WO2010024715A1

WO2010024715A1 - Strain of clostridium acetobutylicum and method of producing organic solvents

Info

Publication number: WO2010024715A1
Application number: PCT/RU2009/000351
Authority: WO
Inventors: Evgeniy Rubenovich Davidov; Petr Sergeevich Kanygin; Kirill Borisovich Filippov; Oleg Anatolievich Frakin; Igor Vladimirovich Cheremnov; Elizaveta Vasilievna Chekasina
Original assignee: Open Joint Stock Company "Corporation Biotechnologies"
Priority date: 2008-08-28
Filing date: 2009-07-13
Publication date: 2010-03-04

Abstract

The present invention relates to a novel strain of butanol, acetone, ethanol-producing bacteria Clostridium acetobutylicum and a method of producing organic solvents using a fermentation process that employs this strain.

Description

Strain of Clostridium acetobutylicum and method of producing organic solvents.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a novel strain of butanol, acetone, ethanol-producing bacteria Clostridium acetobutylicum and a fermentation process for producing organic solvents using this strain.

BACKGROUND OF THE INVENTION

Butanol, acetone and ethanol are important industrial chemicals and are currently used as solvents in making nitro dopes, synthetic resins; as a feedstock chemical in the plastics industry and as a food grade extractant in the food and pharmaceutical industry. The recent trend of using butanol as biofuel has revived interest in acetone/butanol/ethanol (ABE) fermentation.

The fermentation of carbohydrates to acetone, butanol and ethanol by solventogenic microorganisms including Clostridia is known from beginning of the 20^th century.

Traditionally such fermentations were performed (and still are in some cases) by a wild microorganism of Clostridium acetobutylicum isolated from the natural enviroment (soil, lake sediments, etc.), the process involved the random screening of a large number of isolates.

Then the bacteria was subsequently modified by conventional strain improvement strategies, using mutagenesis. Mutations result from a physical change to the DNA of a cell, such as deletion, insertion duplication, inversion and translocation of a pieace of DNA, or a change in the number of copies of an entire gene or chromosome. Spontaneous mutation rates are low, the rate can be greatly increased by using mutagens, which are two types: physical mutagens include ultraviolet, γ and X radiation; and chemical mutagens are compounds such as ethane methane sulphonate (EMS)₅ nitroso methyl guanidine (NTG), nitrous acid, etc. For example, RU 2080382 discloses the strain Clostridium acetobutylicum (VKPM B-5359) that is selected by induced mutagenesis and the following mutant clone selection on selective medium. Strain S-3716 accumulates 22 g/1 solvents including 15.5 g/1 n-butanol and 6.5 g/1 acetone at fermentation for 36-40 h on medium containing bee-sugar molasses and mineral salts. Carbohydrate conversion to the end product is 40%. The mutagen was ethane methane sulphonate or nitroso methyl guanidine.

JP 3058782 discloses mutant strain of Clostridium pasteurianum CA 101 (FERM P- 10817). Clostridium pasteurianum DSM 525 stock as a mother stock of genus Clostridium bacterium is anaerobically cultured in a nitrogen atmosphere at 37 ⁰C for 12 hours and collected by centrifuge, then subjected to mutation treatment with adding mutagen such as N-methyl-N'- nitro-N-nitrosoguanidine, thus resultant mutant is cultured in a medium containing crotonyl alcohol to afford a mutant of genus Clostridium bacterium having colony of crotonyl alcohol- resistant stock and butanol productivity.

US 5192673 discloses a fermentation process for producing butanol using a mutant strain of Clostridium acetobutylicum designated Clostridium acetobutylicum ATCC 55025. The biologically pure asporogenic mutant of Clostridium acetobutylicum is produced by growing sporogenic Clostridium acetobutylicum ATCC 4259 and treating the parent strain with ethane methane sulfonate. There are examples of batch and continuous fermentation.

US 4757010 discloses the Clostridium acetobutylicum mutant IFP 904 (ATCC 39058) obtained by spreading a culture of a strain of Clostridium acetobutylicum at the surface of a solid culture medium containing n-butanol at a specified concentration, growing the strain in the presence of a mutagenic agent and recovering a strain of increased resistance to n-butanol. The specification teaches that the various sugars, including hydrolysates of lignocellulosic material such as corn stalks are metabolized by Clostridium acetobutylicum. The fermentation was run in a batch mode.

