CN101148651A - Clostridium and its culturing method and application - Google Patents
Clostridium and its culturing method and application Download PDFInfo
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- CN101148651A CN101148651A CNA200710121453XA CN200710121453A CN101148651A CN 101148651 A CN101148651 A CN 101148651A CN A200710121453X A CNA200710121453X A CN A200710121453XA CN 200710121453 A CN200710121453 A CN 200710121453A CN 101148651 A CN101148651 A CN 101148651A
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Abstract
The present invention discloses one kind of clostridium and its culture process and application. The clostridium is rumen clostridium H1 in the preservation number of CGMCCNo.2125, and has high culture efficiency and contained cellulose degrading composite enzyme system comprising exocellulase, endo cellulase, xylanase, mannanase, esterase and pectase. The rumen clostridium H1 has capacity of decomposing natural cellulose, esterase activity of decomposing ester, easy medium temperature culture at 39 deg.c and capacity of utilizing pentose. It may find its wide application in cellulose degrading industry.
Description
Technical field
The present invention relates to a kind of clostridium and cultural method and application in the microorganism field.
Background technology
Plant generates organism by photosynthesis, wherein 35% or manyly exist with cellulose substances forms such as crop materials, but the plain class material of this fibrid also fail at present fully to be developed, thereby become important environomental pollution source again.Cellulose substances can be degraded to five-carbon sugar, hexose, and sugar can produce fuel alcohol or other Chemicals raw material by microbial fermentation then.Therefore people are converted into renewable energy source in exploratory development with cellulosic waste always.Generally be to produce sugar at present, produce alcohol fuel with biological fermentation sugar again, but this kind technical costs height never have extensive industrialization with chemistry or physical method hydrocellulose.Come degraded cellulose can improve degradation efficiency with microorganism cells and cellulase, reduce cost.Thereby seek efficiently microorganism cells or cellulase is applied to cellulose degradation industry, will important meaning be arranged to promoting cellulose substances be converted into renewable resources.
Cud is as the digestion organs of ruminating animal, be natural system hitherto known, that the Degradation and Transformation cellulose substances is most effective, the synergy that it depends on rumen microorganism colony becomes a series of Animal nutritions and energy substance with the quick Degradation and Transformation of natural fiber class material.Can think that cud is perfect, a natural stalk fibre degraded digestion source mill and an efficient anaerobic fermentation jar, still can match in excellence or beauty with it without any artificial system at present.
The clostridium of the energy degraded cellulose of report has at present: separate the fiber clostridium, separation is from soil, has cellulolytic ability, but do not have esterase activity (Petitdemange, E., F.Caillet, J.Giallo, andC.Gaudin.1984.Clostridium cellulolyticum sp.nov., a cellulolytic, mesophilic species from decayed grass.Int.J.Syst.Bacteriol.34:155-159); The thermal fiber clostridium, separation is from soil, high temperature (58 ℃) growth, has cellulolytic ability, but can not utilize wherein xylan and wood sugar (Johnson EA, Sakajoh M, Halliwell G, Madia A, Demain AL.1982.Saccharification of Complex Cellulosic Substratesby the Cellulase System from Clostridium thermocelium.Appl EnvironMicrobiol.43 (5): 1125-1132).
Summary of the invention
An object of the present invention is to provide a kind of clostridium, this clostridium has the activity of degraded cellulose and xylan, wood sugar and Ester.
Clostridium provided by the present invention is accredited as and occupies cud clostridium Clostridium ruminocolum, derives from the yak rumen content.
Describedly occupy the cud clostridium and be specially and occupy cud clostridium (Clostridium ruminocolum) H1, it is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on August 6th, 2007, the preservation centre address is No. 3, Da Tun road, Chaoyang District, BeiJing, China city first, and preserving number is CGMCC No.2125.
The cud clostridium H1 that occupies provided by the present invention has following feature:
1) colony characteristics: colony diameter is 1.0-2.0mm, and bacterium colony is rounded, smooth surface, and neat in edge, projection, yellow or white.
2) cell morphological characteristic: cell is shaft-like, and nose-circle is blunt; Gram-negative; Cell dia 0.8-1.0 μ m.
