RU2626544C1 - Kluyveromyces marxianus vkpm y-4290 yeasts strains applied for production of ethanol on cellulose catalytic hydrolysates - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: Kluyveromyces marxianus C1 thermotolerant yeasts strain is able to produce ethanol. The yeasts strain is deposited in the Russian National Collection of Industrial Microorganisms under accession number VKPM Y-4290 and can be used to produce ethanol by fermenting sugars mixtures, being products of the one-stage catalytic hydrolysis-dehydration of microcrystalline cellulose.

EFFECT: invention enables to increase the yield of ethanol.

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Description

The invention relates to the field of biotechnology and is a strain of thermotolerant yeast Kluyveromyces marxianus, deposited in the All-Russian collection of industrial microorganisms FSUE GosNIIGenetika under the number Y-4290. The strain Kluyveromyces marxianus VKPM Y-4290 is designed to produce ethanol by fermentation of sugar mixtures, which are products of catalytic hydrolysis-dehydration of microcrystalline cellulose. The strain Kluyveromyces marxianus VKPM Y-4290 has a high thermal tolerance (up to 50 ° C), a specific growth rate (0.40 ± 0.05 h ^-1 ) and resistance to compounds formed as a result of catalytic hydrolysis-dehydration of cellulose (including 5-hydroxymethyl furfurol, furfural and etc.). When microcrystalline cellulose was cultured on a catalytic hydrolyzate, the ethanol yield was 83.9 ± 5.0% of theoretical.

Biomass is an important source of renewable raw materials for a wide range of materials and chemicals. Its composition includes lignin, hemicellulose and cellulose. Cellulose has found the greatest application as a biopolymer, which is a polysaccharide consisting of monomeric units of anhydro-β-D-glucopyranose (glucose), which are connected by 1,4-glycosidic bonds.

Currently, there are industrial technologies for the production of a number of chemicals obtained by processing lignocellulosic materials, including sugars (glucose, xylose), lignin and furfural. But the greatest application in biotechnology has received ethanol by fermentation of glucose formed during the hydrolysis of cellulose. In this case, hydrolysis is carried out using acids, for example sulfuric acid, and using alkalis, ammonia, or enzymes [1]. A significant drawback of chemical methods of hydrolysis, for example, acidic, is the formation of a certain amount of side compounds, including acids, for example, levulinic 0.1-0.3%, formic 0.03-0.1%, acetic 0.2-0.5%, etc., 5-hydroxymethylfurfural (0.03-0.18 %) and furfural (0.02-0.12%) [2]. Despite the fact that the listed compounds are valuable substances used in chemical synthesis, their small amounts obtained during the process of cellulose hydrolysis do not allow using them as independent products. Another disadvantage of chemical hydrolysis is the formation of a large amount of waste, which is a product of the neutralization of acids and alkalis (gypsum).

The use of enzymatic saccharification of delignified biomass, despite its environmental purity, is complicated by the inhibition of enzymes by products of the previous reaction and their high cost.

An alternative method of cellulose depolymerization is the use of a one-stage hydrolysis-dehydration process using solid acid catalysts, during which the main reaction products are 5-hydroxymethylfurfural (5-GMF) and glucose. 5-GMF can be extracted with high efficiency from a catalytic cellulose hydrolyzate using organic solvents. Thus, the resulting extract contains glucose and other soluble oligosaccharides (fructose, mannose, cellobiose), as well as a small amount of organic acids and furan derivatives (for example, furfural) and can be used for fermentation.

Obtained by homogeneous acidic (and alkaline) hydrolysis of sugar pulp is subjected to fermentation mainly using mesophilic yeast Saccharomyces cerevisiae. Using this type of yeast allows you to get up to 55-58 l of ethanol from 100 kg of fermentable sugars using hydrolysis mixtures [2]. However, significant problems for the use of S. cerevisiae yeast are their low resistance to elevated temperatures and to compounds formed during the hydrolysis of cellulose, including 5-GMP (IC ₅₀ 8 mm) and furfural (IC ₅₀ 5.8 mm) [3] . Basically, Saccharomyces cerevisiae have the ability to produce ethanol at temperatures of 25-37 ° C, which increases the cost of its production.

