PROCESS FOR THE PREPARATION OF XYLITOL FROM XYLOSE BY CULTIVATING CANDIDA GUILLIERMONDII

Abstract: Microbiological method for the preparation of xylitol from xylose containing mixtures. The yeast strain Candida guilliermondii VTT-C-71006 is grown, preferably under conditions employing limited aeration, in a xylose rich medium. A very good xylitol yield is obtained by adding to the medium furfural at an amount of about 0.2 to 1.0 g/l. In the fermentation, either a conventional batch process or a so-called fed-batch-process is used.

Description: Process for the preparation of xylitol from xylose by ^ cultivating Candida guilliermondii. The object of the present invention is a microbiological process for the preparation of xylitol from xylose con¬ taining mixtures using the yeast strain Candida guillier¬ mondii VTT-C-71006. Xylitol is conventionally prepared on a technical scale by hydrolysing hemicellulose containing substances, for example birch wood, whereby a pentose rich hydrolysate is obtained. The xylose of the hydrolysate can thereafter be hydrogenated catalytically to form xylitol. Also microbiological methods for the production of xylitol are prior known. In the publication H. Onishi & T. Suzuki, "The Production of Xylitol, L-Arabinitol and Ribitol by Yeasts", Agr. Biol. Chem., Vol. 30, No. 11, p. 1139 to 1144, 1966, is disclosed the microbiological production of xylitol from a xylose containing solution by the yeast species Candida guilliermondii. The initial xylose con¬ centration of the solution used as raw material was 10 % by weight. The yield of xylitol was 40.3 %. The strain Candida guilliermondii VTT-C-71006 is deposited in the strain collection at National Collection of Yeast Cultures (NCYC), Norwich, England, on 17th December 1986 under no NCYC-1644. Candida guilliermondii VTT-C-71006 exhibits the following morphological and physiological properties. Morphological characteristics; Macroscopic morphology: Soft, shiny, white/light yellow colonies with a diameter of 1 to 2 mm on malt extract (3 g/1)-yeas extract (3 g/l)-glucose (10 g/l)-peptone (5 g/l)-agar (20 g/1) (MYGP-agar) . Microscopic morphology: The cells immobile in MYGP-sol tion, somewhat oval, dimen¬ sions 2-5 x 7-3 /urn. Under some conditions formation of pseudo ycelium. Physiological characteristics: The physiological properties were determined as described in the book N.J.W. Kreger-van Rij, "The yeasts - a taxono- mic study", 3rd edition, Elsevier Science Publishers B.V. Amsterdam 1984, p. 76-. Fermentation: glucose + sucrose + raffinose ? galactose • p maltose lactose xylose Assimilationt glucose + sucrose + maltose + cellobiose + raffinose + xylose + lactose - starch - nitrate - inositol According to the process of the invention, cultivation is carried out by cultivating the strain Candida guilliermon- dii VTT-C-71006 preferably under aerated conditions in a xylose containing medium having a xylose concentration va¬ rying from 5 to 50 % by weight at a temperature of 20 to 40°C for a period ranging from 20 hours to 10 days. After the removal of the yeast the xylitol formed during the cultivation may be recovered by any known manner. Suitable raw materials are any xylose containing mixtures, for example the xylose rich mixtures obtained from the hydrolysis of hemicellulose containing materials. In the fermentation solution as the N-source for the yeast different ammonium salts may be used and as a growth fac¬ tor source for example yeast extract, yeast produced in the process or extract of plant origin. Essential is the restriction of the oxygen supply to the yeast. By adjust¬ ing the stirring and aerating conditions to an optimum, xylitol is obtained at an amount as high as 78 %. The pro¬ cess is slowed down at very high initial xylose concentra¬ tions. The process may, however, be accelerated compared to a normal batch process even by 40 % by using so called fed-batch cultivation, which means that during the pro¬ cess, nutrients are added to the fermenter. In our tests the cultivation was carried out using different xylose concentrations and different aeration efficiences in both a fermenter and in agitated cultures. The composition of the medium was xylose 40 to 300 g/1 yeast extract 10 g/1 Bacto peptone 3 g/1. In our tests it was surprisingly discovered that the addi¬ tion of furfural favourably affected the yield of xylitol. Previously the presence of furfural in microbiological processes has been considered purely disadvantageous as furfural inhibits the growth of yeast. The inhibition of the growth of the yeast is as such desirable in processes of this kind, as the growth of yeast consumes raw materi¬ als which is manifested in a decrease of the amount of xy¬ litol. Surprising was, however, the fact that furfural did not completely inhibit the action of the yeast, but acted as a factor promoting the production of xylitol. According to literature, furfural usually disturbs both the growth and the fermentation process (for example N. Banerjee et al., European J. Appl. Microbiol. Biotechnol. 1981, 11:226-228)) . According to this process it was discovered that the growth of yeast and the subsequent reduction of the xyli¬ tol yield may be limited by adding furfural to the fermen¬ tation solution at an amount of 0.2 - 1.0 g/1. The use of furfural for the promotion of microbiological processes is not limited to the production of xylitol from xylose by means of the process described above. It is evi¬ dent that this observation may be applied also to other microbiological processes, the object of which is not the production of the microbe as such but the production of substances independent of the microbial growth, for examp¬ le in biotransformations. During the cultivation, the yeast concentration was deter¬ mined by measuring the turbidity, the xylose concentration by determining the concentration of reducing substances and the formation of xylitol was determined gualitatively by using thin layer chromatography and quantitatively by using liquid chromatography or an enzymatic method. The process according to the invention exhibits many ad¬ vantages with respect to the known processes. Of these may be mentioned for example: - High xylitol yield, 50 to 78 %, depending on the xylo¬ se concentration. The process mentioned earlier and be¬ longing to the state of art gave a xylitol yield of on¬ ly 40.3 % although the initial xylose concentration was only 10 %. When using our method and starting from the corresponding initial xylose concentration, the yield obtained was 74 %. - It is possible to use high initial xylose concentrati¬ ons. This factor is of great technical and economical importance. With an initial xylose concentration of the raw material of 300 g/1 a xylitol yield as high as 50 % was obtained. - The xylitol produced with this method is pure and is thus quite suitable for use in foodstuffs. Xylitol pro¬ duced by means of catalytic hydrogenation may contain residues of toxic catalyst. The method according to the invention is also more economical, as the catalysts are expensive and are consumed in the process. - This method is also very specific, as the yeast strain Candida guilliermondii VTT-C-71006 converts xylose only to xylitol. In the catalytic hydrogenation, besides xy¬ litol by-products may be obtained, which makes purifi¬ cation more difficult. in the following examples the process according to the in¬ vention is described in more detail. Example 1 The strain Candida guilliermondii VTT-C-71006 is grown aseptically while agitating in a solution volume of 100 ml in a 250 ml vessel. The growth temperature is 28°C. The medium contains 80 g/1 xylose, 10 g/1 yeast extract and 3 g/1 Bacto peptone. The pH of the medium was adjusted to a value of 5.0. As the inoculum 1 ml of a culture grown overnight on MYGP- medium is used. After two days of cultivation, the xylose has been consumed and the xylitol concentration is 50 g/1. The xylose was determined as reducing sugars and the xyli¬ tol using liquid chromatograph . The xylitol yield was thus 62.5 %. Example 2 The method according to the Example 1 was repeated, the initial xylose concentration being 300 g/1. After 6 days the xylose was consumed completely and the xylitol con¬ centration was 150 g/1, which corresponds to a yield of 50 %. Example 3 The method described in the Example 1 was repeated, but furfural was added to the medium at an amount of 0 * 6 g/1. The xylitol concentration after 53 hours cultivation was 61 g/1 (yield 76 %) . Example 4 The strain mentioned in the Example 1 is grown aseptically in a fermenter in a medium containing 100 g/1 xylose, 10 g/1 yeast extract and 3 g/1 Bacto peptone. The pH of the medium was adjusted to a value of 5.0 and the stirring speed to 400 rp and the aeration to about 0.5 1/min. Af¬ ter 50 hours of cultivation the xylose was totally consu¬ med and the xylitol concentration was 74 g/1 (yield 74 %) . Example 5 The process of Example 4 was repeated but using 250 g/1 as the initial xylose concentration. After cultivating for 160 hours, the xylose was completely consumed and the xy¬ litol concentration was 138 g/1 (yield 55 %) . Example 6 The strain mentioned in the previous example is grown in a fermenter as a fed-batch-culture. The initial volume of the culture solution was 60 % of the end volume and its composition was 100 g/1 xylose, 10 g/1 yeast extract and 3 g/1 Bacto peptone. Cultivation was started as in the Example 4, but when the xylose concentration had decreased to a value of 40 g/1, the introduction of a xylose solut¬ ion at a concentration of 350 g/1 was begun. By means of the addition the xylose concentration in the fermenter was kept at 40 g/1. When the final volume was reached the ad¬ dition was stopped. After cultivating for 120 hours the xylose was completely consumed and the xylitol concentrat¬ ion was 164 g/1, which corresponds to a xylitol yield of

