Method for obtaining spore material of bacteria of clostridium type
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FIELD: biotechnologies.
SUBSTANCE: method for obtaining spore material of bacteria of Clostridium type provides for production of inoculum of bacteria in a full synthetic growth medium, seeding of inoculum and cultivation under the corresponding conditions in growth medium including potato, glucose, ammonium sulphate and chalk. During the main fermentation process for 18-28 parts of bacterial culture growth depending on time of occurrence in one field of view of at least 80-100 thickened forms of cells, n-butanol in the amount of 0.16-0.81 wt % is added to growth medium. Number of formed spores is 1.08-2.86·108 in one millilitre of the medium.
EFFECT: increased content of spores in spore material.
1 tbl, 15 ex
The invention relates to the biotechnology industry and the receipt of spore material of bacteria of the genus Clostridium used when getting an organic solvent (n-butanol, acetone and ethanol) microbiological method.
Organic solvents are widely used in many sectors of the economy: production of synthetic rubber, silk, the extraction of raw materials for pharmaceuticals and organic products. The most valuable product of the obtained organic solvent is n-butanol in connection with the emergence of new trends in using n-butanol as a biofuel, alternative hydrocarbon [Debabov VG Biofuels // Biotechnology. - 2008. No. 1. - P.3-14].
For the microbiological production of n-butanol in an industrial scale traditionally used anaerobic solventogenic (i.e. forming solvent: butanol, ethanol and ethanol), bacteria of the genus Clostridium, namely .acetobutylicum, C.beijerinckii, C.saccharoperbutylacetonicum, .saccharobutylicum and C.aurantibutyricum, .thermosaccharolyticum, .thermoaceticum.
For stage acidogenesis use strains of anaerobic bacteria such as Clostridium tyrobutyricum, C.thermobutyricum, C.butyricum. C.felsinium, C.pasteurianum, C.sporogems.
Given the strains differ in their ability to form alcohols and acids and utilize the nutrient subst the ATA.
One of the oldest processes in biotechnology is a microbiological method for producing organic solvent (n-butanol, acetone and ethanol) by bacteria Clostridium acetobutylicum.
In acetone the process of Clostridium acetobutylicum during fermentation of different carbohydrates are synthesized simultaneously three target product: n-butanol, acetone and ethanol, the percentage ratio of 60:30:10. This ratio is not strictly constant, and may significantly vary in the direction of increasing the output of a product of fermentation, depending on the properties of the producer strain, from the technological processes, types of fermentation raw materials and other
Members of the genus Clostridium are anaerobic bacteria which are capable of forming spores. Vegetative cells are in the form of straight rods, single or forming a pair. At the end of the exponential phase of growth, cells begin to accumulate granules and form the extracellular capsule that leads to changes in their shape from rod-shaped to cigar-shaped (i.e. form clostridia). Morphological changes usually associated with switching of metabolism from the synthesis of the acid to the synthesis of neutral products of alcohols and acetone. In the aging cell culture lose their mobility and move to sporoobrazovanie. The resulting disputes have avelinawile spherical shape. Bacterial spores getting into a favorable environment, germinate into vegetative cells that grow and divide, making the substrates in the final products [O. Berezina, Zakharova N., jarocki ST., Zverlov CENTURIES Microbial producers of butanol // Biotechnology. - 2011. No. 4. - P.8-25].
The main stages of development of the bacteria Clostridium, namely Clostridium acetobutylicum (life cycle) is divided into five types: the initial growth of elongated cells (Type I); the disintegration of threads on individual cells (Type II); most active stage of culture (Type III); the beginning of autolysis and education clostridia (Type IV): education dispute (Type V) [Lopatkin I.S. Technology acetone fermentation. M - 1958].
The process of microbiological synthesis of organic solvents begin with the revival of the bacteria Clostridium botulinum, which is stored in the form of spores. The sporulation is a property of bacteria to withstand adverse conditions. In bacteria, Clostridium acetobutylicum, in particular, to sporoobrazovanie passes a small number of cells: usually from 0.03 to 0.1×108cells/ml, which equals to the maximum number of bacteria from 0.1 to 0.3%. Other bacteria in old cultures die and lyse [Lopatkin I.S. Technology acetone fermentation. M - 1958].