An alternative use of the yeast Saccharomyces cerevisiae for ethanol production is the use of thermotolerant yeast, including those belonging to the species Debaryomyces hansenii, Kluyveromyces bulgaricus, Kluyveromyces marxianus, Ogataea polymorpha, Ogataea parapolymorpha, Cryptococcus tepidarius, Pichia thermomethanolica, Candida krabiensis, Candida sithepensis. It is shown that these yeasts have the ability to grow at temperatures above 37-40 ° C, which makes the process of obtaining ethanol economically more profitable. The most promising ethanol producers among them are Kluyveromyces marxianus yeast. Unlike the yeast Saccharomyces cerevisiae, the thermotolerant Kluyveromyces marxianus yeast has the ability to consume xylose, cellobiose and arabinose in addition to glucose, and biomass production rates on substrates such as glucose, fructose and mannose in Kluyveromyces marxianus yeast are higher. Such properties, a high level of consumption of sugars, which are part of the hydrolyzate, as well as high resistance to compounds formed in the process of gyrolysis, are necessary to obtain an effective alternative producer of ethanol.

Previous studies on the production of ethanol on the delignified biomass hydrolysates obtained using homogeneous catalysts (acids and enzymes) showed the possibility of production of this compound by the Kluyveromyces marxianus yeast strains.

For example, in [4], when fermenting with Kluyveromyces sp.IIPE453 yeast on an enzymatically hydrolyzed bagasse cake at a temperature of 45 ° С, ethanol productivity of 0.36 g L ^-1 h ^-1 and 88% of the theoretical yield on a mixture of hexoses was achieved. In [5], fermentation of hydrolysates of Japanese cedar and eucalyptus obtained by enzymatic saccharification of mechanically activated biomass was studied. It was shown that at a temperature of 42 ° C for the Kluyveromyces marxianus DMB1 strain, the ethanol yield is 0.602 ± 0.094 g / g of consumed sugars and is able to grow at 48 ° C. A drop in performance indicators for ethanol productivity is associated with the presence of inhibitors in the hydrolyzate.

US 8906654 [6] describes strains of Kluyveromyces marxianus NRRL Y-50798 and Kluyveromyces marxianus NRRL Y-50799, obtained by UV mutagenesis, capable of fermentation of sugars (pentoses and hexoses) and production of ethanol. Under aerobic conditions, the strains ferment glucose (at 47 ° C) and xylose, and also grow anaerobically on media containing galacturonic acid, pectin, glucose, arabinose, xylose and galactose at 46 ° C. The disadvantage of this strain is the lack of data on its cultivation on hydrolysates of lignocellulose (cellulose).

The yeast strain Kluyveromyces marxianus SSSJ-0 described in US patent 8304219 [7] has the ability to utilize sugars, including cellobiose, glucose, mannose, galactose, or combinations thereof. Using it, ethanol production was shown in an amount of 125 mg / l ethanol on paper processing waste (350 mg) at 43 ° C by direct fermentation with the Kluyveromyces marxianus SSSJ-0 yeast strain. This invention focuses on the use of Kluyveromyces marxianus yeast by fermenting hydrolysates obtained by using homogeneous catalysts (acids or enzymes) to produce ethanol, rather than heterogeneous acid catalysts.

US 8268600 [8] describes a yeast strain Kluyveromyces sp.IIPE453 MTCC 5314, which is capable of fermenting sugars (including glucose, maltose, xylose, sucrose and starch) and fermenting the biomass hydrolyzate obtained using a dilute sulfuric acid solution and pre-purified using ion exchange resins at 50-55 ° C, in ethanol with a yield of 10-58.75%. The disadvantage of the strain used in this invention is that its cultivation is carried out on a hydrolyzate obtained by treating lignocellulosic biomass with a homogeneous catalyst (sulfuric acid), as well as a low yield of ethanol.

EP 1130085 B1 [9] describes an ethanol producing strain Kluyveromyces marxianus CEST 10875 (obtained by chemical mutagenesis), which was used to ferment the hydrolyzate of lignocellulosic biomass obtained by enzymatic treatment of biomass after a steam explosion for 72 hours at 42 ° C. The disadvantage of this invention is the use for cultivation of the strain Kluyveromyces marxianus CEST 10875 enzymatic hydrolyzate of biomass, accompanied by the need to use expensive hydrolytic enzymes.

