CN101245327B - Bacillus licheniformis and method for producing alkali proteinase with sewage sludge as raw material - Google Patents
Bacillus licheniformis and method for producing alkali proteinase with sewage sludge as raw material Download PDFInfo
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- CN101245327B CN101245327B CN 200810026812 CN200810026812A CN101245327B CN 101245327 B CN101245327 B CN 101245327B CN 200810026812 CN200810026812 CN 200810026812 CN 200810026812 A CN200810026812 A CN 200810026812A CN 101245327 B CN101245327 B CN 101245327B
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- Treatment Of Sludge (AREA)
Abstract
The invention discloses a bacillus licheniformis and a method for utilizing the sludge as raw material for producing alkaline protease. The bacillus licheniformis is gram-positive bacteria and is shaped like a straight rod, and the collection number is CGMCC 1970. The method for utilizing the bacillus licheniformis for producing the alkaline protease includes (1) the conditioning of a culture medium; (2) shaking culture; (3) the fermentation by a fermentation tank; and (4) the purification of the protease. The bacterial strain of the invention is utilized for producing the alkaline protease, which can not only dispose sludge, but also obtain the alkaline protease product with high additional value and lower cost. The method uses urban waste sludge to replace the traditional food culture medium for producing the alkaline protease product, which not only provides a new way for the resource disposal of urban sludge, but also can greatly reduce the price of the product and promote the industrial production and application of protease preparations.
Description
Technical field
The present invention relates to novel bacterial screening and zymin production field, specifically, relate to a kind of Bacillus licheniformis and utilize municipal sludge to produce the method for Sumizyme MP as fermentation raw material.
Background technology
Municipal sludge is the throw out that produces in the sewage treatment process, contains materials such as a large amount of organism, abundant nitrogen phosphorus and pathogenic bacteria etc., is important environomental pollution source.At present, the about 8,000,000 tons of dry weights of China's municipal sludge year generation, and along with the increase of sewage work and the raising of wastewater treatment rate, generation will be huge day by day.How properly to dispose sewage plant sludge, prevent its secondary pollution, and it is used as a kind of useful resources, turn waste into wealth, have crucial meaning.
At present, the disposal approach of municipal sludge mainly contains: landfill, burning and soil utilization etc.But they all exist and are difficult to the weakness that overcomes separately.In fact, contain great number of organic matters and nutritive elements such as the nitrogen that enriches, phosphorus in the mud, can satisfy the needs of microorganism growth and propagation.According to the retrieval, existingly utilize municipal sludge to produce the report of microbial pesticide, biological hydrogen and lactic acid etc. for fermenting raw materials.
Sumizyme MP is most important in the world at present industrial enzyme preparation, is mainly used in enzyme-containing detergent industry, and in field widespread uses such as process hides, silk, feed, medicine and environmental protection, its output accounts for 25% of whole zymin.The main mode of production of Sumizyme MP is a microbial fermentation, and mainly producing bacterial strain is genus bacillus such as bacillus pumilus and subtilis.
At present, the main flow production technique of Sumizyme MP is a liquid submerged fermentation, generally adopts analysis for soybean powder, starch, mineral substance etc. as fermentation raw material, and these expensive raw material price cause product price higher.Up to now, do not find as yet to utilize municipal sludge to produce the report of Sumizyme MP for fermentation raw material.
Summary of the invention
The objective of the invention is to remedy weak point of the prior art, and provide a kind of Sumizyme MP that is suitable for the municipal sludge fermentation to produce bacterial strain-Bacillus licheniformis.
Another object of the present invention provides utilizes above-mentioned Bacillus licheniformis, is the method for raw material production Sumizyme MP with mud.
Obtained strains of the present invention is Bacillus licheniformis (Bacillus licheniformis) HM-3, is gram-positive microorganism, and direct rod shape is located away from the thickened sludge that moral sewage work is hunted in the Guangzhou.On March 12nd, 2007, be numbered: CGMCC 1970 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation.