Describes the different ways of getting spore material of bacteria of the genus Clostridium. So 25%potato medium used for Clostidium butyricum [SU 339191]; complex nutrient medium containing hydrolyzed meat, casein, corn extract, yeast, thioglycolic acid for dispute test culture Clostridium sporogenes [SU 1712408]; potato pulp with the addition of ammonium sulfate, corn extract, ferric chloride and chalk receive the spores of Clostridium felsinium of 5 to 6×106in 1 ml of [SU 293045].
A method of obtaining spores, bacterial cells of Clostridium thermosuccharolyticum by adding to the culture medium of divalent cations and slowly metabolizing a carbon source of xylan. In this mode select conditions for obtaining synchronized cells and to conduct the process in such a way as to selectively induce the formation of products of metabolism and spores. As a source of divalent cations use calcium salt [RU 2091483].
Numerous works by the method of obtaining n-butanol and application of new strains of Clostridium acetobutylicum reported storage method of culture of acetone bacteria in the form of spores in liquid nutrient media in sealed tubes at a temperature from + 4°C to room temperature. Almost from source to source quoted the same sentence. Storage of strain in the form of spores in liquid nutrient medium: 6% environment flour, potato-glucose, molasses-flour or other synthetic When specified with the food used for the primary fermentation process, and spore material before sowing pasteurized [EN 2381279, EN 2393213, EN 2404247, EN 2455350, EN 2458118].
Information about the number of spores of the bacteria Clostridium acetobutylicum formed in the used liquid nutrient media not found.
A method of obtaining spore material by fermentation of flour production environments strain of Clostridium acetobutylicum [production schedule for the production of solvents: acetone, butanol and ethanol by the method of fermentation. PR 64-35-89, G. Efremov, 1989].
Preparation of spore material is produced 6%environment flour (corn, rye or wheat). Apply two variants of the workpiece dispute: a) mass seeding and b) the method of spill:
a) when the mass sowing of each tube with cooked fresh sterile environment flour sow themselves and the entire fermentation cycle, ending with the formation of spores, spend in it from beginning to end;
b) in case of a spill fermentation is carried out in a large glass vessel, which at the end of the process the fermented medium is poured into sterile tubes, which are then sealed and stored at room temperature.
After a two-month shelf life activity of spore material is checked by repeated subcultures to fresh nutrient medium after 24 hours (5-7 subcultures). A final check is carried out on the product is only 8%flour environment. The quality of spore material appreciate for the following technical parameters: no starch (24 hours); the total amount of released gases of fermentation (not less than 30 g/l); the content of acetone (at least 6 g/l); the content of non-fermented carbohydrates (not more than 0.3%). The process of checking the activity of spore material is quite laborious and time consuming.
The closest analogue of the claimed method is a method for spore material of the bacteria Clostridium acetobutylicum on potato-glucose medium, of the following composition (wt.%): potatoes - 25.0, glucose and 0.5, ammonium sulphate is 0.15, chalk - 0,2, water - the rest, which is then used as a nutrient medium for the recovery of this spore material [RU 2080382]. Medium containing potato chemical composition richer than containing flour. In potato tubers contain proteins (up to 2%) carbohydrates (starch - 13,1-36,8%), cellulose, pectin, mono - and oligosaccharides: glucose, fructose, sucrose, and vitamins and mineral salts. Basic vitamins - ascorbic acid, the whole complex of b vitamins: B1, B2, B6, folic and nicotinic acid. The composition of the potatoes with more than 20 different chemical elements. Of mineral salts, potassium salts prevail (400-500 mg%) and phosphorus (45-50 mg%), as well as micro - and macro-elements - iron, calcium, magnesium, manganese, Nickel, Koba is before, the iodine. The potato contains all the substances necessary for the growth and development of acetone bacteria.
In the way is the nearest analogue of the number of spores in the composition used spore material of the bacteria Clostridium acetobutylicum is not specified.
The technical task of the present invention to provide a spore material of bacteria of the genus Clostridium with a high content dispute.