The disadvantages of all these inventions that use strains of Kluyveromyces marxianus yeast to obtain ethanol are the lack of data on their use for the fermentation of catalytic hydrolysates of lignocellulose (cellulose), obtained as a by-product of 5-GMF and furfural. In the present invention, the use of such a substrate for cultivation allows (compared with other methods of hydrolysis of biomass) to reduce the amount of waste generated, as well as to process lignocellulose into ethanol using a strain of thermotolerant yeast Kluyveromyces marxianus in high yield.

The invention solves the problem of obtaining a producer strain of thermotolerant yeast for use in the process of producing ethanol by cultivation on a catalytic cellulose hydrolyzate.

The problem is solved by the fact that the proposed strain of thermotolerant yeast Kluyveromyces marxianus VKPM Y-4290, with the ability to grow and produce ethanol on the hydrolyzate obtained by single-stage catalytic hydrolysis-dehydration of microcrystalline cellulose. Compared with the Kluyveromyces marxianus and Saccharomyces cerevisiae strains described above, the use of the Kluyveromyces marxianus VKPM Y-4290 strain is preferable due to its high growth rate, resistance to the compounds included in the resulting hydrolyzate, and high thermal tolerance, which allows it to be effectively used for fermentation of catalytic hydrolysis cellulose to ethanol.

The Kluyveromyces marxianus yeast strain according to the invention is obtained from kefir on YPD medium with 20 μg / ml streptomycin at 30 ° C and isolated in a pure culture at a temperature of 40 ° C. The strain was identified by PCR according to the sequence of 18s rRNA and ITS 1. The strain was deposited in the All-Russian collection of industrial microorganisms of the Federal State Unitary Enterprise State Research Institute of Genetics under number Y-4290. Distinctive features of the strain Kluyveromyces marxianus VKPM Y-4290 are its high thermal tolerance (growth at temperatures up to 50 ° C), which allows it to reduce the cost of cooling during the fermentation process. During cultivation, it is able to ferment most of the sugars (including glucose, mannose, fructose) that make up the catalytic cellulose hydrolyzate. The strain is characterized by a high level of ethanol production, growth rate, and is also highly resistant to compounds that are part of the catalytic cellulose hydrolyzate, including 5-GMF and furfural, which allows the ethanol production process to be carried out with high efficiency.

Cultural and morphological features

When cultured for 3 days at 42 ° C in YPD medium, the culture of the strain Kluyveromyces marxianus VKPM Y-4290 forms round and oval cells 4-8 μm in size, both single cells and arranged in chains are observed. When cultured in suspension culture forms a precipitate. Propagated by budding, forms spores. The color of the colonies on YPD agar is dull cream, the colonies are flat, round with a smooth edge.

Physiological and biochemical characteristics

Ferments D-glucose, D-galactose, sucrose, raffinose.

Assimilates D-galactose, sucrose, cellobiose, raffinose, D-mannitol, ethanol, lactate, succinate, D-mannose, D-ribose, D-xylose, L-sorbose, D-glucuronic acid, lactose, maltose, succinic acid, D arabinose, glycerin. It grows in a vitamin-free environment and in an environment with 50% glucose. It grows at temperatures of 25-50 ° C and at pH 3.0-7.0.

The invention is further illustrated by the following examples, which do not limit the scope and essence of the claims associated with it.

Example 1. The cultivation of the yeast strain Kluyveromyces marxianus VKPM Y-4290 at temperatures of 37-50 ° C.

The overnight culture of the yeast strain Kluyveromyces marxianus VKPM Y-4290 is grown to an optical density of 0.8 - 1.0 at 595 nm in YNB minimal medium with a pH of 4.75 (containing g / l: KH ₂ PO ₄ 1.0, MgSO ₄ 0.5, NaCl 0.1, CaCl ₂ 0.1, (NH ₄ ) ₂ SO ₄ 5.0) with 5.0 g / L enzyme peptone, 3.0 g / L yeast extract and 20 g / L glucose. 20 μl of cells with a final concentration of 5 × 10 ⁶ cells in 1 ml are added to 180 μl of YNB medium and 1 g / l of yeast extract in a well of a 96-well plate with the corresponding monosaccharide (glucose, mannose, sucrose or cellobiose) at a concentration of 20 g / l Cultivation is carried out at 350 rpm for 16 hours at a temperature of 42-48 ° C.

In FIG. 1 shows the values of the optical density of the culture after cultivation. The strain Kluyveromyces marxianus VKPM Y-4290 has the highest growth on media with glucose, mannose and fructose.