G/L is meant the gram number of the target compound that adds in every 1L medium liquid;
% is meant the mass fraction that inserts inoculum in the medium liquid of per 100 parts of quality;
One. gained Bacillus licheniformis of the present invention is obtained by following method screening:
Take by weighing 10 gram thickened sludges and put into the triangular flask (granulated glass sphere is arranged) that the 90mL sterilized water is housed, 28 ℃, 150 rev/mins shaking table shaking culture 15 minutes, sterilized water is diluted to proper concn, draws the 0.1mL diluent and evenly coats the casein substrate flat board, and 28 ℃ of thermostat containers are cultivated.After treating that bacterium colony grows, the bacterium colony that even coating trichoroacetic acid(TCA) solution on culture medium flat plate, picking have an obvious hydrolysis circle is cultivated repeatedly and is verified at the casein food grade flat board, and carries out separation and purification and preservation.Used casein substrate composition is: casein food grade 20g, and agar powder 20g, pH 9.0, distilled water 1L.This substratum is a selective medium, is used for the screening of Sumizyme MP bacterial strain.Used slant preservation substratum is the LB substratum, and composition is (g/L): Tryptones 10g, and yeast extract 5g, NaCl 10g, pH 7.4, and distilled water 1L is used for the preservation of slant strains.
Two. bacterial strain of the present invention has following form and physiology, biochemical characteristic:
1. morphological specificity
Short and small, the direct rod shape of Bacillus licheniformis HM-3 thalline, wide 0.1-0.3 μ m, long 0.5-1.0 μ m; Bacterium colony is oyster white, loose, easy picking, and is opaque, surface irregularity, and the edge is irregular.The gemma end is given birth to, shape ellipse or column, and sporangium does not have obviously expands.
2. physio-biochemical characteristics
B.licheniformis HM-3 strictness is aerobic, has mobility, the Gram-reaction positive, M.R and V.P reacting positive, energy hydrolyzed starch, liquefy gelatin; Can produce proteolytic enzyme, can utilize malonate, can be nitrite with nitrate reduction.Be accredited as Bacillus licheniformis through BIOLOG microorganism identification instrument.
Three, bacterial strain of the present invention is that fermentation raw material is produced Sumizyme MP with the municipal sludge substratum, and step is as follows:
(1) substratum conditioning: regulating the mud solid content is 2～5%, adds 0.1～0.3% bubble enemy, is 7.0～7.5 with acid or adjusting PH with base, stirs, and sterilization is cooled off standby;
(2) shake-flask culture: picking one ring Bacillus licheniformis is inoculated into is equipped with in the LB liquid nutrient medium, and 28～32 ℃, 50～250 rev/mins were carried out shake-flask culture 10～15 hours;
(3) ferment tank: the inoculum size by 1～5% will be shaken bottle bacterium liquid and be moved into the fermentor tank that solid content is 2～5% mud is housed, and carry out fermentation culture;
(4) protease purification: after fermentation was finished, transferring pH was 6.0～7.0, concentrated and made liquid dosage form or cohesion, the Sumizyme MP solid dosage is made in filtration;
(5) quality product detects: the product quality inspection method is carried out with reference to light industry standard " detergent use basic protein zymin " (QB 1806-1993).
In aforesaid method, the described sterilization of step (1) is preferably 0.1KPa pressure and 121 ℃ of following sterilizations 45 minutes.
In aforesaid method, the condition optimization of the described fermentation culture of step (3) is: 200 rev/mins of jar 28～32 ℃ of temperature, tank pressure 0.05MPa, air flow 1: 1.0 (V/V/ branch), stirring velocitys, fermentation time 60～80 hours.
Four, the Sumizyme MP of the present invention's preparation has following character:
The suitableeest action pH is 10.0, and 40 ℃ of optimum temperatures are more stable in pH 6.0-11.0,50 ℃ of scopes; Ca
2+Can improve the vigor of proteolytic enzyme, Mg
2+, Na
+To the obviously influence of enzyme nothing alive, Cu
2+, Hg
2+The severe inhibition enzyme activity; Inhibitor EDTA has the obvious suppression effect to this enzyme enzyme work, and PMSF then suppresses this enzymic activity fully; Resistance of oxidation is stronger, with commercially available stain remover consistency is preferably arranged.
Know-why of the present invention is: the high reactivity of separation screening alkalescence protease-producing bacterium from municipal sludge, the characteristic of utilizing it to be suitable for the mud growth and to produce proteolytic enzyme, with the contained abundant organic carbon of municipal sludge, nitrogen and inorganic salt is nutritive substance, under the condition of appropriate pH, temperature and air flow, grown and propagation, and in fermented liquid secreted alkaline proteolytic enzyme, fermented liquid is made the basic protein enzyme product through certain processing.
