention include amino acids, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels, or other organic polymers, acids, such as succinic acid or succinate, itaconic acid or itaconate, fumaric acid or fumarate, citric acid or citrate, malic acid or malate and lactic acid, and alcohols such as ethanol, including fuel ethanol, 1-butanol, 2,-butanol, isobutanol and isoamylalcohol, The term "ethanol" is being understood to include ethyl alcohol or mixtures of ethyl alcohol and water.

Specific value-added products that may be produced by the methods of the invention include, but not limited to, biofuels (including ethanol and butanol and a biogas); lactic acid; a plastic, a chemical, e.g. caprolactam; a specialty chemical; an organic acid, including citric acid, succinic acid, fumaric acid, itaconic acid and maleic acid; 3-hydoxy-propionic acid, acrylic acid; acetic acid and salts thereof; 1 ,3-propane- diol; ethylene, glycerol; a solvent; an animal feed supplement; a pharmaceutical, such as a β-lactam antibiotic or a cephalosporin; vitamins; an amino acid, such as lysine, methionine, tryptophan, threonine, and aspartic acid; 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; and a chemical feedstock. Preferred fermentation products are ethanol, butanol (1-butanol, 2,-butanol, isobutanol) and succinic acid. Ethanol obtained by the process of the invention may be used as, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits, or industrial ethanol, including fuel additive.

Distillation

The process of the invention may further comprise recovering of the fermentation product, e.g. an alcohol, preferably ethanol; hence the alcohol may be separated from the fermented material and purified. Following the fermentation the mash may be distilled to extract the ethanol. Ethanol with a purity of up to e.g. about 96 vol. % ethanol can be obtained by the process of the invention.

Thus, in one embodiment, the process of the invention further comprises a step of distillation to obtain the ethanol, wherein the fermentation step and the distillation is carried out simultaneously or sequentially, optionally followed by one or more process steps for further refinement of the ethanol.

The following examples illustrate the invention.

EXAMPLES

Example 1

Yeast strain CEN.PK1 13-7D was cultured in YEP-medium (1% w/v yeast extract, 2% w/v peptone) with 2% ethanol, 2% glycerol and 0.05% glucose as carbon sources. Strain CEN. PK1 13-7D was inoculated in the same medium. Cells were grown overnight in a rotary shaker at 30⁰C and 280 rpm. The optical density at 600 nm (OD₆oo) was measured and cells were harvested by centrifugation, followed by washing. Fresh cultures of YNB-medium without amino acids (Difco; 6.7 g/l w/v) supplied with either 2% w/v isomaltulose, 2% w/v sucrose or 1 % w/v isomaltulose and 1 % w/v sucrose were inoculated with the washed yeast suspensions at a starting OD600 of 0.2. Total volume amounted to 100 ml.

The OD₆Oo was measured in time as an indication of yeast growth. At the same time, a sample was taken for analysis of residual sugars and product formation. The results are set out in Figure 1. Surprisingly, it was observed that CEN.PK1 13-7D was able to grow on isomaltulose. Surprisingly strain CEN. PK1 13-7D was also very well capable of utilizing isomaltulose as a carbon-source; at least equally well as sucrose. Also with this strain, co-fermentation of sucrose and isomaltulose occured.

Example 2 As a follow-up to Example 1 and in order to rule out the possibility that the utilization of isomaltulose is a peculiarity of CEN.PK1 13-7D, other strains were included in a subsequent analysis. Addition, this second set of experiments was executed under both aerobic and anaerobic conditions, in order to verify whether or not isomaltulose consumption and ethanol production could take place under anaerobic conditions.

Strains CEN.PK1 13-7D, BIE101 (Ethanol Red®, commercially available from Fermentis, USA, Product No. 42138), BIE105 (a bakers' yeast strain) and BIE1 19 (a wine yeast strain) were used to inoculate 25 ml of YNB-medium without amino acids (6.7 g/l w/v) with 2% ethanol, 2% glycerol and 0.05% glucose as carbon source. After overnight growth, the OD₆oo was measured; cells were washed with 0.9% (w/v) NaCI- solution and resuspended in the same.

Flasks containing a medium based on Verduyn et al. Yeast 8, 501-517, 1992 (2 g/l ureu (w/v), 2 g/l KH₂PO₄ (w/v), 0.5 g/l MgSO₄.7H₂0 (w/v), and trace elements, vitamins and tween/ergosterol as described by Verduyn et al.[5]) supplied with either 3.1 % isomaltulose or 2.5% sucrose and 2.5% isomaltulose, were inoculated with the washed yeast cell suspensions in duplicate. The starting OD₆oo was 0.2. The pH was 4.0 and the temperature 30⁰C, One of each duplicate flask was provided with a water lock, in order to provide anaerobic conditions after the oxygen dissolved in the fluids had been exhausted. The second duplicate flask was incubated as such, with a cotton plug (as in Example 1 ), allowing the yeast cells to grow in the presence of oxygen, although the yeast cells will produce ethanol under these conditions as a consequence of the Crabtree effect (see e.g. Postma et al. Applied and Environmental Microbiology 55(2), 468-477, 1989 and references therein).