EP 0973929 discloses the fermentation process for producing butanol, acetone and ethanol comprising steps of anaerobically culturing a culture of Clostridium beijerinckii BAlOl in a nutrient medium with assimilable carbohydrates and recovering butanol, acetone and ethanol, the amount of butanol recovered is from about 18 g/1 to about 21 g/1. There are experiments of batch and continuous fermentation.

However mutagenesis methods generally have rather limited use as they primarly achieve either loss of an undesirable characteristic or increasing production of a product, due to impairment of a control mechanism. Mutant strains are unlikely to survive well in nature, as they often have altered regulatory controls that create metabolic imbalances. Also they must then be maintained on specific media that select for, and help retain, the special characteristic(s) (Michael J. Waites et al., Industrial Microbiology: Introduction, Wiley-Blackwell, 2005, pp. 79- 81). Also there is some risk in use of mutant strains obtained after a particular mutagenic treatment because of disjoining and loss of properties. Unfortunately there is no information about the mutant stability in the cited references. The use of such strains in industrial scale and continuous processes requires the strict control for the strain performance.

Several processes developed in the last 20 years have involved recombinant microorganisms and genetic engineering technology has increasingly been used to improve established strains. However some workers have believed that (Eric J. Steen et al., Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol, Microbial Cell Factories 2008, 7:36) Clostridium are not ideal because of the relative lack of genetic tools to manipulate their metabolism, their slow growth. Neverless recombinant DNA technology has allowed specific gene sequences to be transferred from one organism to another and allows additional methods to be introduced into strain improvement schemes. This can be used to increse the product yeld by removing metabolic bottle-necks in pathways and by modifying specific metabolic steps (US 2007259410, WO 2007050671). However, these methods have not totally replaced traditional mutagenesis methods and the strain isolation from the natural enviroment. The key factor in the development of new strains is their stability (Michael J. Waites et al., Industrial Microbiology: Introduction, Wiley-Blackwell, 2005, pp. 79-81).

The economics of butanol has been studied extensively and the cost of substrate/raw material is one of the most influential factors impacting the economics of fermentation-derived liquid fuels. In order to reduce production cost, it has been attempted to produce butanol from renewable agricultural resources including wheat, corn and sugar beet (SU 1604852, RU 2080382), but the production of biofuels from edible substrates, such as starch or sucrose, would not be appropriate because of food shortage.

Prominent among the economic factors is the current surplus of agricultural waste, waste wood, waste of dairy industry, waste of biodiesel production that can be utilized as inexpensive fermentation substrates. Billiones of tonnes of these materials currently go to waste each year, which could be converted into chemical energy or useful fermentation products.

However lignocellulosic feedstock causes a number of difficulties with regard to preparation and fermentation of the fermentolysates and hydrolysates by Clostridium acetobutylicum. Lignocellulose is the most abundant renewable resource and is recognized as having great potential as a substrate for fermentation, provided that the hemicellulose and cellulose components can be degraded and utilized efficiently. The cellulose insolubility in water and heterogeneity cause the low possibility of the acid catalyst action and make cellulose resistent to fermentative hydrolisis. Therefore any lignocellulosic material especially such as coniferous sawdust requires pretreatment to obtain sugars that then be metabolized by Clostridium acetobutylicum. Pretreatment process includes dilute acid, steam explosion, hydrothermal processes, "organosolv" processes involving organic solvents in an aqueous medium, strong alkali processes using a base such as, ammonia, NaOH, mechanical treatment, etc.

Cheese whey attracts interest as an alternative substrate for ABE fermentation because of its disposal problem, lactose content, and availability in many countries. Biodiesel refers to a non-petroleum- based diesel fuel consisting of a mix of mono- alkyl esters of long-chain fatty acids. Biodiesel is commonly produced by transesterification of the vegetable oil or animal fat feedstock. A by-product of the transesterification process is the production of glycerol. For every 1 tonne of biodiesel that is manufactured, 100 kg of glycerol are produced. With the increase in global biodiesel production there is an enormous problem of this crude glycerol utilization. However as it turned out this glycerol can be used as fermentation substrate for producing acetone, butanol, ethanol.