Compared with prior art, the present invention has following beneficial effect:
(1) screening efficient alkaline protease-producing bacterium from municipal sludge, i.e. Bacillus licheniformis HM-3, this bacterial strain is directed to mud, is suitable for growing multiplication in the mud environment, many, the product enzyme efficient height of bacterium number in mud.
(2) compare with existing Sludge Disposal Techniques, utilize bacterial strain of the present invention to produce Sumizyme MP, but disposing sludge not only, and can obtain added value height, lower-cost basic protein enzyme product.
(3) compare with existing Sumizyme MP production technology, the present invention is with waste---and municipal sludge replaces traditional grain substratum to produce the basic protein enzyme product, not only the disposal of resources for municipal sludge provides brand-new approach, and can reduce the price of product significantly, promote the suitability for industrialized production and the application of protease preparation.The basic protein enzyme product of gained can be applicable to industries such as washing, process hides, and is environmentally safe harmless.
Description of drawings
Fig. 1 is a Bacillus licheniformis HM-3 stereoscan photograph.
Embodiment
Separation, the purifying of embodiment 1. bacterial classifications
Take by weighing 10 gram thickened sludges and put into the triangular flask (granulated glass sphere is arranged) that the 90mL sterilized water is housed, 28 ℃, 150 rev/mins shaking table shaking culture 15 minutes, sterilized water is diluted to 10
-5, to draw the 0.1mL diluent and evenly coat the casein substrate flat board, 28 ℃ of thermostat containers were cultivated 3 days.Evenly be coated with trichoroacetic acid(TCA) solution on culture medium flat plate, picking has single bacterium colony of obvious hydrolysis circle to cultivate repeatedly at the casein food grade flat board, and carries out separation and purification and preservation.Used casein substrate composition is: casein food grade 20g, and agar powder 20g, pH 9.0, distilled water 1L.Used slant preservation substratum is the LB substratum, and composition is (g/L): Tryptones 10g, and yeast extract 5g, NaCl 10g, pH 7.4, distilled water 1L.
Embodiment 2. strain identification
(1) physio-biochemical characteristics are identified
B.licheniformis HM-3 physio-biochemical characteristics qualification result sees Table 1.
The physio-biochemical characteristics of table 1.HM-3 bacterial strain
Annotate :+expression can utilize;-expression can not utilize
(2) BIOLOG identifies
The qualification result of automatic microbe assessing instrument (BIOLOG) shows that HM-3 is Bacillus licheniformis (Bacillus licheniformis).Bacillus licheniformis HM-3 stereoscan photograph is seen Fig. 1.
Embodiment 3. Bacillus licheniformis HM-3 are that fermenting raw materials is produced Sumizyme MP with mud
(1) substratum conditioning: regulating the municipal sludge solid content is 3%, adds 0.2% bubble enemy, and to transfer pH with NaOH be 7.0, stirs, and 0.1KPa pressure was sterilized 45 minutes down with 121 ℃, cooled off standby.
(2) shake-flask culture: picking one ring lawn is inoculated into the triangular flask that the LB liquid nutrient medium is housed from the HM-3 slant medium, and 30 ℃, 100 rev/mins are carried out shake-flask culture 12h.
(3) fermentation: the inoculum size with 3% will be shaken bottle bacterium liquid and be moved into the fermentor tank that solid content is 3% mud is housed, and carry out fermentation culture.Condition is: jar 30 ℃ of temperature, tank pressure 0.005MPa, air flow 1: 1.0 (V/V/ branch), 200 rev/mins of stirring velocitys, fermentation time 64 hours.
(4) protease purification: after fermentation was finished, transferring pH was 7.0, made the Sumizyme MP solid dosage by cohesion, filtration.
(5) quality product detects: the product quality inspection method is carried out with reference to light industry standard " detergent use basic protein zymin " (QB 1806-1993).Assay shows that this protease preparation meets the specification of quality of Sumizyme MP, and product is qualified.
Embodiment 4. Sumizyme MP property testings
Adopt embodiment 3 fermentation process to ferment.After fermentation is finished, sampling, 3000-6000 rev/min of centrifugal 15min, supernatant liquor is crude enzyme liquid.Borax-NaOH damping fluid dilution enzyme liquid with pH 10.5 adds each metal ion species and inhibitor mixing, makes metal ion and inhibitor reach required concentration, in 40 ℃ of water bath heat preservation 10min, measures residual protein enzyme enzyme and lives.Not add metal ion is contrast.The results are shown in Table 2, show Ca
2+Can improve the vigor of proteolytic enzyme, Mg
2+, Na
+To the obviously influence of enzyme nothing alive, Cu
2+, Hg
2+The severe inhibition enzyme is lived; Work has the obvious suppression effect to inhibitor EDTA to enzyme, the then complete inhibitory enzyme activity of PMSF.
