CS270333B1 - Method of producing yeast products from methanol - Google Patents

Abstract

The solution relates to the field of microbiology, biotechnology, feed industry and possibly the production of special chemicals by microbial synthesis such as alcohol oxidase. formaldehyde and formate dehydrogenase, dihydroxyacetone synthesis, catalase, reduced glutathione, reduced nicotinamide adenine dinucleotide and adenosine triphosphate. Microbial biomass and the above products are obtained by cultivating methylotrophic yeast strains in a medium containing methanol in a concentration of 0.3 to 1% by weight and xylose in a concentration of 0.1 to 0.3% by weight, at a temperature of 30 to 40 °C and a pH of 3.0 to 3.5. Cultivation is carried out under sterile conditions using yeast strains Candida boidinii llBh, C. boidinii 1, Hansenula polymorpha, Pichia or their mixtures or under non-sterile conditions using acid-tolerant yeast strains C. boidinii 2 and Hansenula sp. 5. Waste xylose contained in lignocellulose hydrolysates can replace up to 50% by weight of the raw material methanol and thus reduce the costs of producing feed yeast and other specific products. The yeast biomass obtained in this way is rich in proteins, essential amino acids and vitamins B^ and B^.

Description

The invention relates to a method for producing yeast products from methanol.

Xylose, especially in the form of waste lignocellulosic hydrolysates, serves as a cheap carbon source for the production of feed yeast using yeasts other than methylotrophic yeasts (author's certificate No. 1888457 and δ. 244335)* However, xylose can be used as a raw material, either alone or in combination with methanol for the growth of methylotrophic yeasts to obtain high-value yeast biomass usable as protein and vitamin-rich feed for livestock and as a source for the preparation of specific products such as alcohol oxidase, formaldehyde and formate dehydrogenase, dihydroxyacetone synthesis, catalase, reduced glutathione (GSH), reduced nicotinamide adenine dinucleotide (NAS) and adenosine triphosphate (ATP). '

Unlike foreign processes, where pure methanol and sterile cultivation conditions are used for the production of biomass and specific products such as alcohol oxidase (German Patent 2557623, Unilever PLC, NSC Patent Application 823035), glutathione (Japan Kokai 77, 156, 994) and ATP (Agr. Biol. Chem. 49, 637, 1985), it is possible, according to the invention, to replace methanol by up to 50% by weight with waste xylose contained in lignocellulosic hydrolysates while maintaining the same quality of yeast biomass and content of specific products as from methanol itself. and when using acid-tolerant strains of methylotrophic yeast, non-sterile cultivation conditions can also be used. This makes it possible to reduce the economic costs of both the raw material methanol and the energy required for sterilization of the medium, equipment and cultivation management.

The subject of the invention is a method for producing yeast products from methanol, the essence of which is that methylotrophic yeast strains are cultivated in a medium containing methanol in a concentration of 0.3 to 1% by weight, and xylose in a concentration of 0.1 to 0.3% by weight, at a temperature of 30 to 40 °C and a pH of 3.0 to 3.5.

As methylotrophic yeast strains, Candida boidinii HBh, C. boidinii 1, Hansenula polymorpha, Píchla or mixtures thereof under sterile cultivation conditions or acid-tolerant methylotrophic yeast strains Candida boidinii 2 and Hansenula sp. 3 under non-sterile cultivation conditions are used.

The production method according to the invention is made possible by the fact that methylotrophic yeast strains are capable of synthesizing enzymes specific for methanol metabolism during growth on xylose in the presence of methanol, unlike glucose or ethanol, in an amount reaching up to 100% of the level of enzymes synthesized in cells grown on methanol alone. This fact is conditioned by the fact that xylose, unlike glucose and ethanol, is a weak catabolic repressor of the synthesis of enzymes specific for methanol metabolism and in an amount of 0.1 to 3 g per liter of medium, in the presence of even a small amount of methanol as an inducer, does not repress the synthesis of C^ specific enzymes at all.

Since xylose is utilized independently and simultaneously with methanol during the cultivation of methylotrophic yeasts with maximum biomass yields, the resulting amount of yeast biomass produced on xylose and methanol mixtures is additive. The production of all products that are produced from methanol using biomass is therefore additive.

The ability of methylotrophic yeasts to produce specific products and biomass on xylose with methanol of the same quality as on methanol alone makes it possible to save the raw material methanol and increase the yield of methylotrophic yeast biomass based on the consumed methanol from 40% by weight, which is the maximum for methanol, up to 85% by weight.

