CN105567623A - Method for producing Bacillus mucilaginosus liquid microbial inoculant from lignocellulose - Google Patents
Method for producing Bacillus mucilaginosus liquid microbial inoculant from lignocellulose Download PDFInfo
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- CN105567623A CN105567623A CN201610144171.0A CN201610144171A CN105567623A CN 105567623 A CN105567623 A CN 105567623A CN 201610144171 A CN201610144171 A CN 201610144171A CN 105567623 A CN105567623 A CN 105567623A
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- bacillus cereus
- colloid bacillus
- bacillus mucilaginosus
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Abstract
The invention provides a method for producing a Bacillus mucilaginosus liquid microbial inoculant from lignocellulose. The method comprises the following steps: (1) pulverizing a lignocellulose raw material into 40-60-mesh particles, and carrying out hydrothermal treatment to obtain a suspension; (2) adding cellulase into the suspension obtained in the step (1), carrying out enzymolysis, and centrifugating to remove residues, thereby obtaining a glucose-containing enzymolysis solution; (3) adding culture medium components, inoculating Bacillus mucilaginosus, and culturing at 37 DEG C at the rate of 200 rpm under the pH value of 7.0 to obtain the Bacillus mucilaginosus cells; and (4) adding the Bacillus mucilaginosus cells obtained in the step (3) into an inorganic salt buffer system to obtain the Bacillus mucilaginosus liquid microbial inoculant. After the hydrothermal technique is adopted to pretreat the lignocellulose raw material, the degradation capacity of the cellulose is enhanced. The enzymolysis solution is utilized to perform the fermentation production of the Bacillus mucilaginosus microbial inoculant, thereby enhancing the viable count and implementing the value-added utilization of the lignocellulose.
Description
Technical field
The application relates to the field of biomass recycling use and agricultural wastes comprehensive utilization, is specifically related to a kind of method utilizing lignocellulosic material to produce colloid bacillus cereus liquid bacterial agent.
Background technology
China is agriculture production big country, has abundant lignocellulosic material, and only agricultural crop straw will produce more than 700,000,000 ton every year, and wherein maize straw, wheat stalk and rice straw are the three major types stalks of China.Except small part is utilized, there is the stalk of several hundred million tons directly burned or become solid waste every year, cause the wasting of resources and environmental pollution.
In lignocellulosic material, Mierocrystalline cellulose accounts for 35-45%; hemicellulose accounts for 20-40%; according to appropriate technology, they are degraded into fermentable sugar; then microorganism further fermentation to produce biological base chemical is utilized; no matter to the recycling of agricultural resource waste; alleviating energy crisis, or reduce the aspect such as environmental pollution all tool be of great significance.Utilize lignocellulosic material fermentative production as ethanol, butanols, acetone, hydrogen, L-glutamic acid, citric acid etc. in prior art.
In lignocellulose pre-treatment, generally adopt mechanical process, diluted acid method, alkaline process, biologic enzymolysis method etc. at present.Diluted acid method or alkaline process can cause forming a large amount of acid-base waste fluids, cause environmental pollution, and can form the fermentation inhibitors such as hydroxymethylfurfural in treating processes, and then the transformation efficiency of affecting glucose.
Colloid bacillus cereus (Bacillusmucitaginosus), also known as silicate bacteria, can be grown on the mineral such as feldspar, mica, phosphatic rock, and decomposition discharges the nutritive element such as potassium, phosphorus, the soluble phosphorus & potassium improved in soil utilizes for plant-growth, metabolism can produce the nutritive substance such as organic acid, amino acid being conducive to plant-growth simultaneously, strengthen the activity of soil saccharase, urase and Phosphoric acid esterase, improve soil ecology, increasing soil fertility and reach effect of increasing production, is a kind of good bio-feritlizer production bacterial strain.But the problems such as viable count is not high, gemma productive rate is unstable that traditional colloid bacillus cereus microbial inoculum exists in producing.