Samples were taken every 24 hours for determination of the growth (OD₆oo reading) and determination of residual sugar concentration and the concentration of products (NMR analysis).

The results are set out in Figure 2. It is clear that all strains could grow on a mixture of isomaltulose and sucrose. The flasks without waterlock essentially yielded the same graph, although higher OD₆oo-values were obtained, due to the presence of oxygen.

Surprisingly, all strains tested except BIE119 (a wine yeast) is showing growth on isomaltulose as sole carbon source (figure 3). Again, without a waterlock, essentially the same graph was obtained, but a higher OD₆oo was obtained. The OD₆oo development in time matches well with the disappearance of isomaltulose from the medium. The results are given in tables 1 and 2. Table 1 : Conversion of isomaltulose for the strains of example 2a, with isomaltulose and sucrose (each 25g/l), with waterlock, at start (t=0) and after 24h of fermentation (t=24h) in percentage of amount of isomaltulose at start.

Table 2: Conversion of isomaltulose for the strains of example 2b, with isomaltulose as single sugar (31 g/l), with waterlock, at start (t=0) and after 24h of fermentation (t=24h) in percentage of amount of isomaltulose at start.

The results are graphically given in Figures 4 and 5. All strains except for BIE119 (herein a comparative experiment strain) consumed isomaltulose. At 48h, all isomaltulose has been consumed, even if sucrose is present (see Figure 4). The cultures without waterlock gave similar results (data not shown).

In Figures 6 and 7, the ethanol production (in g/l) has been plotted against time.

In Figure 6, it can be seen that strains BIE101 , CEN.PK1 13-7D, BIE105 and CBS8841 allowed similar levels of ethanol to be achieved. At 48 hours, the fermentation appeared to be (near) complete, since the ethanol concentration at 72 hours did not increase, or only increased minimally, as compared with the concentration at 48 hours. Strain BIE119 produced some ethanol, which must have been made from the sucrose, since BIE119 did not utilize isomaltulose.

Strain BIE101 , which consumed isomaltulose at the highest rate in Figure 4, was also the fastest ethanol producer (see Figure 7). All other strains consumed isomaltulose and excreted ethanol at similar rates, except for strain BIE1 19, which did not consume isomaltulose and hence did not produce ethanol.

Calculation of the Specific growth rate on isomaltulose UMAX (h^~ )and maximum specific fermentation rate QSMAX (g substrate/g biomass h)

From the growth data of figure 1 the maximum specific growth rate (mumax or μ_maχ ) could be derived on the different growth substrates by plotting the LN(OD) versus time and when OD was smaller than 1.2; between 1.5 and 6.5 hrs. The regression line had a correlation quotient R² of equal or >0.99 in all cases. This clearly indicates that the strain BIE104 has a specific growth rate of 0.26 h^"1. Growth on the sugar mixture sucrose + palatinose, which will be relevant in the application of the invention in production of biooethanol is higher than on palatinose alone and approaches the maximum specific growth rate on sucrose as the sole carbon source.

The maximum specific isomaltulose uptake rate can be derived from the maximum specific growth rate by dividing the maximum specific growth rate by the maximum yield of biomass on substrate under fermentative conditions of 0.1 g biomass dry weight per g of substrate consumed. At 100 g/l of biomass as used in cell recycle systems the production rate of ethanol can be as high as in the sucrose fermentation. In the examples, the cells operate far below their maximum sugar uptake rates.

Table 3: Specific growth rate on isomaltulose μ_MAx (h^"1), Regression coefficient R² and maximum specific isomaltulose uptake rate QS_MAX (g substrate/g biomass h) of the example 1 based on data in figure 1.

The surprisingly high growth rate on isomaltulose also allows rapid growing of the yeast in the propagation process to provide the ethanol factory with seed material in a rapid and economic way.

The data from figure 7 indicate that the ethanol production levels are >10 g/L on isomaltulose. Using 31 g/L isomaltulose in the medium this means that a Yield of > 0.33 gr ethanol/gr isomaltulose could be produced.

The examples show according to the invention, the conversion of isomaltulose is effective and there is a high activity towards conversion of isomaltolose. It is possible to simultaneously convert isomaltulose and to produce ethanol, in a single process step. Conversion of isomaltulose as a sole hydrocarbon source is possible. The strains used in the examples are non-genetically modified (non-GMO).

The yeast strains referred to in these Examples were all deposited at the CBS (Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands) on 5 August 2008. The deposits have been accorded the following deposit/accession numbers:

C BS 123313 Saccharomyces cerevisiae B I E 101

CBS 123316 Saccharomyces cerevisiae BIE104 (=CEN.PK1 13-7D)

C BS 123317 Saccharomyces cerevisiae B I E 105

CBS 123331 Saccharomyces cerevisiae BIE119

Literature [1] L. G. Wu, R. G. Birch, Doubled sugar content in sugarcane plants modified to produce a sucrose isomer, Plant Biotech. J. 5 (2007) 109— 1 17.