The task is solved by developing a method of producing spore-forming material of the bacteria Clostridium by inoculation inoculate the bacteria Clostridium botulinum grown in complete synthetic medium, followed by cultivation in suitable conditions in a nutrient medium consisting of potato, glucose, ammonium sulfate and calcium carbonate, which is in the process of primary fermentation at 18-28 o'clock in the development of a culture of bacteria depending on the time of occurrence in a single field of view of at least 80-100 thickened form cells of clostridia, add n-butanol in the amount of 0.16 is 0.81 wt.%.
One of the main features of the proposed method is to use in experiments, cultures of micro-organisms in a specific physiological age.
The method in General
The inventive method is as follows. Cook the potato medium of the following composition (wt.%): potatoes (starch content of 17-19%) - 20,0, glucose and 0.5, sulfuric am one - 0,15, chalk - 0,2, water - the rest, sterilized at 0.8 ATM for 20-25 minutes, cooled to a temperature of 37°C. Then the medium inoculated with inoculum of bacteria of the genus Clostridium, pre-grown in complete synthetic environment, in particular the PSS environment the following composition, wt.%: K2HPO4- 0,07; KH2PO4- 0,07; MgSO4·7H2O - 0,01; MnSO4·H2O - 0,002; FeSO4·7H2O - 0,0015; NaCl - 0,001; glucose - 2,0; ammonium acetate to 0.3; yeast extract - 0.1; bakterien - 0.1; cysteine - 0,05, water the rest at 37°C for 24 hours (EN 2080382), or on the environment MSS (medium synthetic structure) of the following composition, wt.%: K2HPO4- 0,1; KH2PO4- 0,08; MgSO4·7H2O - 0,1; FeSO4·7H2O - 0,005; ammonium acetate to 0.3; yeast extract and 0.5; cysteine-HCl - 0,05; para-aminobenzoic acid is 0.001, glucose - 2, water the rest at 37°C for 24 hours (EN 2455350), or on the environment SOL (solution) of the following composition, wt.%: K2HPO4- 0,07; KH2PO4- 0,07; MgSO4·7H2O - 0,01; MnSO4·H2O - 0,002; FeSO4·7H2O - 0,0015; NaCl - 0,001; ammonium acetate to 0.3; yeast extract - 0.1; peptone - 0.1; cysteine - 0.05; starch - 0,1; glucose - 1,0; vitamin solution 1 ml/l (para-aminobenzoic acid and 0.0001; thiamine - 0,0001; Biotin - 1 µg); resazurin is 0.0001, water the rest at 37°C for 24 hours (EN 2458118). Environment MSS, MSS and SOL prepared as follows: sample components with the food consistently transfer into the tank, dissolved in distilled water, poured into special bottles with a total volume of 120 cm for anaerobic cultivation of bacteria, boil on a water bath for 25 min, saturated with nitrogen (to create anaerobic conditions), closed with rubber stoppers and screw up metal lids. Then sterilized by autoclaving at 116°C for 20 minutes.
The process of anaerobic fermentation conducted on potato environment within 18-28 hours at 37°C. Starting with 18 hours of fermentation, the culture of the bacteria examined under a microscope for the purpose of recording the appearance of thickened form cells of clostridia. If the same field of view at least 80-100 clostridia in medium was added n-butanol in the amount of 0.16 is 0.81 wt.%. The process then continues for up to 48 hours. At the end of the fermentation, the fermented medium counted under a microscope using a counting chamber Goryaeva the number of the resulting dispute. The inventive method allows to obtain spore material containing up to 3.6×108spores/ml.
Storage of seed material of bacteria of the genus Clostridium carried out by placing in a special glass test tubes with rubber stoppers and screw caps for storage anaerobic cultures in liquid medium at +4°C or freeze-dried for storage in analiticheskoy condition.
Example 1. The receipt of STRs is a new material for traditional industry method for flour environments [production schedule for the production of solvents: acetone, butanol and ethanol by the method of fermentation. PR 64-35-89, G. Efremov, 1989].