3) physiological and biochemical property: anaerobic growth; The catalase feminine gender; Do not produce indoles; Urease negative; Not hydrolysis polychrom; Liquefy gelatin not; Do not produce H
2S; Do not reduce nitrate; VP tests negative; Can utilize cellobiose, Mierocrystalline cellulose, maltose, wood sugar, xylan, lactose as carbon source, can not utilize pectinose, fructose, semi-lactosi, glucose, glycogen, pyruvic acid, sucrose, trehalose, starch, amygdaloside, erythritol, glycerine, inositol, lactic acid, N.F,USP MANNITOL, melizitose, melibiose, raffinose, rhamnosyl, ribose, saligenin, urobenzoic acid; Can utilize ammonium salt as nitrogenous source.
4) the 16S rRNA gene order length that occupies cud clostridium H1 is 1547bp, and its nucleotide sequence is shown in sequence table sequence 1.
Another object of the present invention provides the method that a kind of cultivation occupies cud clostridium H1.
Cultivation provided by the present invention occupies the method for cud clostridium H1, is to be inoculated in the RC substratum with occupying cud clostridium H1, cultivates under 25-45 ℃, the condition of pH 6-10.
In the above-mentioned RC substratum of 1000ml, contain: rumen fluid, 200-600ml; Inorganic salt solution I, 100-200ml; Inorganic salt solution II, 100-200ml; Carbon source material, 1-4g; Nitrogen source, 0.3-1.0g; Surplus is a water.
Wherein, described inorganic salt solution I is that concentration is the K of 0.2-0.4%
2HPO
4Solution; Described inorganic salt solution II is for containing 0.2-0.4%KH
2PO
4, 0.4-0.8% (NH
4)
2SO
4, 0.4-0.8%NaCl, 0.04-0.08%MgSO
4, 0.04-0.08%CaCl
2The aqueous solution.Described percentage composition is the quality percentage composition.
Wherein, carbon source material can be selected from one or more in cellobiose, Mierocrystalline cellulose, maltose, wood sugar, xylan and the lactose, and nitrogen source can be in cysteine hydrochloride and the ammonium salt one or more, specifically can decide according to practical situation.
For the RC substratum is maintained in the more stable pH value scope, also in substratum, add the NaHCO of 5-15g
3
Above-mentioned RC substratum also comprises oxidation-reduction indicator, specifically can be resazurin, and its content can be 0.2-0.6mg.
In the cultural method of the present invention, required anaerobic environment is by injecting CO
2Gas obtains, the general 1 atmospheric amount that reaches of injecting.Wherein, the suitableeest culture temperature is 35-41 ℃, and optimal pH is 6.5-7.5.
The cud clostridium H1 that occupies provided by the present invention easily cultivates, and the culture efficiency height can reach 2.5 ± 1g/L.
The cud clostridium H1 that occupies of the present invention separates to obtain from the cud of yak, be a kind of can degraded cellulose and the microorganism of xylan, wood sugar and ester class.The prozyme system of containing degraded cellulose in this microorganism, comprise circumscribed cellulase, endo cellulase, zytase, mannase, esterase, pectinesterase, it is 4.1 ± 1.19U/mg, 118.7 ± 4.5U/mg, 167.36 ± 22.9U/mg, 45.9 ± 3.52U/mg, 4 ± 0.5U/mg, 35.3 ± 1.77U/mg that enzyme work is respectively.Compare with separating the fiber clostridium, occupy cud clostridium H1 except that having the ability of decomposing natural cellulose among the present invention, also have and decompose the wherein esterase activity of ester; Compare with the thermal fiber clostridium, occupy temperature (39 ℃) growth among the cud clostridium H1, easily cultivate, and have the ability of utilizing pentose.By the research that the enzyme that the present invention is occupied the degraded of cud clostridium H1 participation cellulose substances is, explore and utilize its cell or cellulase to set up the cellulose substances transformation system of high efficiency, can provide technical support for realizing that cellulose substances efficiently utilizes.Therefore occupying cud clostridium H1 and can be used widely in degraded cellulose class material among the present invention can have broad application prospects in cellulose degradation industry.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Basic composition is of employed RC liquid nutrient medium in the experiment (/ liter):
Rumen fluid: 400ml; Inorganic salt solution I:150ml; Inorganic salt solution II:150ml; Cellobiose: 2.5g; Cysteine hydrochloride: 0.5g; Resazurin: 0.4mg; NaHCO
3: 10g; Distilled water is settled to 1000ml.
