RU2560585C1 - STRAIN OF BACTERIA Bacillus stratosphericus INTENDED TO PRODUCE ETHANOL FROM LIGNOCELLULOSIC BIOMASS - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of bacteria Bacillus stratosphericus Sol. NK 2, having the ability to produce ethanol, is deposited in RNCM under the registration number RNCM B-11678. The strain can be used for industrial production of ethanol, as well as in food industry, medicine, agriculture for feed production.

EFFECT: invention enables to extend the range of strains having the ability of processing lignocellulosic biomass to ethanol.

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Description

The invention relates to microbiology and biotechnology and can be used for the industrial production of ethanol, as well as in various industries, for example, medicine, food industry and agriculture for the production of feed.

Unlike starch, which contains homogeneous and easily hydrolyzable polymers, lignin cellulosic biomass is not homogeneous in nature. In addition to easily convertible cellulose and hemicellulose, it also contains lignin and other components that cannot be easily converted to fermentable sugars.

To date, various microorganisms known to produce ethanol are known.

The most common ethanol producer is yeast - Saccharomyces cerevisiae. These microorganisms are able to withstand high concentrations of alcohol and sugar, grow at a high concentration of cells, which allows them to achieve a significant rate of ethanol production. However, all representatives of this species are not capable of fermenting pentasugars, which makes their use unprofitable for processing lignocellulosic biomass.

For example, the known strain of yeast Saccharomyces cerevisiae B-3855 - producer of ethyl alcohol. The strain is thermo-tolerant, capable of efficiently fermenting molasses culture medium using non-phenolic class geothermal water with a carbohydrate concentration of 21.0 g / 100 cm ³ at temperatures of 30-32 ° C (patent RU 2492229 C1, op. 10.09.2013). However, this microorganism has a number of significant drawbacks: low growth rate and exactingness to the substrate.

Attempts have been made to use other types of yeast capable of fermenting xylose to ethanol. Three types of yeast have been most studied in this direction: Pichia stipites (application WO 2009004273 No. 1, op. 08.01.2009); Pachysolen tannophilus (application WO 1982003874 A1, op. 11.11.1982) and Candida lusitaniae (application WO 2010072093 A1, op. 01.07.2010). The main disadvantage of these microorganisms is their sensitivity to the effects of inhibitory agents resulting from the hydrolysis of lignocellulosic biomass.

A known method of producing ethanol by fermentation of pentose hydrolyzates of corn stalks with yeast Pachysolen zannophilus (Besic S. Grba S. Fermentation of hydrolyzate of corn stover hemicellulose to ethanol by Pachysolen tannophlus. Kem. Ind. 1990, v. 39, N 2, p. 57- 67), however, this method is characterized by a low yield of ethanol from wood hydrolysates, since hexose (prevailing in wood) and pentose are effectively fermented by different types of yeast under various process conditions.

Another common producer of ethyl alcohol is the bacterium Zymomonas mobilis. These bacteria are capable of producing ethanol in high concentrations (application WO 1986004357 A1, op. 07.31.1986). The disadvantage is their inability to ferment pentasugars.

It is known that bacteria of the genus Clostridium synthesize ethanol during the fermentation of various carbohydrates. So, the strain of Clostridium acetobutylicum VKM B-2512D is a producer of n-butyl alcohol, acetone and ethanol (patent RU 2393213 C1, op. 27.06.2010).

Also known is a strain of Clostridium thermocellum (application WO 2012109578 A2, op. 16.08.2012), capable of producing ethyl alcohol. This strain carries a mutant genotype with a deletion in the gene of the enzyme formate pyruvate lyase, which leads to the cessation of the production of formate, respectively, to reduce the yield of by-products.

An urgent task today is the search for new strains of bacteria that can efficiently assimilate pentasugar, which can account for up to 40% of all sugars contained in the plant biomass of fast-growing herbaceous plants. Such plants are the most promising sources of biomass, which can be used as a renewable source of energy and substances, cellulose-containing raw materials.