Summary of the invention
This application provides a kind of method utilizing lignocellulosic material to produce colloid bacillus cereus liquid bacterial agent, comprise the steps: that lignocellulosic material is ground into 40-60 object particle by (1), carry out hydrothermal treatment consists, obtain suspension liquid; (2) add cellulase, enzymolysis, centrifugal segregation residue in the suspension liquid obtained to step (1), obtain the enzymolysis solution containing glucose; (3) add nutrient media components, access colloid bacillus cereus, in 37 DEG C, cultivates under the condition of pH7.0,200rpm, obtains colloid bacillus cereus somatic cells; (4) the colloid bacillus cereus somatic cells that step (3) obtains is added in inorganic salt buffer system, obtain colloid bacillus cereus liquid bacterial agent.In some embodiments, in described step (1), the condition of described hydrothermal treatment consists is 140-180 DEG C, 0.8-1.2MPa, 10-20min.In some embodiments, in described step (2), the condition of described enzymolysis is: add cellulase according to the consumption of 15FPU/g lignocellulosic material, in 55 DEG C, reacts 40-50h under the condition of pH4.5-4.8,100-150rpm.In some embodiments, in described step (2), in the enzymolysis solution of acquisition, the concentration of glucose is 50 ~ 60g/L.In some embodiments, in described step (3), described nutrient media components and concentration thereof are: dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.05g/L, ammonium sulfate 5g/L, yeast extract paste 0.5g/L.In some embodiments, in described step (4), the composition of described inorganic salt buffer system is: potassium primary phosphate 5g/L; Dipotassium hydrogen phosphate 8g/L; Magnesium sulfate 0.5g/L; Ferrous sulfate 0.02g/L; Calcium chloride 0.05g/L; Manganous sulfate 0.01g/L; Zinc sulfate 0.005g/L, pH7.0-7.2.In some embodiments, described lignocellulosic material comprises maize straw, wheat stalk and/or rice straw, preferred maize straw.
Present invention also provides the colloid bacillus cereus liquid bacterial agent produced according to the method for the application.
The application proposes a kind of method utilizing lignocellulosic material to produce colloid bacillus cereus liquid bacterial agent, and for achieving the above object, the application takes following technical scheme:
1, the pre-treatment of lignocellulosic material
Lignocellulosic material pulverizer is ground into particle, puts into hydrothermal reaction kettle, be made into suspension liquid, process for some time under suitable condition after, lower the temperature stand-by.
2, cellulase degradation
In the suspension liquid that step 1 obtains, add cellulase according to suitable consumption, react for some time under suitable condition, centrifugal segregation residue, obtain enzymolysis solution.
3. the fermentation of colloid bacillus cereus
In the enzymolysis solution that step 2 obtains, after adding the components such as dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate, yeast extract paste, access colloid bacillus cereus, cultivate, when thalli growth enters stationary phase, and when gemma productive rate reaches more than 80% in suitable condition bottom fermentation, fermentation ends, collected by centrifugation thalline.
4. microbial inoculum preparation
By the colloid bacillus cereus thalline of step 3 gained, be added in inorganic salt buffer system according to suitable concentration, colloid bacillus cereus liquid bacterial agent can be obtained.
Beneficial effect
The application takes a kind of hydro-thermal treatment method, in conjunction with biologic enzymolysis method, compared with the lignocellulose pretreatment method of prior art, improves cellulosic hydrolysis efficiency, realizes the efficiency utilization of lignocellulose.
The application adopts the enzymolysis solution of lignocellulose, substitutes the composition such as starch, dregs of beans in traditional zymotic substratum, improves the gemma productive rate of colloid bacillus cereus, and the increment realizing lignocellulosic material utilizes.
The application adopts hydrothermal technology to carry out the pre-treatment of lignocellulosic material, time is short, efficiency is high, cellulose hydrolysis is more thorough, and hydrolysising by-product is few, and use it for the fermentative production of colloid bacillus cereus, improve the utilising efficiency of lignocellulosic material.
Embodiment
Next, by embodiment, the application is illustrated in further detail, but the application is not limited only to these embodiments.Should be appreciated that and can adopt other embodiments, and suitable change can be made and do not depart from the spirit or scope of the application.In order to avoid for enabling those skilled in the art put into practice details unnecessary the application, specification sheets may eliminate some known to those skilled in the art information.Therefore, below describe in detail and should not understand with restrictive meaning, and the scope of the application is only defined by claims.
The each raw material used in following embodiment, except particularly pointing out, all can by commercially available acquisition.
Experimental technique
(1) method of counting of viable count:
Previously prepared nutrient agar culture dish.After sampling, aseptically, gradient dilution is carried out to fermented liquid, get the bacterium liquid spread plate of 100 μ L acceptable diluent degree, after 30 DEG C of constant temperature culture are inverted cultivation 24-48h, calculate viable count.Concrete grammar is as follows: get the test tube that 7 are equipped with 4.5mL sterilized water, with 10
-1-10
-7number consecutively, draws 0.5mL with liquid-transfering gun from fermention medium, puts into numbering 10
-1centrifuge tube, rocks mixing, makes 10
-1bacterium liquid, the rest may be inferred, until the 7th pipe numbering 10
-7centrifuge tube, make different dilution bacterium liquid.Then draw the different dilution bacteria suspension of 0.1mL in the plate of sterilizing, be coated with glass slicker, each dilution substratum does 3 repetitions, is inverted after cultivating and counts.
The calculation formula of viable count is:
Viable count in 1mL original fermented solution=same extent of dilution more than three repeats mean number × extension rate × 10 of plate bacterium colony
(2) method of calculation of gemma productive rate
Gemma counting process refers to that be placed in rapidly cold water and cool, then dilution spread, concrete grammar is the same by fermented liquid after constant temperature 80 DEG C of water-bath 10min.