Cook flour environment by mixing rye flour (starchy 58%) with water at the rate of 6 wt.% by weight. The resulting mass razvarivat for 60 min in a boiling water bath water and sterilized at 1.5 ATM for 1.5 hours. Next cool environment to a temperature of 37°C and inoculated with inoculum, representing an industrial strain of bacteria Clostridium acetobutylicum VKPM b-43 61-grown flour environment, in the amount of 5% by volume environment. The fermentation is carried out at a temperature of 37°C for 60 hours. The number of the resulting dispute was 0.3×108in ml After fermentation spore material is left for storage. By the end of the month party dispute subjected to the test, conducting the test ferment for 6 and 8% flour environment by replanting from one test tube to another with a freshly prepared 6%flour medium. The first replanting carried out after 24 hours, and the remaining 14 hours. The last audit to 8 wt.% flour the environment.
The result is a standard bacterial spores for industrial applications, the quality of spore material judged by the amount of released gases of fermentation and accumulation of acetone in the environment.
Example 2. Getting spore material strain Clostridium acetobutylicum In PMBC-5359 on potato medium without added n-butanol (control to C the show method) using inoculum, grown on the PSS environment
Use a strain of Clostridium acetobutylicum In PMBC-5359 stored in lyophilized form in a sealed glass ampoules. To obtain inoculum (inoculum), the ampoule is opened, add 500 ál of sterile distilled water, leave for swelling, then 100 µl of the cell suspension is transferred with a syringe in a sealed vial with a nutrient medium PSS and cultured at 37°C for 24 hours.
Environment for producing inoculum prepared as follows: sample components PSS consistently transfer in chemical beaker 1 l (placed on the magnetic stirrer), dissolved in 1000 liters of distilled water, poured into 60 ml in special flasks for anaerobic cultivation, boil on a water bath for 25 min, after boiling the environment is saturated with nitrogen, closed with rubber stoppers and screw up metal lids. Then sterilized by autoclaving at 116°C for 20 minutes.
To obtain spore material used potato starch content of 19%. The environment is prepared as follows: a portion of potatoes 200 g (pre-cleaned) are ground on a fine grater, transfer in chemical beaker 1 l, add 600 ml of distilled water, placed into a water bath and stirring constantly, heat on the gelatinization of potato and razvarivat within 30 minutes. Sample of glucose (5 g), (NH4)2SO4(1.5 g) and caso3(2.0 g), dissolved in 100 ml of water and transferred to klasterizovannykh potatoes. Razvarivat for 20 minutes, then the volume of the medium was adjusted to 1 liter, poured into 70 ml glass vials (special for anaerobic fermentation) and sterilized by autoclaving at 116°C for 20 minutes. After that, the medium is cooled to a temperature of 37°C, then in a prepared potato Wednesday contribute inoculum obtained on the environment operates in an amount of 5% of the volume of the environment; the process of fermentation is carried out at a temperature of 37°C for 48 hours. After fermentation fermented environment count the number of spores formed, the content of which after fermentation is 1.06×108in ml.
Example 3. Getting spore material strain Clostridium beijerinckii VKPM B-9600 on potato medium without added n-butanol (control to the claimed method) using inoculum grown on the PSS environment
Repeat the procedure described in example 2, but using a strain of Clostridium beijerickii VKPM B-9600 stored in lyophilized form in a sealed glass ampoules. After fermentation fermented environment count the number of spores formed, the content of which after fermentation is 0.98×108in ml.
Primer. Getting spore material strain Clostridium saccharoperbutylacetonicum VKPM B-9602 on potato medium without added n-butanol (control to the claimed method) using inoculum grown on the PSS environment
Repeat the procedure described in example 2, but using a strain of Clostridium saccharoperbutylacetonicum VKPM B-9602 stored in lyophilized form in a sealed glass ampoules. After fermentation fermented environment count the number of spores formed, the content of which after fermentation is 1.18×108in ml.
Example 5. Getting spore material strain Clostridium butyricum VKPM B-9619 on potato medium without added n-butanol (control to the claimed method) using inoculum grown on the PSS environment
Repeat the procedure described in example 2, but using a strain of Clostridium butyricum VKPM B-9619 stored in lyophilized form in a sealed glass ampoules. After fermentation fermented environment count the number of spores formed, the content of which after fermentation accounts for 0.86×108in ml.
