CN105505781A - Method for controlling aspergillus filamentous fungi to form mycelium pellets - Google Patents
Method for controlling aspergillus filamentous fungi to form mycelium pellets Download PDFInfo
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- CN105505781A CN105505781A CN201410558748.3A CN201410558748A CN105505781A CN 105505781 A CN105505781 A CN 105505781A CN 201410558748 A CN201410558748 A CN 201410558748A CN 105505781 A CN105505781 A CN 105505781A
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- filamentous fungus
- aspergillus
- mycelium pellet
- czapek
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Abstract
The invention discloses a method for controlling aspergillus filamentous fungi to form mycelium pellets. The method includes steps of culturing spores, preparing spore suspension, controlling the aspergillus filamentous fungi to form the mycelium pellets, and the like. The mycelium pellets of the filamentous fungi are beneficial to mass transfer, low in fermentation broth viscosity, high in product yield, and the like. Other single-cell microorganisms can be harvested easily by the mycelium pellets. The control conditions of the method include spore concentration, pH, and sugar concentration in culture medium. The method has the advantages that the forming conditions of the mycelium pellets can be controlled effectively, and a reliable method is provided for the wide application of the mycelium pellets of the filamentous fungi.
Description
Technical field
The present invention relates to micro-raw engineering, particularly a kind of method controlling Aspergillus filamentous fungus formation mycelium pellet.
Technical background
Aspergillus filamentous fungus is extensively distributed on cereal, air, soil and various article.Relatively common are aspergillus niger, aspergillus oryzae, Aspergillus nidulans etc.They have played very large effect in biotech development process, and a lot of product comes from the primary and secondary meta-bolites of filamentous fungus.Aspergillus niger has been used for producing citric acid and a lot of enzyme, and such as, on market, common amylase, cellulase, hemicellulase, glucolase, glucuroide, glucose oxidase, peroxidation oxygenase, tilactase, zytase, naringinase, tannase, lipase, phytase, proteolytic enzyme, polygalacturonase etc. can obtain from Aspergillus filamentous fungus.Aspergillus filamentous fungus can produce the combination of enzyme not of the same race according to the difference of substratum, as cellulase, zytase and polygalacturonase.Food and drug administration (FDA) has assert that the multiple product from Aspergillus filamentous fungus is safe, so the product of Aspergillus filamentous fungus is widely used in food grade products.
In deep drainpipe process, Aspergillus filamentous fungus can form mycelium pellet under certain condition.Filamentous fungus has the advantages such as fermentation broth viscosity is low, mass transfer good, Product yields is high, thalline recovery is simple with the growth of the form of mycelium pellet.And control the energy consumption that certain mycelium pellet size can also avoid dissolved oxygen restriction, raising Product yields, attenuating bio-reactor further.So the size controlling filamentous fungus mycelium pellet is an important technology.The object that filamentous fungus mycelium pellet can also adsorb metal ion in waste water, dyestuff reaches process waste water.Filamentous fungus can also be total to balling-up with other unicellular microorganism, and such as aspergillus niger and multiple marine alga form simple harvesting method after being total to balling-up.The method utilizing mycelium pellet to gather in the crops has saved the unicellular microorganism water treatment large-scale energy consumption equipment required when gathering in the crops, and reduces cost.
Summary of the invention
Object of the present invention, controls to provide a kind of the method that Aspergillus filamentous fungus forms mycelium pellet exactly.
In order to realize object of the present invention, present invention employs following technical scheme: a kind of method controlling Aspergillus filamentous fungus formation mycelium pellet, is characterized in that, comprise the following steps:
The cultivation of the first step, spore
Be inoculated on solid medium by Aspergillus filamentous fungus, in 22 DEG C of-32 DEG C of incubators, cultivate 48-240h, Aspergillus filamentous fungus forms spore;
The preparation of second step, spore suspension
Utilize sterilized water or phosphoric acid buffer washing spore, obtain spore suspension, be dispensed in the tubule after sterilizing, preserve stand-by at 2-6 DEG C;
3rd step, control Aspergillus filamentous fungus form mycelium pellet
Be inoculated into by spore suspension in czapek's solution, inoculating spores concentration is 7.23 × 10
5/ L ~ 8.68 × 10
8/ L; Then, be placed in rotary shaker and cultivate, culture condition is: pH3.0 ~ 8.0, temperature 22-35 DEG C, shaking speed 100-200rpm; Cultivate 48h in czapek's solution after, Aspergillus filamentous fungus forms mycelium pellet.