Inorganic salt solution I: mass percent concentration is 0.3% K
2HPO
4Solution.
Inorganic salt solution II: contain 0.3%KH
2PO
4, 0.6% (NH
4)
2SO
4, 0.6%NaCl, 0.06%MgSO
4, 0.06%CaCl
2The aqueous solution.
Described percentage composition is the quality percentage composition.
Employed RC solid medium is to add 15g agar in above-mentioned RC liquid nutrient medium in the experiment, and making its final concentration is 1.5%.
The separation preparation of embodiment 1, bacterial strain H1
Get the yak rumen content, be inoculated in it with the filter paper fibre element and be the cultivation of going down to posterity in the RC liquid nutrient medium of substrate, obtain enriched substance; As inoculation source, it is inoculated in the cellobiose is the RC solid medium of substrate with enriched substance, separates obtaining with the Hungate rolling tube technique.
It is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), and its deposit number is CGMCC No.2125.
Embodiment 2, identification of strains
One, form and physiological and biochemical property
Bacterial strain H1 (CGMCC No.2125) is inoculated in the RC solid medium that cellobiose is a substrate (contain the quality percentage composition be 1.5% agar), is 7.0 at pH, and temperature is that solid rolls pipe and cultivated 2 days under 39 ℃ the condition.
Observe the morphological specificity of bacterium colony and cell, and carry out following Physiology and biochemistry experiment: the test of 1 gramstaining, 2 catalase tests, 3 produce indole test, 4 urease tests, the test of 5 hydrolysis polychroms, the test of 6 liquefy gelatins, 7 produce H
2The S test, 8 reduction nitrate tests, 9 VP test, the test of 10 substrate utilizations, 11 nitrogenous source utilizations experiment.
Observations and experimental result are as follows:
1) colony characteristics: colony diameter is 1.0-2.0mm, and bacterium colony is rounded, smooth surface, and neat in edge, projection, yellow or white.
2) cell morphological characteristic: cell is shaft-like, and nose-circle is blunt; Gram-negative; Cell dia 0.8-1.0 μ m.Produce terminal spore, cyst expands.
3) physiological and biochemical property: anaerobic growth; The catalase feminine gender; Do not produce indoles; Urease negative; Not hydrolysis polychrom; Liquefy gelatin not; Do not produce H
2S; Do not reduce nitrate; VP tests negative; Can utilize cellobiose, Mierocrystalline cellulose, maltose, wood sugar, xylan, lactose as carbon source, can not utilize pectinose, fructose, semi-lactosi, glucose, glycogen, pyruvic acid, sucrose, trehalose, starch, amygdaloside, erythritol, glycerine, inositol, lactic acid, N.F,USP MANNITOL, melizitose, melibiose, raffinose, rhamnosyl, ribose, saligenin, urobenzoic acid; Can utilize ammonium salt as nitrogenous source.
Two, the pcr amplification and the sequencing of the 16S rRNA gene of bacterial strain H1 (CGMCC No.2125)
1) extracts DNA
To occupy cud clostridium H1 (CGMCC No.2125) and be inoculated in the cultivation of RC liquid nutrient medium; Get and grow to the logarithm fermented liquid in late period, 5000 rev/mins centrifugal 5 minutes, remove supernatant liquor; With TES (50mM Tris, 50mM EDTA-Na
2, 50mM NaCl, pH 8.0-8.2) and solution washes 3 times; With the thalline mixing, add an amount of N,O-Diacetylmuramidase with 0.5mL TES solution, 37 ℃ are incubated 2 hours; Add 0.2mL 20%SDS, 60 ℃ are incubated 10 minutes; Add 0.3mL 5M NaClO
4, mixing; Add equal-volume phenol-chloroform-primary isoamyl alcohol (25: 24: 1), shake up gently about 5 minutes, centrifugal (5000 rev/mins, 5 minutes) draw supernatant liquor, use phenol-chloroform-primary isoamyl alcohol (25: 24: 1) to handle again one time; Use successively then chloroform-primary isoamyl alcohol (24: 1, v/v) handle twice, up to there not being protein film to occur; Supernatant liquor adds 37 ℃ of insulations of 20 μ l 0.2%RNA enzymes 30 minutes, and chloroform-primary isoamyl alcohol (24: 1, v/v) handle one time; Supernatant liquor adds 20 μ l Proteinase Ks (50-70 μ g/mL), and 37 ℃ are incubated 1 hour, and chloroform-primary isoamyl alcohol (24: 1, v/v) handle one time; Supernatant liquor is iced ethanol sedimentations with 2 times of volumes, 70% ice alcohol solution dipping 5 minutes; Be dissolved in after drying in the TE solution as template DNA.