Table 2 metal ion and inhibitor are to the influence of enzyme activity
Claims (5)
1. a Bacillus licheniformis is gram-positive microorganism, direct rod shape, on March 12nd, 2007 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, be numbered: CGMCC 1970.
2. the method for utilizing the described Bacillus licheniformis of claim 1 to produce Sumizyme MP is characterized in that comprising the steps:
(1) substratum conditioning: regulating the mud solid content is 2～5%, adds 0.1～0.3% bubble enemy, is 7.0～7.5 with acid or adjusting PH with base, stirs, and sterilization is cooled off standby;
(2) shake-flask culture: picking one ring Bacillus licheniformis is inoculated into is equipped with in the LB liquid nutrient medium, 28～32 ℃, 50～250 rev/mins shake-flask culture 10～15 hours;
(3) ferment tank: the inoculum size by 1～5% will be shaken bottle bacterium liquid and be moved into the fermentor tank that solid content is 2～5% mud is housed, and carry out fermentation culture;
(4) protease purification: after fermentation was finished, transferring pH was 6.0～7.0, concentrated and made liquid dosage form or cohesion, the Sumizyme MP solid dosage is made in filtration.
3. method as claimed in claim 2 is characterized in that the described sterilization of step (1) is 0.1KPa pressure and 121 ℃ of following sterilizations 45 minutes.
4. method as claimed in claim 2 is characterized in that the described LB liquid culture of step (2) based formulas is: yeast extract 5g, and Tryptones 10g, NaCl 5g, water 1000mL, pH 7.2～7.6.
5. method as claimed in claim 2 is characterized in that the condition of the described fermentation culture of step (3) is: 28～32 ℃ of jar temperature, fermentation time 60～80 hours.
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| CN101805772B (en) * | 2010-03-26 | 2012-07-25 | 东华大学 | Method for extracting protein from excess sludge |
| CN102010109B (en) * | 2010-10-29 | 2012-09-19 | 孙祥章 | Application of bio-enzyme catalysis in residual sludge treatment |
| CN103451121B (en) * | 2013-06-03 | 2015-06-03 | 华南理工大学 | Bacillus licheniformis and application thereof |
| CN105779423A (en) * | 2015-11-17 | 2016-07-20 | 济南诺能生物工程有限公司 | Compound protease as well as production method and applications thereof |
| CN108179140A (en) * | 2018-03-12 | 2018-06-19 | 广州勇达生物科技有限公司 | A kind of preparation method of neutral proteinase |
| CN112280768B (en) * | 2020-10-26 | 2021-07-06 | 林小丽 | Alkaline protease low-temperature mutant and application thereof in sludge treatment |
| CN112458070B (en) * | 2020-10-26 | 2021-07-06 | 林小丽 | Enzyme preparation for sludge treatment |
-
2008
- 2008-03-14 CN CN 200810026812 patent/CN101245327B/en not_active Expired - Fee Related
Non-Patent Citations (6)
| Title |
|---|
| Siriporn Yossan et al..Purification and characterization of alkaline protease fromBacillus megaterium isolated from Thai fish sauce fermentationprocess.ScienceAsia32 4.2006,32(4),377-383. |
| Siriporn Yossan et al..Purification and characterization of alkaline protease fromBacillus megaterium isolated from Thai fish sauce fermentationprocess.ScienceAsia32 4.2006,32(4),377-383. * |
| 包巨南等.微生物源碱性蛋白酶研究进展.酿酒34 3.2007,34(3),50-52. |
| 包巨南等.微生物源碱性蛋白酶研究进展.酿酒34 3.2007,34(3),50-52. * |
| 王春铭等.城市污泥模拟堆肥过程中高温菌群的筛选、鉴定及降解效果.环境科学学报27 6.2007,27(6),979-986. |
| 王春铭等.城市污泥模拟堆肥过程中高温菌群的筛选、鉴定及降解效果.环境科学学报27 6.2007,27(6),979-986. * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106676024A (en) * | 2015-11-05 | 2017-05-17 | 中国科学院天津工业生物技术研究所 | Bacillus amyloliquefaciens for high yield of alkali protease and application of bacillus amyloliquefaciens |
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