Example 6. Getting spore material strain of Clostridium tyrobutyricum In PMBC-9615 on potato medium without added n-butanol (control to the claimed method) using inoculum grown on the PSS environment
Repeat the procedure, is written in example 2, but use a strain of Clostridium tyrobutyricum In PMBC-9615 stored in lyophilized form in a sealed glass ampoules. After fermentation fermented environment count the number of spores formed, the content of which after fermentation is 0.78×108in ml.
Example 7. Getting spore material of the inventive method with the use of a strain of Clostridium acetobutylicum In PMBC-5359 using inoculum grown on the PSS environment
The process is carried out according to example 2. In the fermentation process, since 18 hours, under a microscope, examine the culture for the purpose of recording the appearance of clostridia. After discovering them in the amount of 100 in the same field of view in the environment type of 0.41 wt.% n-butanol and hold the process up to 48 hours. At the end of the fermentation process in the environment the number of the resulting dispute is to 2.75×108in ml, which is 2.6 times more than in the control.
Example 8. Getting spore material of the inventive method with the use of a strain of Clostridium beijerinckii VKPM B-9600 using inoculum grown on the PSS environment
The process is carried out according to example 7, but using strain Clostridium beijerinckii VKPM B-9600. At the end of the fermentation process in the environment the number of the resulting dispute is of 2.68×108in ml that 2.73 times higher than in the control.
Example 9. Getting spore material Appl the subject method using strain Clostridium saccharoperbutylacetonicum VKPM B-9602 using inoculum, grown on the PSS environment
The process is carried out according to example 7, but add to 0.62 wt.% n-butanol using a strain of Clostridium saccharoperbutylacetonicum VKPM B-9602.
At the end of the fermentation process in the environment the number of the resulting dispute is 2,54×108in ml, that of 2.15 times higher than in the control.
Example 10. Getting spore material of the inventive method using a strain of Clostridium butyricum VKPM B-9619 using inoculum grown on the PSS environment
The process is carried out according to example 9, but using strain Clostridium butyricum VKPM B-9619. At the end of the fermentation process in the environment the number of spores formed around 1.98×108in ml, which is 2.3 times more than in the control.
Example 11. Getting spore material of the inventive method using a strain of Clostridium tyrobutyricum In PMBC-9615 using inoculum grown on the PSS environment
The process is carried out according to example 7, but using a strain of Clostridium tyrobutyricum In PMBC-9615.
At the end of the fermentation process in the environment the number of the resulting dispute is of 1.85×108in ml, which is 2.4 times more than in the control.
Example 12. Getting spore material strain Clostridium acetobutylicum In PMBC-5359 on a potato medium without the addition of n-butanol using inoculum grown on medium MSS (control to the claimed method)
Use the strain Costridium acetobutylicum VKPM B-5359, stored in lyophilized form in a sealed glass ampoules. To obtain inoculum (inoculum), the ampoule is opened, add 500 ál of sterile distilled water, leave for swelling, then 100 µl of the cell suspension is transferred with a syringe in a sealed vial with a nutrient medium MSS and cultured at 37°C for 24 hours.
Environment for producing inoculum prepared as follows: sample components MSS consistently transfer in chemical beaker 1 l (placed on the magnetic stirrer), dissolved in 1000 liters of distilled water, poured into 60 ml in special flasks for anaerobic cultivation, boil on a water bath for 25 min, after boiling the environment is saturated with nitrogen, closed with rubber stoppers and screw up metal lids. Then sterilized by autoclaving at 116°C for 20 minutes.
To obtain spore material used potato starch content of 19%. The medium is prepared as in example 2. In a prepared potato Wednesday contribute inoculum obtained on the MSS environment, in the amount of 5% of the volume of the environment; the process of fermentation is carried out at a temperature of 37°C for 48 hours. After fermentation fermented environment count the number of spores formed, the content of which after the end the of fermentation is 1.08×10 8in ml.
Example 13. Getting spore material of the inventive method using a strain of Clostridium acetobutylicum In PMBC-5359 using inoculum grown on medium MSS
The process is carried out according to example 12. In the fermentation process, since 18 hours, under a microscope, examine the culture for the purpose of recording the appearance of clostridia. After discovering them in the amount of 100 in the same field of view in the environment type of 0.41 wt.% n-butanol and hold the process up to 48 hours. At the end of the fermentation process in the environment the number of the resulting dispute was -2,78×108in ml, which is 2.57 times higher than in the control.