Gemma productive rate is the per-cent of the gemma number that obtains of viable bacteria counting method and total count.
Reagent source and type as follows:
(1) yeast extract paste, Oxide, biochemical reagents;
(2) starch, sucrose and dregs of beans effluent north Lingshan crane vegetable-protein Manufacturing Co., Ltd provides;
(3) dipotassium hydrogen phosphate, potassium primary phosphate, magnesium sulfate, ferrous sulfate, ammonium sulfate, calcium chloride, sulfuric acid, manganous sulfate, zinc sulfate are domestic analytical reagent, arbitrary producer.
The pre-treatment of embodiment 1 lignocellulosic material
Take maize straw as raw material, first the particle of (280-400um) about being ground into 40-60 order with pulverizer, the suspension liquid that concentration is 20% (w/v%) is made into water, (model is CJF-20L to put into hydrothermal reaction kettle, the sensible reactor factory in Dalian) in, in 180 DEG C, under the condition of 1.2MPa, process 10min, the fibrous texture of stalk is made to become loose, so that subsequent disposal.
The cellulase degradation of embodiment 2 lignocellulosic material
Cellulase (SDC-A is added according to the consumption of 15FPU/g stalk in the suspension liquid that embodiment 1 obtains, Ningxia Sunson Industrial Group Co., Ltd.), in 55 DEG C, pH4.8,48h is reacted under the condition of 100rpm, centrifugal segregation residue, obtaining containing glucose concn is the enzymolysis solution of 50-60g/L.
Embodiment 3 lignocellulose enzymolysis liquid fermentative production colloid bacillus cereus
In the maize straw enzymolysis solution that embodiment 2 obtains, after adding dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate, yeast extract paste component, consumption access colloid bacillus cereus according to 10% is (purchased from Chinese agriculture Microbiological Culture Collection administrative center (AgriculturalCultureCollectionofChina, ACCC), preserving number is ACCC19749), in 37 DEG C, pH7.0, the condition bottom fermentation of 200rpm is cultivated, and when 24h is carried out in fermentation, viable count can reach 3.67 × 10
8individual/mL, now gemma productive rate is more than 85%, fermentation ends, collected by centrifugation thalline.
The preparation of embodiment 4 colloid bacillus cereus liquid bacterial agent
According to following formulated inorganic salt buffer system, specific as follows:
Potassium primary phosphate 5g/L; Dipotassium hydrogen phosphate 8g/L; Magnesium sulfate 0.5g/L; Ferrous sulfate 0.02g/L; Calcium chloride 0.05g/L; Manganous sulfate 0.01g/L; Zinc sulfate 0.005g/L, pH7.0.
Colloid bacillus cereus thalline embodiment 3 obtained adds in above-mentioned buffer system, makes its viable count final concentration be 2 × 10
8individual/mL, according to after the specification packaging of 10L/ bag (wrapping machine model is GHR-G16, Heze Wei Fute wrapping mechanism company limited), can obtain colloid bacillus cereus liquid bacterial agent.
Embodiment 5 is with other lignocellulosic material fermentative production colloid bacillus cereus
The concrete grammar of reference embodiment 1 and embodiment 2, carries out pre-treatment and enzymolysis to wheat stalk and rice straw, obtains wheat stalk enzymolysis solution and rice straw enzymolysis solution.Then the fermentative production of colloid bacillus cereus is carried out according to the concrete grammar of embodiment 3.Result shows, when fermenting with wheat stalk enzymolysis solution, when proceeding to 24h, viable count can reach 3.46 × 10
8individual/mL; When fermenting with rice straw enzymolysis solution, when proceeding to 24h, viable count can reach 3.28 × 10
8individual/mL, the viable count (3.67 × 10 when all fermenting a little less than maize straw enzymolysis solution
8individual/mL).
Lignocellulose enzymolysis after comparative example's 1 Ordinary pulverization+acidolysis process
By maize straw, through Ordinary pulverization and acid treatment, (actual conditions is: sulfuric acid 10%, temperature 100-180 DEG C, pressure 0.8-1MPa, time 5min) lignocellulose that obtains, carry out enzymolysis according to the condition of embodiment 2, the concentration that reaction terminates glucose in the enzymolysis solution of rear acquisition is only 30-40g/L.By comparison, the enzymolysis solution that embodiment 2 obtains is 50-60g/L containing glucose concn.