2) pcr amplification of 16S rRNA gene and order-checking
The forward primer that is used for pcr amplification be 5 '-AGAGTTTGATCCTGGCTCAGAACGAACGCT-3 ' (nt8-37), reverse primer is 5 '-TACGGCTACCTTGTTACGACTTCACCCC-3 ' (nt 1479-1506), corresponds respectively to the base of 8-37 and 1479-1506 position in the colibacillary 16S rRNA gene.PCR reaction system (50 μ l) is: 10 * buffer, 5 μ l; 25mmol/L MgCl
24 μ l; 10mmol/L dNTPs 1 μ l; Each 1 μ l of 30pmol/L primer; DdH
2O 36 μ l; Taq DNA enzyme 1 μ l; Template 1 μ l.The PCR reaction conditions is: 95 ℃ of 10min, 95 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min, 4 ℃ of preservations.
ABI BigDye3.1 sequencing kit (Applied Biosystems) and automatic dna sequencer (model ABI3730 are adopted in the order-checking of PCR product; Applied Biosystems).
Sequencing result shows that the 16S rRNA gene order length of bacterial strain H1 (CGMCC No.2125) is 1547bp, and its nucleotide sequence is shown in sequence table sequence 1.
16S rRNA gene order is carried out the homology compare of analysis in GenBank, the result shows that the similarity of the 16S rRNA gene order of its sequence and Clostridium lentocellum is 94%.
With reference to " Bergey ' s Manual of Systematic Bacteriology " (second edition), according to its morphological specificity and physiological and biochemical property, and according to the Search Results of its 16S rRNA gene order in GenBank, bacterial strain H1 (CGMCC No.2125) is accredited as novel species and occupies cud clostridium (Clostridiumruminocolum).
Embodiment 3, the cultivation that occupies cud clostridium H1
1) will occupy cud clostridium H1 and be inoculated in 50ml RC liquid nutrient medium with 5% ratio, be to cultivate under 6 the condition at 25 ℃, pH.
The composition of RC liquid nutrient medium (/ liter) as follows:
Rumen fluid: 200ml; Inorganic salt solution I:100ml; Inorganic salt solution II:l00ml; Mierocrystalline cellulose: 1.0g; Cysteine hydrochloride: 0.3g; NaHCO
3: 5g; Resazurin: 0.2mg; Distilled water is settled to 10000ml.
Inorganic salt solution I: mass percent concentration is 0.2% K
2HPO
4Solution.
Inorganic salt solution II: contain 0.2%KH
2PO
4, 0.4% (NH
4)
2SO
4, 0.4%NaCl, 0.04%MgSO
4, 0.04%CaCl
2The aqueous solution.
Described percentage composition is the quality percentage composition.
Cultivating required anaerobic environment is by injecting CO
2Gas obtains, until reaching 1 normal atmosphere (1.01 * 10
5Pa) till.
Three repetitions are established in experiment, and average cell concentration reaches 0.5 ± 0.3g/L.
2) will occupy cud clostridium H1 and be inoculated in 50ml RC substratum with 5% ratio, be to cultivate under 6.5 the condition at 35 ℃, pH.
The composition of RC substratum (/ liter) as follows:
Rumen fluid: 300ml; Inorganic salt solution I:125ml; Inorganic salt solution II:125ml; Maltose: 2.0g; Cysteine hydrochloride: 0.4g; NaHCO
3: 7g; Resazurin: 0.3mg; Distilled water is settled to 1000ml.
Inorganic salt solution I: mass percent concentration is 0.25% K
2HPO
4Solution.
Inorganic salt solution II: contain 0.25%KH
2PO
4, 0.5% (NH
4)
2SO
4, 0.5%NaCl, 0.05%MgSO
4, 0.05%CaCl
2The aqueous solution.
Described percentage composition is the quality percentage composition.