[2] B.A.R. Lina, D. Jonker, G. Kozianowski, lsomaltulose (Palatinose (R)): a review of biological and toxicological studies, Food Chem. Toxicol. 40 (2002) 1375-1381.

[3] J. Gressel, Review: Transgenics are imperative for biofuel crops, 3 december 2007 Crop Biodiversity, Elsevier Plant Science www.sciencedirect.com.

[4] CC. Emeis and S. Windisch, Palatinose Vergaerung durch Hefen, Z.eitschrift fuer die Zuckerindustrie, 58(1960), 248-249.

[5] Verduyn, C, Postma, E., Scheffers, W.A. and van Dijken, J. P. (1992) Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation.

[6] Postma et al. Applied and Environmental Microbiology 55_(2)_(1989), 468-477.

I Applicant's or agent's file reference number 26852-WO-PCT | International application No

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

A. The indications made below relate to the microorganism referred to in the descπption first mentioned on page 16 line 21 (PDF document)

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet X

Name of depositary institution

CENTRAAL BUREAU VOOR SCHIMMELCULTURES

Address of depositary institution (including postal code and country) Uppsalalaan 8 P O Box 85167 NL-3308 AD Utrecht The Netherlands

Date of deposit 05 August 2008 Accession Number CBS 123313

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet

We inform you that the availability of the microorganism identified above, referred to Rule 13bis PCT, shall be effected only by issue of a sample to an expert nominated by the requester until the publication of the mention of grant of the national patent or, where applicable, for twenty years from the date of filing if the application has been refused, withdrawn or deemed to be withdrawn

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This sheet was received with the international application

Authorized officer

Wallentin, Marko

Form PCT/RO/134 (July 1992) I Applicant's or agent's file reference number 26852-WO-PCT | International application No

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

A. The indications made below relate to the microorganism referred to in the descπption first mentioned on page 16 line 22 (PDF document)

Name of depositary institution

CENTRAAL BUREAU VOOR SCHIMMELCULTURES

Address of depositary institution (including postal code and country) Uppsalalaan 8 P O Box 85167 NL-3508 AD Utrecht The Netherlands

Date of deposit 05 August 2008 Accession Number CBS 123316

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Wallentin, Marko

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

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Name of depositary institution

CENTRAAL BUREAU VOOR SCHIMMELCULTURES

Date of deposit 05 August 2008 Accession Number CBS 123317

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

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INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

A. The indications made below relate to the microorganism referred to in the descπption first mentioned on page 16 line 24 (PDF document)

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet

Name of depositary institution

CENTRAAL BUREAU VOOR SCHIMMELCULTURES

Date of deposit 05 August 2008 Accession Number CBS 123331

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This sheet was received with the international application

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Wallentin, Marko

Form PCT/RO/134 (July 1992)

Claims

1. A process for the fermentation of isomaltulose, wherein isomaltulose obtained from plant material is contacted with cells of Saccharomyces.

2. Process according to claim 1 wherein the Saccharomyces is Saccharomyces cerevisiae.

3. Process according to claim 1 or 2, wherein the Saccharomyces is a baker's yeast.

4. Process according to any of claims 1-3, wherein the specific growth rate on isomaltulose is 0.025 h^"1 or more, under anaerobic conditions.

5. Process according to claim 4, wherein the specific growth rate on isomaltulose is 1.00 h^"1 or more, under anaerobic conditions.

6. Process according to any of claims 1-5, wherein the fermentation has a yield of ethanol of 0.3 or more g ethanol/g sugar.

7. Process according to any of claims 1-6, wherein the specific isomaltulose uptake of the cells QS_MAX is 0.25 g isomaltulose/(g biomass^*h) or more.

8. Process according to any of claims 1-10, wherein the Saccharomyces cerevisiae is Ethanol Red®, commercially available from Fermentis, USA, Product No.

42138.

9. Process according to any of claims 1-1 1 , wherein the fermentation is a co-fermentation of isomaltulose with one or more other sugars.

10. Process according to claim wherein the one or more other sugars is chosen from the group: fructose, glucose, sucrose, threhalulose.

1 1. Process according to claim 9, wherein the one or more other sugars is sucrose.

12. Process for the production of fermentation product, wherein a plant material comprising isomaltulose is subjected to extraction and the extraction product is converted by the process of any of claims 1-1 1 , whereby a fermentation product is produced.

13. Process according to claim 12, wherein the plant material is sugar cane, sugar beet, sweet sorghum and/or sweet corn.

14. Process according to claim 12 or 13, wherein the fermentation product is chosen from the lsit including: amino acids, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels, or other organic polymers, acids, such as succinic acid or succinate, itaconic acid or itaconate, fumaric acid or fumarate, citric acid or citrate, malic acid or malate and lactic acid, and alcohols such as ethanol, including fuel ethanol, 1-butanol, 2,-butanol, isobutanol and isoamylalcohol,

15. Process according to claim 14, wherein the fermentation product is ethanol.