Comparative example 2 is with traditional starch sucrose medium fermentative production colloid bacillus cereus
Colloid bacillus cereus is (the same, purchased from Chinese agriculture Microbiological Culture Collection administrative center) inoculum size with 10% accesses traditional starch sucrose medium (starch 7g/L, dregs of beans 0.7g/L, sucrose 2g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate 2g/L, calcium carbonate 1g/L, iron trichloride 0.28g/L, ammonium sulfate 0.6g/L, yeast extract paste 0.8g/L, pH7.0-7.2) in, in 37 DEG C, pH7.0, the condition bottom fermentation of 200rpm is cultivated, and when 24h is carried out in fermentation, viable count can reach 2.07 × 10
8individual/mL, now gemma productive rate is more than 80%, fermentation ends.
Therefore, compared with the starch sucrose medium of prior art, adopt the lignocellulose enzymolysis liquid fermentative production colloid bacillus cereus of the application can significantly improve its viable count, (time in embodiment 3 with cellulase hydrolyte fermented liquid, the viable count of fermentation termination is 3.67 × 10 to improve 77.3%
8individual/mL, when fermenting with starch sucrose medium in comparative example 2, the viable count of terminal is 2.07 × 10
8individual/mL, viable count improves (3.67-2.07) × 10
8)/2.07 × 10
8× 100%=77.3%).
In sum; these are only the preferred embodiment of the application, be not intended to limit the protection domain of the application, therefore; the any amendment done within all spirit in the application and principle, equivalent replacement, improvement etc., within the protection domain that all should be included in the application.
Claims (8)
1. utilize lignocellulosic material to produce a method for colloid bacillus cereus liquid bacterial agent, comprise the steps:
(1) lignocellulosic material is ground into 40-60 object particle, carries out hydrothermal treatment consists, obtain suspension liquid;
(2) add cellulase, enzymolysis, centrifugal segregation residue in the suspension liquid obtained to step (1), obtain the enzymolysis solution containing glucose;
(3) add nutrient media components, access colloid bacillus cereus, in 37 DEG C, cultivates under the condition of pH7.0,200rpm, obtains colloid bacillus cereus somatic cells;
(4) the colloid bacillus cereus somatic cells that step (3) obtains is added in inorganic salt buffer system, obtain colloid bacillus cereus liquid bacterial agent.
2. method according to claim 1, wherein: in described step (1), the condition of described hydrothermal treatment consists is 140-180 DEG C, 0.8-1.2MPa, 10-20min.
3. method according to claim 1, wherein: in described step (2), the condition of described enzymolysis is: add cellulase according to the consumption of 15FPU/g lignocellulosic material, in 55 DEG C, 40-50h is reacted under the condition of pH4.5-4.8,100-150rpm.
4. method according to claim 1, wherein: in described step (2), in the enzymolysis solution of acquisition, the concentration of glucose is 50 ~ 60g/L.
5. method according to claim 1, wherein: in described step (3), described nutrient media components and concentration thereof are: dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.05g/L, ammonium sulfate 5g/L, yeast extract paste 0.5g/L.
6. method according to claim 1, wherein: in described step (4), the composition of described inorganic salt buffer system is: potassium primary phosphate 5g/L; Dipotassium hydrogen phosphate 8g/L; Magnesium sulfate 0.5g/L; Ferrous sulfate 0.02g/L; Calcium chloride 0.05g/L; Manganous sulfate 0.01g/L; Zinc sulfate 0.005g/L, pH7.0-7.2.
7. method according to claim 1, wherein: described lignocellulosic material comprises maize straw, wheat stalk and/or rice straw, preferred maize straw.
8. the colloid bacillus cereus liquid bacterial agent that the method according to any one of claim 1-7 is produced.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1467180A (en) * | 2002-07-12 | 2004-01-14 | 陈五岭 | Method for producing biotechnical fulvic acid composite biological bacteria fertilizer by microorganism fermentation of sugar grass straw |
| CN1618772A (en) * | 2003-11-20 | 2005-05-25 | 郭伟光 | High efficiency biological organic fertilizer and its production technology |
-
2016
- 2016-03-14 CN CN201610144171.0A patent/CN105567623A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1467180A (en) * | 2002-07-12 | 2004-01-14 | 陈五岭 | Method for producing biotechnical fulvic acid composite biological bacteria fertilizer by microorganism fermentation of sugar grass straw |
| CN1618772A (en) * | 2003-11-20 | 2005-05-25 | 郭伟光 | High efficiency biological organic fertilizer and its production technology |
Non-Patent Citations (4)
| Title |
|---|
| 刘五星等: "硅酸盐细菌肥料的研究进展", 《天津农业科学》 * |
| 杜英等: "利用玉米秸秆酶解液发酵胶质芽胞杆菌 WY120 的研究", 《中国农业科技导报》 * |
| 王雪等: "胶质芽孢杆菌培养条件及发酵工艺的研究进展", 《过程工程学报》 * |
| 胡秋龙等: "木质纤维素生物质预处理技术的研究进展", 《中国农学通报》 * |
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Application publication date: 20160511 |