Cultivating required anaerobic environment is by injecting CO
2Gas obtains, until reaching 1 normal atmosphere (1.01 * 10
5Pa) till.
Three repetitions are established in experiment, and average cell concentration reaches 2.0 ± 1g/L
3) will occupy cud clostridium H1 and be inoculated in 50ml RC substratum with 5% ratio, be to cultivate under 7.0 the condition at 38 ℃, pH.
The composition of RC substratum (/ liter):
Rumen fluid: 400ml; Inorganic salt solution I:150ml; Inorganic salt solution II:150ml; Cellobiose: 2.5g; Cysteine hydrochloride: 0.5g; NaHCO
3: 10g; Resazurin: 0.4mg; Distilled water is settled to 10000ml.
Inorganic salt solution I: mass percent concentration is 0.3% K
2HPO
4Solution.
Inorganic salt solution II: contain 0.3%KH
2PO
4, 0.6% (NH
4)
2SO
4, 0.6%NaCl, 0.06%MgSO
4, 0.06%CaCl
2The aqueous solution.
Described percentage composition is the quality percentage composition.
Cultivating required anaerobic environment is by injecting CO
2Gas obtains, until reaching 1 normal atmosphere (1.01 * 10
5Pa) till.
Three repetitions are established in experiment, and average cell concentration reaches 2.5 ± 1g/L.
4) will occupy cud clostridium H1 and be inoculated in 50ml RC substratum with 5% ratio, be to cultivate under 7.5 the condition at 41 ℃, pH.
The composition of RC substratum (/ liter):
Rumen fluid: 500ml; Inorganic salt solution I:175ml; Inorganic salt solution II:175ml; A kind of in xylan and the wood sugar or two kinds: 3.5g altogether; (NH
4)
2SO
4: 0.7g; NaHCO
3: 12g; Resazurin: 0.5mg; Distilled water is settled to 1000ml.
Inorganic salt solution I: mass percent concentration is 0.35% K
2HPO
4Solution.
Inorganic salt solution II: contain 0.35%KH
2PO
4, 0.7% (NH
4)
2SO
4, 0.7%NaCl, 0.07%MgSO
4, 0.07%CaCl
2The aqueous solution.
Described percentage composition is the quality percentage composition.
Cultivating required anaerobic environment is by injecting CO
2Gas obtains, until reaching 1 normal atmosphere (1.01 * 10
5Pa) till.
Three repetitions are established in experiment, and average cell concentration reaches 2.0 ± 1g/L.
5) will occupy cud clostridium H1 and be inoculated in 50ml RC substratum with 5% ratio, be to cultivate under 10 the condition at 45 ℃, pH.
The composition of RC substratum (/ liter):
Rumen fluid: 600ml; Inorganic salt solution I:200ml; Inorganic salt solution II:200ml; Lactose: 4g; NH
4NO
3: 1.0g; NaHCO
3: 15g; Resazurin: 0.6mg; Distilled water is settled to 1000ml.
Inorganic salt solution I: mass percent concentration is 0.4% K
2HPO
4Solution.
Inorganic salt solution II: contain 0.4%KH
2PO
4, 0.8% (NH
4)
2SO
4, 0.8%NaCl, 0.08%MgSO
4, 0.08%CaCl
2The aqueous solution.
Described percentage composition is the quality percentage composition.
Cultivating required anaerobic environment is by injecting CO
2Gas obtains, until reaching 1 normal atmosphere (1.01 * 10
5Pa) till.
Three repetitions are established in experiment, and average cell concentration reaches 0.3 ± 0.1g/L.
6) will occupy cud clostridium H1 and be inoculated in 50ml RC substratum with 5% ratio, be to cultivate under 7.0 the condition at 46 ℃, pH.
The composition of RC substratum (/ liter):
Rumen fluid: 400ml; Inorganic salt solution I:150ml; Inorganic salt solution II:150ml; Cellobiose: 2.5g; Cysteine hydrochloride: 0.5g; NaHCO
3: 10g; Resazurin: 0.4mg; Distilled water is settled to 1000ml.
Inorganic salt solution I: mass percent concentration is 0.3% K
2HPO
4Solution.
Inorganic salt solution II: contain 0.3%KH
2PO
4, 0.6% (NH
4)
2SO
4, 0.6%NaCl, 0.06%MgSO
4, 0.06%CaCl
2The aqueous solution.