Quantitative determination of the growth parameters of the strain Kluyveromyces marxianus VKPM Y-4290 is carried out by aerobic cultivation in deep 96-well plates in the Microflask system (Applikon Biotechnology). Cells are pre-grown in YNB medium with 5 g / L enzyme peptone, 3 g / L yeast extract and 20 g / L glucose, then they are separated by centrifugation at 500 × g for 10 minutes, washed twice, YNB medium with 2% glucose and 1 is added g / l of yeast extract to a concentration of 5 × 10 ⁵ cells in 1 ml The cultivation is carried out at 400 rpm for 8-10 hours, at a temperature of 37-50 ° C, in parallel, the optical density is determined at 595 nm and the amount of glucose in the culture fluid is measured by the glucose oxidase method.

They calculate the specific growth rate (μ h ^-1 ), glucose biomass yield (Y _{b / s} , g g ^-1 ), biomass (g L ^-1 ), the results are shown in table 1.

The Kluyveromyces marxianus VKPM Y-4290 strain has the maximum specific growth rate (μ) and biomass production at 42 ° C.

Example 2. The resistance of the yeast strain Kluyveromyces marxianus VKPM Y-4290 to fermentation inhibitors.

Aerobic cultivation of yeast in 96-well plates is carried out, as described in example 1, in the presence of compounds that are part of the catalytic cellulose hydrolyzate or formed during the fermentation process, including 5-GMF, furfural, acetic acid, formic acid, lactic acid, glycolic acid, tartaric acid, ethanol, at 42 ° C for 8-10 hours upon reaching the stationary phase of growth. The inhibitory effect of substances is evaluated as an IC ₅₀ index corresponding to a substance concentration corresponding to a 50% decrease in biomass growth, the data are shown in table 2.

The strain Kluyveromyces marxianus VKPM Y-4290 is highly resistant to 5-GMF and furfural compared to the values obtained for Saccharomyces cerevisiae yeast [3], as well as the strain is resistant to other compounds that are part of the catalytic cellulose hydrolyzate.

Example 3. The production of ethanol and biomass of the yeast strain Kluyveromyces marxianus VKPM Y-4290 on mixtures of sugars that are part of the catalytic hydrolysates.

For fermentation, a yeast culture is grown on a dense agar medium containing 20 g / l enzyme peptone, 10 g / l yeast extract, 20 g / l glucose, at 42 ° C for 2 days, one colony is added to 10 ml of pH medium 4.75, containing 5.0 g / l of enzymatic peptone, 3.0 g / l of yeast extract and a mixture of sugars (glucose 20 g / l, mannose 1.6 g / l, fructose - 0.8 g / l, cellobiose 1.3 g / l), salts from YNB medium, yeast extract 1.0 g / l and enzyme peptone 5 g / l are added, then cultivated aerobically at 42 ° C, 250 rpm until the optical density is reached and 1.0 at 565 nm. Then the cells are separated by centrifugation at 500 × g for 10 min and bring them in an amount corresponding to 1.0 g / l of dry biomass in 10 ml of the same medium, but not containing enzymatic peptone. Fermentation is carried out anaerobically with stirring at 100 rpm, at 42 ° C. During the experiment, the OD of the cells is evaluated at 595 nm and the composition of the culture fluid is analyzed. The composition of the products in the samples was determined by HPLC using a Shimadzu LC-20AD XR LC chromatograph on a Rezex ROA-Organic Acid H + 300 × 7.8 mm column (Phenomenex). As the mobile phase, 0.005 N sulfuric acid in water is used. The flow rate of the mobile phase is 0.6 ml min ^-1 , the analysis time is 50 min, and the column temperature is 65 ° C.

The results are shown in FIG. 2.

When cultivating the Kluyveromyces marxianus VKPM Y-4290 strain, the ethanol yield for fermentable sugars (glucose, mannose, fructose) is 0.44 ± 0.02 g / g, and the specific growth rate is 0.29 ± 0.01 h ^-1 . The ethanol yield as a percentage of theoretical (the maximum theoretical ethanol yield was taken as 0.521 g / g of fermentable sugars, including glucose, mannose and fructose) is 84.0 ± 4.2%.