Closest to the claimed strain, the prototype, is a bacterial strain Clostridium acetobutylicum, which is a producer of butanol, acetone and ethanol from cellulose-containing raw materials (patent RU 2381270 C1, op. 10.02.2010). The strain is deposited in the All-Russian collection of microorganisms, IBPM them. G.K. Scriabin RAS under the number B-2531D. For the cultivation of the strain, nutrient media containing an autolysate of feed yeast, biotin and para-aminobenzoic acid are used. Fermentation is carried out by incubation at 36-37 ° C for 40 hours. The ethanol yield is 0.01-1.35%.

The disadvantages of the known strain are its anaerobicity, which requires additional equipment and complicates the cultivation conditions, as well as the need of the strain for growth factors (the need to add food raw materials, for example flour or bran, as well as mineral additives) to the nutrient media.

The objective of the invention is to obtain a bacterial strain that is a producer of ethanol, capable of converting cellulose-containing raw materials into ethanol and resistant to toxic components of lignocellulosic hydrolyzate.

The task is achieved by obtaining a strain of Bacillus stratosphericus, with the ability to produce ethanol from lignocellulosic biomass.

EFFECT: simplification of cultivation conditions of the strain and expansion of the assortment of producer strains for processing lignocellulosic biomass into ethanol.

The proposed strain was isolated from the natural material of the bottom sediment of Lake Solenoe, Novosibirsk Region (Kupinsky District) as a result of a targeted search.

The obtained strain of Bacillus stratosphericus was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) of the Federal State Unitary Enterprise of the State Research Institute of Genetics and Breeding of Industrial Microorganisms (GosNIIgenetika) under registration number VKPM B-11678.

The strain of Bacillus stratosphericus VKPM B-11678 is characterized by the following features.

Cultural and morphological characteristics: gram-positive bacilli, grow on LB-broth, LB-arap, Getchinson medium with lignocellulosic biomass. When grown on an agar medium, LB forms irregular white colonies. The profile of the colonies is slightly convex; sizes vary from 3 to 5 mm. Growth in liquid media (LB-broth) is characterized by even turbidity, the sediment is easily sedimented.

Physiological and biochemical signs: aerob, mesophile. The optimum growth temperature is 30-37 ° C. It grows well in the range of pH from 6.5 to 7.5. The strain is characterized by the ability to use peptone, a yeast extract as substrates. It does not utilize citrate, mannitol, malonate, inositol, adonitol, sorbitol, dulcite, pyruvate, sugar (glucose, cellobiose, sucrose, trehalose, rhamnose, melibiosis, raffinose) and amino acids (arginine, tryptophan, phenylalanine). Weakly decarboxylates lysine and ornithine. Not capable of hydrolysis of urea.

The strain does not have an infectious and general toxic effect.

The strain is non-pathogenic and is not included in the lists given in the sanitary rules of SP 1.3.2322-08; the strain does not carry dangerous genetic constructs.

The strain was identified based on sequencing of a fragment of the 16S gene of ribosomal RNA.

The strain is stored in Getchinson’s medium with glycerol at a temperature of -70 ° C.

For the cultivation of the strain used medium of the following composition:

LB medium (g / l): sodium chloride - 5.0; tryptone - 10.0; yeast extract - 5.0.

Hetchinson's medium (g / l): NaNO ₃ - 2.5, K ₂ HPO ₄ - 1, (NH ₄ ) ₂ SO ₄ - 0.15, MgCl ₂ - 0.15, NaCl - 0.1, CaCl ₂ - 0.1, lignocellulosic biomass - 15, FeCl ₃ * 6H ₂ O - 50 mcg.

The proposed bacterial strain Bacillus stratosphericus, with the ability to process lignocellulosic biomass into ethanol, has several advantages, which are as follows.

1. Bacillus bacteria have part of the enzymes of the cellulolytic complex and, therefore, an ethanol producer based on Bacillus strains can be a good basis for subsequent work aimed at creating a strain capable of direct conversion of lignocellulosic biomass to ethanol.

2. Based on bacteria of the genus Bacillus, it is possible to create effective complexes of microorganisms that degrade lignocellulosic biomass and produce ethanol.

3. Bacillus bacteria have a rather high growth rate and the rate of catabolic processes, therefore, the rate of lignocellulosic biomass conversion into ethanol will be higher than that of other organisms.