Described percentage composition is the quality percentage composition.
Cultivating required anaerobic environment is by injecting CO
2Gas obtains, until reaching 1 normal atmosphere (1.01 * 10
5Pa) till.
Three repetitions are established in experiment, and thalline is not all grown in three repetitions.
Experimental result shows: bacterial strain H1 anaerobic growth, growth temperature are not grown optimum growth temperature 35-41 ℃ for 46 ℃ from 25 ℃ to 45 ℃; PH growth scope 6-10, the suitableeest growth pH is 6.5-7.5.Under optimum growing condition, cell concentration can reach 2.5 ± 1g/L.
Embodiment 4, occupy the enzyme activity determination of the enzyme of several degraded celluloses among the cud clostridium H1CGMCC No.2125
One, preparation enzyme liquid
To occupy cud clostridium H1 CGMCC No.2125 and be inoculated in the RC liquid nutrient medium, pH is 7.0, and culture temperature is 39 ℃, cultivates after 3 days centrifugal collecting cell; Cell precipitation is suspended in the PBS damping fluid as enzyme liquid.
The RC substratum consists of (/ liter):
Rumen fluid: 400ml; Inorganic salt solution I:150ml; Inorganic salt solution II:150ml; Cellobiose: 2.5g; Cysteine hydrochloride: 0.5g; NaHCO
3: 10g; Resazurin: 0.4mg; Distilled water is settled to 1000ml.
Inorganic salt solution I: mass percent concentration is 0.3% K
2HPO
4Solution.
Inorganic salt solution II: contain 0.3%KH
2PO
4, 0.6% (NH
4)
2SO
4, 0.6%NaCl, 0.06%MgSO
4, 0.06%CaCl
2The aqueous solution.
Described percentage composition is the quality percentage composition.
Cultivating required anaerobic environment is by injecting CO
2Gas obtains, until reaching 1 normal atmosphere (1.01 * 10
5Pa) till.
Two, enzyme activity determination
1) enzyme activity determination reaction system (500 μ l) comprising: 1% reaction substrate (quality percentage composition), 250 μ l; 500mM ethane sulfonic acid morpholine (morpholine ethanesulfonic acid, MES)-the NaOH damping fluid, 100 μ l, pH are 6.0; Enzyme liquid, 150 μ l.Temperature of reaction is 37 ℃, the reducing sugar amount that adopts the DNS colorimetric method for determining to produce.
An enzyme activity unit (1U) is defined as the enzyme amount that per minute discharges 1 μ mol reducing sugar.
2) measure different enzymes respectively with above-mentioned enzyme activity determination reaction system and live, three repetitions are established in each experiment:
A, circumscribed cellulase: substrate is a Microcrystalline Cellulose, reacts 12 hours, and three times are repeated on average to record enzyme work is 4.1 ± 1.19U/mg.
B, endo cellulase: substrate is carboxymethyl cellulose (CMC), reacts 30 minutes, and three times are repeated on average to record enzyme work is 118.7 ± 4.5U/mg.
C, zytase: substrate is an xylan, reacts 30 minutes, and three times are repeated on average to record enzyme work is 167.36 ± 22.9U/mg.
D, mannase: substrate is locust bean gum (locust bean gum), reacts 30 minutes, and three times are repeated on average to record enzyme work is 45.9 ± 3.52U/mg.
E, pectinesterase: substrate is a pectin, reacts 30 minutes, and three times are repeated on average to record enzyme work is 35.3 ± 1.77U/mg.
F, esterase: substrate is a Ferulic acid methylester, reacts 30 minutes, and three times are repeated on average to record enzyme work is 4 ± 0.5U/mg.
Embodiment 5, utilize the cud clostridium H1 degraded filter paper fibre element that occupies of the present invention
One, preparation substratum
Whatman I filter paper is cut into strip, adds with 3% quality percentage composition and do not contain in the RC substratum of substrate, (/ liter) composed as follows of the final RC substratum that forms:
Rumen fluid: 400ml; Inorganic salt solution I:150ml; Inorganic salt solution II:150ml; WhatmanI filter paper: 2.5g; Cysteine hydrochloride: 0.5g; NaHCO
3: 10g; Resazurin: 0.4mg; Distilled water is settled to 1000ml.
Inorganic salt solution I: mass percent concentration is 0.3% K
2HPO
4Solution.