One problem encountered in production of butanol by the fermentation process is the long time before the microorganism begins to produce appreciable quantities of solvent. It is obvious that the earlier the microorganism begins to produce solvents, the more it produces for the time period.

EP 0111683 discloses a biologically pure culture of Clostridium acetobutylicum ATCC 39236 that produces butyl alcohol at an accelerated rate in a batch fermentation and reaches a steady state of solvent production in a continuous fermentation in a much shorter time than does the parent strain. However in accordance with the invention there was no acetone production at 24 hours of the fermentation with the strain, the butanol concentration was 0.18 grams in 100 ml. At 72 hours the butanol concentration was 1.55 grams in 100 ml, acetone concentration was 0.46 grams in 100 ml.

Summarizing the aforesaid, a need exists for a novel stable strain with the following qualities: utilization of a wide range of low-cost and readily available carbon sources

(which in many cases are obtained from wastes); being easily cultured and developed for commercial purposes; genetic stability; has the high speed of growth and solvent production.

BRIEF SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a microorganism of Clostridium acetobutylicum 3108 useful for the production of acetone, butanol and ethanol by fermentation. The strain has been deposited with AU-Russian Collection of Microorganisms VKM in G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms Russian Academy of Sciences and assigned number B-2531D. The strain is a naturally occurring isolate that does not require any genetic manipulations, to be effective. The strain advantage is use of cheap substrates, the ease of its mass cultivation, speed of growth and solvent production. Further in accordance with the present invention, there is provided a method of producing organic solvents which comprises anaerobically culturing a biologically pure culture of Clostridium acetόbutylicum 3108 in a nutrient medium with assimilable sources of carbon, nitrogen, and inorganic substances and then recovering the organic solvents. In one embodiment the assimilable source of carbon is a fermentolysate of soft- and hardwood sawdust. In one embodiment the assimilable source of carbon is a cheese whey. In one embodiment the assimilable source of carbon is a waste of biodiesel production.

Other advantages of the invention will be apparent to those skilled in the art from description of the strain, process and the examples.

DETAILED DESCRIPTION OF THE INVENTION

The strain is prepared by a method of analytic selection of isolates from rice bran (hand threshing). The isolates were selected on the subsequent repeated seeding and growing at the selective mediums, containing the different carbohydrates. Step selection was carried out depending on the fermentation of several types of carbohydrates. From about 0.5 g/1 to 1.0 g/1 of butanol was added to the medium after sterilization to increase the butanol resistance. The fermentation medium was spread at agar medium with butanol, grown anaerobically, selected and reseeded at the liquid nutrition mediums that are different on carbohydrate component, nitrogen source, butanol quantity. The fermentation mediums were analyzed and examined on the accumulated butanol quantity, the unfermented sugars, and the fermentation gas. The isolates were selected, the most active was the isolate No 8 (version 31), that proves its high coefficients at the repeated inoculation and was selected as Clostridium acetobutylicum No 3108.

In the preferred method of the invention, the strain is employed in a process configuration which comprises anaerobically culturing a biologically pure culture of Clostridium acetobutylicum 3108 in a nutrient medium with assimilable sources of carbon, nitrogen, and inorganic substances and then recovering the organic solvents.

The strain has the following cultural, morphologic and physiologic properties.

PROPERTIES OF THE STRAIN OF CLOSTRIDIUM ACETOBUTYLICUM

Morphology: Vegetative bacterium cells with good motion, large rods, isolated or in pairs, short chains depending on the phase growth, Gram positive. Produces endospores. The spore formation type is bacillary; the spores are oval, diameter 2-2.5 μm.

The cell size: at 5-8 h of growth d=1.3-1.5 μm, chains, long rods.

1= 12-20 μm; at 20-32 hours of growth d=1.3-1.5 μm, moveable rods (sometimes in pair) 1=5-8 μm. Morphology in the different nutrient medium:

Agar potato medium with glucose.