Example 4. Production of biomass by the yeast strain Kluyveromyces marxianus VKPM Y-4290 on a catalytic microcrystalline cellulose hydrolyzate.

For the hydrolysis of mechanically activated microcrystalline cellulose, the Sibunit-4 catalyst oxidized with moist air is used [10]. The hydrolysis reaction is carried out at a temperature of 180 ° C and an argon pressure of 1 MPa, in an autoclave with stirring at 1500 rpm for 5 hours. The resulting solution is pre-filtered, then evaporated at a temperature of 70 ° C, after which one volume of isobutanol is added, mix thoroughly and the aqueous portion is taken, the extraction is repeated three times. The resulting solution was re-evaporated, the solution was neutralized to a pH of 4.75. After neutralization, salts of YNB medium and yeast extract are added to a concentration of 1 g / L. To neutralize using ammonia, NaOH or Ca (OH) ₂ . In the case of Ca (OH) _2, neutralization is carried out by adding Ca (OH) ₂ to a pH of 9.0, then titrated with hydrochloric acid to a pH of 4.75.

The results of determining the efficiency of biomass growth with various methods of neutralization are shown in table 3.

Example 5. The production of ethanol by the yeast strain Kluyveromyces marxianus VKPM Y-4290 on catalytic microcrystalline cellulose hydrolysates.

Obtaining a catalytic cellulose hydrolyzate is carried out as in example 4, except that ammonia is used to neutralize. Cultivation is carried out anaerobically in 96-well deep plates, in the Microflask system (Applikon Biotechnology). Cells are pre-grown in YNB medium with 5 g / l of enzymatic peptone and 3 g / l of yeast extract with 2% glucose, separated by centrifugation at 500 × g for 10 min, then washed twice, 1 ml of neutralized hydrolyzate with 1 g / l of yeast extract is added to a cell concentration of 1 and 2 g / l. Cultivation is carried out at 400 rpm for 48 hours at a temperature of 42 ° C. At the end of the cultivation, the optical density was determined at 595 nm and the products were analyzed by HPLC.

The data are shown in table 4.

The maximum ethanol yield of 83.9 ± 5.0% of theoretical is observed at an initial cell density of Kluyveromyces marxianus VKPM Y-4290 1.0 g / L.

Literature

[1] Gromov N.V., Taran O.P., Sorokina K.N., Mishchenko T.I., Utandi S., Parmon V.N. New methods for one-stage processing of polysaccharide components of lignocellulosic biomass (cellulose and hemicelluloses) into valuable products. Part 1. Biomass activation methods // Catalysis in industry. 2016. V. 16. No. 1. R. 74-83.

[2] Kholkin, Yu. I. Technology of hydrolysis production / - Moscow: "Forest industry", 1989. - 496 p.

[3] Jayakody LN, Horie K., Hayashi N., Kitagaki H. Molecular mechanisms for detoxification of major aldehyde inhibitors for production of bioethanol by Saccharomyces cerevisiae from hotcompressed water-treated lignocellulose Mendez-Vilaz A. Material and process for energy: communicating current research and technological developments. Formatex Research Center, Badajox, Spain. 2013.P. 302-311.

[4] Dasgupta D., Ghosh P., Ghosh D., Suman S.K., Khan R., Agrawal D., Adhikari D.K. Ethanol fermentation from molasses at high temperature by thermotolerant yeast Kluyveromyces sp.IIPE453 and energy assessment for recovery // Bioprocess Biosyst Eng. 2014 V. 37 No. 10. P. 2019-2029.

[5] Goshima T, Tsuji M, Inoue H, Yano S, Hoshino T, Matsushika A. Bioethanol production from Lignocellulosic biomass by a Kluyveromyces marxianus II Biosci. Biotechnol Biochem. 2013. V. 77. No. 7. P. 1505-1510.

[6] Patent US 8906654, C12N 1/16, 09/09/2014.

[7] Patent US 8304219, C12N 1/16, 11/06/2012.

[8] Patent US 8268600, C12N 1/16, 09/18/2012.

[9] Patent EP 1130085, B1. C12P 7/10, C12N 1/16, 10/05/2005

[10] Patent RU2583953, C07C 307/40, 05/01/2016.

Claims (1)

The strain of thermotolerant yeast Kluyveromyces marxianus VKPM Y-4290, used to obtain ethanol by fermentation of extracted catalytic microcrystalline cellulose hydrolysates.

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