Inorganic salt solution II: contain 0.3%KH
2PO
4, 0.6% (NH
4)
2SO
4, 0.6%NaCl, 0.06%MgSO
4, 0.06%CaCl
2The aqueous solution.
Described percentage composition is the quality percentage composition.
Cultivating required anaerobic environment is by injecting CO
2Gas obtains, until reaching 1 normal atmosphere (1.01 * 10
5Pa) till.
That two, will grow in the RC liquid nutrient medium that with the cellobiose is substrate occupies cud clostridium H1 as the experiment thalline, when cell concentration reaches 2.5g/L, being inoculated in 4ml its volume ratio with 1/10 above-mentioned is the RC substratum of substrate with WhatmanI filter paper, cultivates under 38 ℃, the condition of pH 7.0.
Three, filter paper is weightless measures
Utilize nitric acid-Ethanol Method to measure and cultivate the remaining filter paper quality in back, compare the filter paper weightlessness that the quality of minimizing causes for degraded with the filter paper quality before cultivating.
Originally the quality of filter paper is 0.12g, cultivates that the quality of filter paper is 0.06g after 8 days.
Four, degradation rate:
The calculation formula of degradation rate is:
Degradation rate=(quality of the quality of filter paper before cultivating-cultivation back filter paper)/quality of filter paper before cultivating
Degradation rate in this experiment=(0.12g-0.06g)/0.12g=50%
Sequence table
<160>1
<210>1
<211>1547
<212>DNA
＜213〉fusobacterium occupies cud clostridium (Clostridium ruminocolum)
<400>1
agagtttgat?acctggctca?ggctggatac?acctccttag?agtttgatca?tggctcaggc 60
tgaacgctgg?cggcgtgctt?aacacatgca?agtcgaacga?actgaggaga?gcttgctctc 120
caaagttagt?ggcggacggg?tgagtaacgc?gtgggtaacc?tgcctcatgc?agggggataa 180
cgtttggaaa?cgaacgctaa?taccgcataa?actatggatt?accgcatggt?aattatagca 240
aagatttatc?ggcatgagat?ggacccgcgt?tggattagct?agttggtgag?ataacagccc 300
accaaggcga?cgatccatag?ccggcctgag?agggtgaacg?gccacattgg?gactgagaca 360
cggcccaaac?tcctacggga?ggcagcagtg?gggaatattg?cacaatgggc?gcaagcctga 420
tgcagcgacg?ccgcgtgaag?gaagaaggtc?ttcggattgt?aaacttctat?cagcagggaa 480
gaaaaaaatg?acggtacctg?actaagaagc?tccggctaac?tacgtgccag?cagccgcggt 540
aatacgtagg?gagcgagcgt?tatccggatt?tactgggtgt?aaagggtgcg?taggcggcat 600
ggtaagtcag?aagtgaaatt?ttggggctca?actccagagc?tgctctgaaa?ctgctgtgct 660
agagtgtggg?agaggaaagt?ggaattccga?gtgtagcggt?gaaatgcgta?gagattcgga 720
ggaacaccag?tagcgaaggc?ggctttctgg?accataactg?acgctgaggc?acgaaagcgt 780
ggggagcaaa?caggattaga?taccctggta?gtccacgccg?taaacgatga?atgctaggtg 840
tcggccctta?cgggggtcgg?tgccgaagtt?aacacattaa?gcattccacc?tgggaagtac 900
gatcgcaaga?ttgaaactca?aaggaattga?cggggacccg?cacaagcggt?ggagcatgtg 960
gtttaattcg?aagcaacgcg?aagaacctta?cctaaacttg?acatccttct?gaccggtctt 1020
taatcggact?tttcggtgct?tgcactgacg?gaagtgacag?gtggtgcatg?gttgtcgtca 1080
gctcgtgtcg?tgagatgttg?ggttaagtcc?cgcaacgagc?gcaaccccta?tccttagtag 1140
ccatcattaa?gttgggcact?ctagggagac?tgccagggat?aacttggagg?aaggtgggga 1200
tgacgtcaaa?tcatcatgcc?ccttatgttt?agggctacac?acgtgctaca?atggctgcta 1260
caaagggaag?caatgccgcg?aggccgagcg?aacctcaaaa?aagcagtccc?agttcggatt 1320
gtagtctgca?actcgactac?atgaagttgg?aatcgctagt?aatcgcgaat?cagaatgtcg 1380
cggtgaatac?gttcccgggt?cttgtacaca?ccgcccgtca?caccatggga?gttgaggggg 1440
cccaacgtcg?gtgacccaac?ccgtaaggga?gggagccgcc?taaggcaaaa?tcaataactg 1500
gggtgaagtc?gtaacaaggt?agccgtatcg?gaaggtgcgg?ctggatc 1547
Claims (10)
1. occupy cud clostridium (Clostridium ruminocolum) H1 CGMCC No.2125.