4. The bacterial strain Bacillus stratosphericus is resistant to the content of acetate and ethanol in the medium, which makes it promising for use in the production environment.

5. The strain is an aerobic and a mesophile, in connection with which the conditions for its cultivation are simplified, since additional heating and maintaining a high temperature of the culture medium are not required.

6. The strain exhibits more active growth on lignocellulosic biomass than on rich nutrient media (meat-peptone agar, Luria-Bertani broth), which will reduce the cost of plant biomass processing technology.

7. The strain is resistant to toxic components of lignocellulosic hydrolyzate, which will simplify the processing technology of plant biomass.

Since the proposed strain was obtained for the first time and it is characterized by a unique ability to process lignocellulosic biomass into ethanol, we can conclude that the proposed strain meets the criteria of the invention of "novelty" and "inventive step".

The invention is illustrated by examples of specific performance.

Example 1. Growing a strain of Bacillus stratosphericus and determining the ethanol content in the culture fluid.

To obtain biomass and determine the content of bioethanol in the culture fluid, the culture of the producer strain was scattered in streaks on plates with agarized Hatchinson medium with lignocellulosic biomass so that separate colonies were obtained. Inoculated plates were cultured in a dry-air thermostat for 24-48 hours at a temperature of 37 ° C. Then, 20 ml of Gatchinson’s medium with lignocellulosic biomass filtrate were inoculated with one colony of the produced strain. Cultivated at 37 ° C for 12 hours on an orbital shaker at a stirring speed of 200 rpm. After that, to 200 ml of Hetchinson’s medium with lignocellulosic biomass filtrate in a liter conical flask was added 4 ml of the accumulated culture. Cultivation was carried out at 37 ° C on an orbital shaker at a stirring speed of 250 rpm until an optical density of OD 600 ~ 0.90-0.95 was reached. Ethanol content was determined in the culture fluid thus obtained.

The ethanol content in the culture fluid was determined by gas chromatography with a mass spectrometric detector on an Agilent Technologies 6890N chromatograph with an Agilent Technologies 5973N quadrupole mass spectrometer and a DB-1 quartz column. The carrier gas is helium with a constant flow of 1 ml / min. Column temperature programming mode: initial temperature of the column thermostat 50 ° C, holding at the initial temperature of 5 minutes, then heating to 250 ° C at a speed of 25 ° C / min, holding at the final temperature of 7 minutes. The injection of the sample was carried out using a microsyringe, the volume of injected sample was 1 μl, the temperature of the injector was 250 ° C. Electron impact ionization (70 eV). After 15 hours of cultivation, 350 μl of culture fluid was taken into a test tube (1.7 ml) and 1050 μl of 0.5 Μ TOA (trioctylamine) in TBP (tributyl phosphate) was added. The extraction was carried out by stirring on a shaker (15 minutes). Then, 1 ml of the organic solvent with the extract was taken into a glass box for chromatograph. Ethanol concentration was determined by comparing the peak areas of the experimental samples with calibration solutions. The ethanol yield was 0.4%.

Example 2. The production of ethanol by a strain of Bacillus stratosphericus.

To determine the amount of ethanol produced by the Bacillus stratosphericus strain, the strain was cultured in a Vortex-5 vortex bioreactor with a programmable control unit (Center for Vortex Technologies, Russia) as follows.

The pre-investigated culture was grown on Hutchinson's nutrient medium with lignocellulosic biomass filtrate at a temperature of 37 ° C in 1/10 of the expected volume of the culture fluid in the bioreactor. After 12 hours, the inoculum was transferred to a bioreactor filled with 3 liters of Hutchinson medium with lignocellulosic biomass filtrate. During cultivation, the temperature was maintained at 37 ° C and pH 7.0. The initial stage of cultivation was carried out under aerobic conditions. Stirring was carried out in a vortex manner using an air mixer (2600 rpm). The ethanol content in the obtained culture fluid was determined analogously to example 1. The ethanol yield was 0.5%.

Using the proposed strain will simplify the process of culturing the latter and expand the range of strains with the ability to process lignocellulosic biomass into ethanol.

Claims (1)

The bacterial strain Bacillus stratosphericus, deposited under the registration number VKPM B-11678, designed to produce ethanol from lignocellulosic biomass.