Round, smooth colonies with round ends of 1-2.2 mm diameter were formed in 48 hours of growth under strictly anaerobically conditions. The structure of colonies is uniform, from light-gray to white color, non-transparent, the surface is smooth, glossy.

Molasses flour medium.

Round, smooth colonies of 1-2.0 mm diameter were formed in 48 hours of growth under anaerobic conditions, round ends, uniform structure, gray-white color, transparent, and raised.

Medium of fermentolysate of sawdust and additives.

Colonies of 1.0 mm diameter were formed in 48 hours of growth under anaerobic conditions at 37 ⁰C, white, non-transparent, round, flat ends, the surface is glossy.

Physiology and biochemistry.

Obligate heterotrophic anaerobe.

Gelatine: fluidifies

On meat-and-peptone (plain) agar (seeded with a syringe) - low growth, breaks with fermentation gas.

Carbon-containing substrates:

Starch, maltose, glucose, saccharose, fructose, xylose, galactose, mannite, mannose, arabinose, lactose, dextrin, cellobiose, glycerol: utilized.

Raffinose: not utilized, dulcite: not utilized.

Other properties: sulfites: reduced.

Nitrogen source: assimilates nitrogen compounds in reductive form.

Phosphorous source: assimilates

Growth factors: needs biotin, p-amino benzoic acid

Temperature: grows at 30-38 ° C; optimum is 36-37 ° C.

It grows at pH 4.5-7.0, optimal pH 5.5-6.5.

Spores were stored in a liquid potato-glucose medium, a molasses-flour medium or a fermentolysate of sawdust in lyophilized state.

The properties of the strain have led to its identification as a Clostridium acetobutylicum strain.

EXAMPLE 1

Method of isolation of Clostridium acetobutylicum 3108.

The bacterial strains were isolated from the rice bran (hand threshing) obtained from the Ail-Russian research institute of rice (RF, Krasnodar). 0.3 g of rice bran were placed in 30 test tubes, containing 5 ml potato medium. The tubes were closed with cotton stopper and placed in the anaerobic chamber where the tubes were incubated for 7 days at 36 ⁰C. As a result 14 tubes with intensive foam and gas formation were used for further study.

Petri dishes with agar-containing potato medium were prepared in the anaerobic chamber. Serial 10-fold dilutions of the suspensions were made and 1 ml from the suspensions (dilution 10^"4) were spread on Petri dishes. Dishes were incubated in the anaerobic chamber at 36 ⁰C for 7 days. Dishes were examined microscopically every 2 days for new colony formation. Colonies that had the morphology typical of Clostridium (small rods with round ends) were picked. Such colonies were placed in tubes with 5 ml of potato medium. The tubes were incubated for 7 days at 36 ⁰C. As a result 5 tubes with intensive foam and gas formation, high butanol production were selected.

100 ml seed flasks (the operating volume is 50 ml) with potato medium were inoculated with material from the tubes. The culture was pasteurized for 1 minute on the boiled water bath before inoculation. The seed flasks with inoculated medium were incubated for 7 days.

The results indicate that the culture in the flask No 8 (mother flask) gave the best characteristics (gas and acid formation, solvent productivity, butanol tolerance, morphology). This isolate has been deposited with All-Russian Collection of Microorganisms VKM in G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms Russian Academy of Sciences and assigned number B-2531D.

EXAMPLE 2

Production of solvents with the obtained strain at fermentolysate of soft- and hardwood sawdust.

Spores of the resultant strain Clostridium acetobutylicum 3108 are pasteurized on the boiled water bath for 1 minute. Cell suspension was inoculated in 150 ml of sterile liquid potatoes-glucose medium containing: potatoes -37 g/1, glucose - 0,75 g/1, CaCθ3 - 0,3 g/1, (NRi)₂ SO₄ - 0,225 g/1, the pH of the medium was adjusted to 6.2, the medium was sterilized by heating in an autoclave at 120 ⁰C for 40 minutes. The medium was incubated at 37 ⁰C in thermostat.

In 12 hours of incubation at 36.5 ⁰C (exponential phase of growth) seed culture (5% of volume of fermented medium) was inoculated at the sterile nutrient medium in 4-liter fermenter (the operating volume was 3 liters).