CS 270333 Bl

Example 1

In a laboratory mechanically stirred fermenter with a total volume of 5 liters and a filling of 2.5 liters, a single cultivation of the yeast Candida boidinii 2 on a mineral medium with xylose was carried out. The composition of the mineral medium was: 1 liter of tap water, 3 g NH ₄ C1; 7 g KH ₂ PO ₄ ;0.5 g MgS0 ₄ .7H ₂ 0; 0.1 g NaCl;

1.00 mg of yeast extract and 10 µg of biotin. Xylose was used in an amount of 3 g in 1 liter of medium. The pH of the medium during cultivation was automatically maintained at a constant pH of 3.0 by dosing 3% by weight of KOH or NaOH and the temperature at 30°C. The concentration of dissolved oxygen in the medium was maintained in the range of 50 to 70% air saturation. The inoculum was yeast cells C. boidinii 2 pre-cultured on a medium with xylose and methanol, preferably on a medium with 1% by weight of methanol.

By utilizing xylose, yeast biomass was obtained with a 43% yield based on the consumed xylose. The specific alcohol oxidase activity level (determined on methanol) was detected in the obtained biomass, which was 40 to 50% rel. to the enzyme activity level in cells grown on methanol medium.

Example 2

In a laboratory mechanically stirred fermenter with a volume of 5 liters, a single cultivation of the yeast C. boidinii 2 was carried out as in example 1, with the difference that the carbon source was xylose mixed with methanol in a ratio of 1 : 1, i.e. 3 g xylose and 3 g methanol in 1 liter of medium. The level of specific alcohol oxidase activity in the obtained cells reached 100% rel. The yield of yeast biomass from both sources was additive. When converted to the consumed methanol, it was 85%.

For comparison, another 3 cultivations were performed with the ratio of xylose and methanol as shown in Table δ. 1 , .

Table 1

Alcohol oxidase activity and biomass yield of C. boidinii 2 yeast grown on xylose-methanol mixtures

Fermenter

3 4

methanol g/1 3 3 3 3 . xylose g/1 1 2 3 ' 7 1« sp. alcohol oxidase activity rel. % 100 100 100 40 biomass yield based on methanol % 56 . 70 85 85 Example 3 ’

In a 75-liter, 40-liter, fully operational, mechanically stirred fermenter, a single cultivation of the yeast C. boidinii 2 was carried out as in Example 2, with the difference that 25 to 50 wt. % xylose-methanol mixture was added to the fermenter separately from the medium in a feed-through manner depending on the concentration of dissolved oxygen in the medium during fermentation. The concentration of dissolved oxygen was maintained automatically in the range of 20 to 40% of medium saturation. The pH of the medium was maintained at a constant level of 3.0 to 3.2 by automatic dosing of 50 wt. % KOH, NaOH

CS 270333 31 preferably ammonia water and the temperature at 30 ° C. The level of specific alcohol oxidase activity and the biomass yield were as in example no. 2. The protein content in the biomass was 55%.

Example 4

The procedure was the same as in Example No. 3, with the difference that the yeast strain Hansenula sp. 3 was used at a cultivation temperature of 37 to 40 °C.

Example 5'

The procedure was the same as in Example No. 3, with the difference that xylose was added to the mixture in the form of a hydrolysate of lignocellulosic waste, preferably straw hydrolysate (AO 188457) or waste xylose extracts from the wood processing industry.

Example 6

In a 75-liter, mechanically quenched fermenter as in Example No. 3, continuous cultivation of the yeast C. boidinii 2 was carried out under non-sterile conditions. The carbon source was xylose with methanol in a mixture as in Example No. 3 or as in Example No. 5, with the difference that xylose flowed into the fermenter together with the mineral medium and methanol was dosed separately in a feed-in manner depending on the dilution rate of the medium and the concentration of dissolved oxygen in the medium as in Example 3. The dilution rate of the medium was in the range D 0.05 to 0.1h~\, preferably at D 0.05 to 0.08. The level of specific activity of alcohol oxidase, but also catalase, in the cells was 100% rel. and the yield based on the consumed methanol was 85% The protein content in the yeast biomass was 60% by weight.

Example 7 . .

The procedure was the same as in Example 6, with the difference that the yeast strain Hansenula sp. 3 was used at a temperature of 37 to 40°C and a medium shaking rate of preferably 0.05 to 0.1.h- ¹ .

The yeast biomass obtained by the method according to the invention is rich in proteins, essential amino acids and vitamins B1 and B2 and can be advantageously used as a valuable protein and vitamin feed for livestock and as a source for the production of specific products such as alcohol oxidase, formaldehyde and formate dehydrogenase, dihydroxyacetone synthase, as well as catalase, NADE, G8H and AIP.

Claims (3)

1. A method for producing yeast products from methanol, characterized in that methylotrophic yeast strains are cultivated in a medium containing methanol in a concentration of 0.3 to 1% by weight and xylose in a concentration of 0.1 to 0.3% by weight, at a temperature of 30 to 40 °C and a pH of 3.0 to 3.5. ' ' 2. The method according to item 1, characterized in that the methylotrophic yeast strains used are Candida boidinii llBh, Candida boidinii 1, Hansenula polymorpha, Pichia or mixtures thereof under sterile conditions and/or acid-tolerant methylotrophic yeast strains Candida boidinii 2 and Hansenula sp. 3 under non-sterile culture conditions.