2. the described cultural method that occupies cud clostridium H1 CGMCC No.2125 of claim 1 is to be inoculated in the RC substratum with occupying cud clostridium H1 CGMCC No.2125, cultivates under 25-45 ℃, the condition of pH6-10.
3. cultural method according to claim 2 is characterized in that: described RC substratum, contain in 1000ml: rumen fluid, 200-600ml; Inorganic salt solution I, 100-200ml; Inorganic salt solution II, 100-200ml; Carbon source material, 1-4g; Nitrogen source, 0.3-1.0g; Surplus is a water;
Described inorganic salt solution I is that concentration is the K of 0.2-0.4%
2HPO
4Solution;
Described inorganic salt solution II is for containing 0.2-0.4%KH
2PO
4, 0.4-0.8% (NH
4)
2SO
4, 0.4-0.8%NaCl, 0.04-0.08%MgSO
4, 0.04-0.08%CaCl
2The aqueous solution;
Described percentage composition is the quality percentage composition.
4. cultural method according to claim 3 is characterized in that: described carbon source material is one or more in cellobiose, Mierocrystalline cellulose, maltose, wood sugar, xylan and the lactose.
5. according to claim 3 or 4 described cultural methods, it is characterized in that: described nitrogen source is one or more in cysteine hydrochloride and the ammonium salt.
6. according to claim 3 or 4 described cultural methods, it is characterized in that: the NaHCO that also contains 5-15g in the described RC substratum
3
7. according to claim 3 or 4 described cultural methods, it is characterized in that: the resazurin that also contains 0.2-0.6mg in the described RC substratum.
8. according to claim 2 or 3 or 4 described cultural methods, it is characterized in that: described culture temperature is 35-41 ℃.
9. according to claim 2 or 3 or 4 described cultural methods, it is characterized in that: described pH is 6.5-7.5.
10. claim 1 is described occupies the application of cud clostridium H1 CGMCC No.2125 in degraded cellulose class material.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864091A (en) * | 2012-01-15 | 2013-01-09 | 河南科技大学 | One-bacterium multiple-enzyme bacterial strain as well as screening method and application thereof |
| CN103947904A (en) * | 2014-05-15 | 2014-07-30 | 邵素英 | Preparation method of glucose precursor for improving dairy cow rumen flora |
| CN104000038A (en) * | 2014-05-15 | 2014-08-27 | 北京东方联鸣科技发展有限公司 | Glucose precursor for improving cow rumen flora and application thereof |
| CN110029123A (en) * | 2019-04-03 | 2019-07-19 | 江西农业大学 | The method and application of Yeast expression carrier building and expression circumscribed-type cellulase |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100523171C (en) * | 2006-12-26 | 2009-08-05 | 华中农业大学 | Clostridium butyricum active bacteria agent production method |
-
2007
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864091A (en) * | 2012-01-15 | 2013-01-09 | 河南科技大学 | One-bacterium multiple-enzyme bacterial strain as well as screening method and application thereof |
| CN103947904A (en) * | 2014-05-15 | 2014-07-30 | 邵素英 | Preparation method of glucose precursor for improving dairy cow rumen flora |
| CN104000038A (en) * | 2014-05-15 | 2014-08-27 | 北京东方联鸣科技发展有限公司 | Glucose precursor for improving cow rumen flora and application thereof |
| CN103947904B (en) * | 2014-05-15 | 2017-11-28 | 宁夏锐盛明杰知识产权咨询有限公司 | A kind of preparation method for improving cow rumen flora glucose precursor thing |
| CN110029123A (en) * | 2019-04-03 | 2019-07-19 | 江西农业大学 | The method and application of Yeast expression carrier building and expression circumscribed-type cellulase |
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