Claims: Claims 1. Process for the preparation of xylitol from xylose con¬ taining mixtures, c h a r a c t e r i z e d in that the yeast strain Candida guilliermondii VTT-C-71006 is culti¬ vated, advantageously under conditions of limited aerat¬ ion, in a xylose containing medium at a temperature of 20 to 40°C for a period which varies between 20 hours and 10 days, whereafter the xylitol formed in the medium is reco¬ vered. 2. Process according to the claim 1, c h a r a c t e r ¬ i z e d in that in order to restrict the growth of the yeast and thus to improve the xylitol yield, furfural at an amount of 0.2 to 1.0 g/1 is added to medium. 3. Process according to the claim 1 or 2, c h a r a c t¬ e r i z e d in that as the medium is used a xylose con¬ taining, pentose rich mixture of sugars obtained from the hydrolysis of hemicellulose containing materials. 4. Process according to any of the previous claims, c h a r a c t e r i z e d in that in the fermentation a fed-batch process is used.

Inventors: LINKO MATTI, OJAMO HEIKKI, YLINEN LASSE, LINKO, MATTI, OJAMO, HEIKKI, YLINEN, LASSE
Applicants: FARMOS OY, FARMOS-YHTYMAE OY
Application year: 1987
Main classification: C12P7
IPC codes: C12P7, C12R1