Medium components: fermentolysate of sawdust - 2,85 1 (ratio glucose: xylose as 8:2, concentration of carbohydrate is 5,4%); yeast autolysate - 3 g/1; biotin~ 3 mg/l; p-amino benzoic acid - 0.003 g/1.

The fermentolyzate sterilization (2.85 1) was carried out through the prepared membrane filter with pore diameter no more than 0.22 μm. 200 ml previously sterilized at 115 ⁰C solution, containing yeast autolizate, biotin, p-amino benzoic acid, was introduced into the sterile fermenter. The fermentation was run at 36.5 ° C and it was completed in 40 hours. The concentration of unfermented carbohydrates was 0.28 g/1. The solvent recovery may carry out by activated sorbents, membranes and distillation. The results obtained when the fermentation was run at the fermentolysate of sawdust are shown in Table 1.

EXAMPLE 3

Production of solvents with the obtained strain at cheese whey.

Spores of the resultant strain Clostridium acetobutylicum 3108 are pasteurized on the boiled water bath for 1 minute. Cell suspension was inoculated in sterile medium consisting of molasses and flour (ratio equivalent of fermented sugars as 1:1). In 12 hours of incubation at 36.5 ⁰C seed culture (5% of volume of fermented medium) was inoculated at the sterile nutrient medium in 4-liter fermenter (the operating volume was 3 liters).

Medium components: cheese whey — 2,85 1 (lactose content — 20g/l); sucrose - 100 g; wheat germ meal - 3 g/1;

The carbohydrate concentration is 5.4%.

Sucrose and pulverized wheat germ meal were introduced into the cheese whey and the medium was sterilized at 110 ⁰C for 30 minutes. The fermentation was run at 36.5 ⁰C. The concentration of unfermented carbohydrates was 0.25 g/1. The solvent recovery may carry out by activated sorbents, membranes and distillation. The results obtained when the fermentation was run at the cheese whey are shown in Table 1.

Table 1

Solvent production by the strain Clostridium acetobutylicum 3108 at the different mediums (g/1)

These results indicate that the resultant strain has ability to ferment both the fermentolysate of soft- and hardwood sawdust and the cheese whey. Therefore a variety of renewable resources can be employed for the production of butanol, acetone, and ethanol.

The following experiment shows that the resultant strain has the production rate better than the known strain.

EXAMPLE 4

The rate of solvent production with the strain obtained.

The rate of solvent production was determined in fermenter containing aqueous synthetic medium containing per liter: (NKt)₂SO₄ 3 g, K₂HPO₄ 0.5 g, MgSO₄*7H₂O 10 mg; CaCO3 - 3 g, yeast extract 4 g; glucose 20 g. The substrate was thus glucose. The culture was effected in conventional conditions of sterility and in anaerobic conditions. The results are given in the following Table 2:

Table 2

Production of acids and solvents by the strain Clostridium acetobutylicuni 3108 (g in 100 ml).

Results presented in Table 2 clearly indicate that the obtained strain begins to produce solvents at the 6^th hour of the fermentation and within 24 hours the butanol concentration is 0.91 g and acetone concentration is 0.30 g in 100 ml of broth. This example shows that the obtained strain produces solvents more rapidly and in better yield than does the known strain ATCC4259 (see EPOl 11683, within 24 hours the butanol concentration was 0.18 g in 100 ml and there was not acetone hi broth).

Therefore the strain Clostridium acetobutylicum 3108 was obtained from the naturally occurring isolates, the strain has ability to ferment a wide range of low-cost and readily available carbon sources (which in many cases are obtained from wastes); it is easily cultured and developed for commercial purposes.

Claims

1. A biologically pure culture of Clostridium acetobutylicum 3108.

2. A method of producing organic solvents which comprises anaerobically culturing a biologically pure culture of Clostridium acetobutylicum 3108 in a nutrient medium with assimilable sources of carbon, nitrogen, and inorganic substances and then recovering the organic solvents.

3. The method of claim 2 wherein the assimilable source of carbon is a fermentolysate of soft- and hardwood sawdust.

4. The method of claim 2 wherein the assimilable source of carbon is a cheese whey.

5. The method of claim 2 wherein the assimilable source of carbon is a waste of biodiesel production.

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