Example 14. Getting spore material strain Clostridium acetobutylicum In PMBC-5359 on a potato medium without added n-butanol (control to the claimed method) using inoculum grown on medium SOL
Use a strain of Clostridium acetobutylicum In PMBC-5359 stored in lyophilized form in a sealed glass ampoules. To obtain inoculum (inoculum), the ampoule is opened, add 500 ál of sterile distilled water, leave for swelling, then 100 µl of the cell suspension is transferred with a syringe in a sealed vial with a nutrient medium SOL and cultured at 37°C for 24 hours.
Environment for producing inoculum prepared as follows: sample component is s environment SOL consistently transfer in chemical beaker 1 l (placed on the magnetic stirrer), dissolved in 1000 liters of distilled water, poured into 60 ml in special flasks for anaerobic cultivation, boil on a water bath for 25 min, after boiling the environment is saturated with nitrogen, closed with rubber stoppers and screw up metal lids. Then sterilized by autoclaving at 116°C for 20 minutes.
To obtain spore material used potato starch content of 19%. The medium is prepared as in example 2. In a prepared potato Wednesday contribute inoculum obtained on the environment SOL in an amount of 5% of the volume of the environment; the process of fermentation is carried out at a temperature of 37°C for 48 hours. After fermentation fermented environment count the number of spores formed, the content of which after fermentation is 1.12×108in ml.
Example 15. Getting spore material of the inventive method using a strain of Clostridium acetobutylicum In PMBC-5359 using inoculum grown on medium SOL
The process is carried out according to example 14. In the fermentation process, since 18 hours, under a microscope, examine the culture for the purpose of recording the appearance of clostridia. After discovering them in the amount of 100 in the same field of view in the environment type of 0.41 wt.% n-butanol and hold the process up to 48 hours. At the end of the fermentation process in the environment the number formed what I dispute was -2,86×10 8in ml, that of 2.55 times higher than in the control.
| Table | |
| Characteristics of the proposed method | |
| Used strain | The number of spores in the spore material, n×108in ml |
| Fermentation without n-butanol | Fermentation with n-butanol |
| Clostridium acetobutylicum In PMBC-5359 | 1,06-1,12 | 2,75-2,86 |
| Clostridium beijerinckii VKPM B-9600 | 0,98 | 2,68 |
| Clostridium saccharoperbutylacetonicum VKPM B-9602 | 1,18 | 2,54 |
| Clostridium butyricum VKPM B-9619 | 0,86 | 1,98 |
| Clostridium tyrobutyricum In PMBC-9615 | 0,78 | 1,85 |
Thus, the inventive method allows to obtain spore material bacteria Clostridium containing up to 3.6×108spores/ml, which is significantly (more than 2 times) to increase the number of spores in the spore material of different species the bacteria Clostridium.
The use of this seed material for planting fermentable substrates after storage in liquid or freeze-dried state does not require pasteurization and multistage validation activity, which significantly simplifies the process of microbiological synthesis of organic solvents, in particular n-butanol.
Sources of information
1. The Debabov VG Biofuels // Biotechnology. - 2008. No. 1. - P.3-14.
2. Berezina O., Zakharov, NV, jarocki SV, Zverlov CENTURIES Microbial producers of butanol // Biotechnology. - 2011. No. 4. - P.8-25.
3. Lopatkin I.S. Technology acetone fermentation. M - 1958.
4. Production schedule for the production of solvents: acetone, butanol and ethanol by the method of fermentation. PR 64-35-89, G. Efremov, 1989.
The method of obtaining the spore material of bacteria of the genus Clostridium, by planting the inoculum and cultivation in suitable conditions in a nutrient medium, including potatoes, glucose, ammonium sulphate and chalk, wherein the inoculum of bacteria of the genus Clostridium get into a full synthetic environment, and in the process of primary fermentation at 18-28 h development of a culture of bacteria depending on the time of occurrence in a single field of view of at least 80-100 thickened form cells of clostridia in culture medium was added n-butanol in the amount of 0.16 is 0.81 wt.%.