Described Aspergillus filamentous fungus comprises aspergillus niger, aspergillus oryzae or Aspergillus nidulans.
Described solid medium comprises PDA nutrient agar, containing the czapek's solution of 2% agar or YPD solid medium.
The incubation time of above-mentioned 3rd step in czapek's solution is 48-168h.
The making method of described czapek's solution is as follows: get glucose25.0g, NaNO
33.0g, K
2hPO
41.0g, KCl0.5g, MgSO
47H
2o0.5g and FeSO
47H
2o0.01g, adds deionized water to 1L; PH is regulated to be 5.5 with 1mol/LHCl and 1mol/LNaOH again.
The present invention obtains mycelium pellet by controlling culture condition cultivation Aspergillus filamentous fungus in czapek's solution, has and improves output, raising mass transfer, the simple advantage of recovery thalline.Filamentous fungus is that a class can form fine hair shape, netted or cotton-shaped mycelial fungi.The mycelia of Aspergillus filamentous fungus mutually can be wound around under certain condition in liquid culture, forms spherical or oval mycelium pellet.The aliment security level product that U.S. food and drug administration (FDA) ratify from aspergillus niger, the multiple metabolite of aspergillus oryzae (organic acid etc.).And it has applicating history for a long time in food.Aspergillus mycelium pellet is Adsorption of Cu when pH4-6
2+, Zn
2+, Ni
2+.Wherein, aspergillus niger ferments in the mode of mycelium pellet the generation of the generation that is conducive to citric acid and other multiple metabolite.The another one advantage of filamentous fungus mycelium pellet is the results being conducive to thalline.The diameter great majority of mycelium pellet, at more than 0.5cm, just can be gathered in the crops mycelium pellet with simple sieve, reduce the cost of results process.The method utilizing mycelium pellet to gather in the crops has saved large-scale energy consumption equipment, reduces cost, and avoids using chemical floc in sewage treatment process, decreases the harm to environment.It is only based on the czapek's solution of organic carbon source containing glucose that the present invention adopts, and is optimized the spore concentration of aspergillus niger, the initial pH of substratum, glucose concn.Result shows that aspergillus niger just can form mycelium pellet clearly when cultivating 24h, and within the time subsequently, mycelium pellet keeps stable always, does not occur the phenomenon of mycelium pellet cracking.In czapek's solution, aspergillus niger is 7.23 × 10 at spore concentration
3/ L ~ 3.62 × 10
7mycelium pellet can be formed between/L, initial pH3.5 ~ pH7.5.After reducing the glucose concn of czapek's solution, aspergillus niger still can form mycelium pellet.Above result of study shows that aspergillus niger has wider mycelium pellet formation condition, for the widespread use of aspergillus niger is provided convenience.
Accompanying drawing explanation
Fig. 1 is the impact of different spore concentration on aspergillus niger balling-up in czapek's solution;
Fig. 2 is the impact of spore concentration on the mycelium pellet of Aspergillus filamentous fungus in czapek's solution;
Fig. 3 is the impact of different pH value on aspergillus niger balling-up in czapek's solution;
Fig. 4 is the mycelium pellet of aspergillus niger in czapek's solution of different spore concentration;
Fig. 5 is aspergillus niger balling-up in the czapek's solution of different glucose concn;
Fig. 6 is the mycelium pellet of aspergillus niger in the czapek's solution of different glucose concn.
Specific implementation method
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1
Be inoculated into by aspergillus niger on PDA nutrient agar, cultivate 96-144h in 22 DEG C of-32 DEG C of incubators after, filamentous fungus forms spore.
Utilize sterilized water to wash filamentous fungus spore, by the spore suspension that obtains after microscopic counting, be dispensed in the tubule after sterilizing, be stored in 2-6 DEG C, stand-by.
Be inoculated in czapek's solution by the spore suspension after preparation, biomass is 7.23 × 10 at spore concentration
3/ L ~ 8.68 × 10
6/ L.Culture condition is: pH5.5, temperature 30 DEG C, mixing speed 200rpm, and czapek's solution is after 72h cultivates, and spore concentration is shown in Fig. 1 to the impact that Aspergillus filamentous fungus grows in czapek's solution.Along with the increase of spore concentration, biomass increases gradually.Biomass is 4.34 × 10 at spore concentration
6/ L ~ 8.68 × 10
6higher biomass (14.27g/L ~ 15.70g/L) is obtained between/L.Residual sugar concentration in nutrient solution fluctuates between 9.61g/L ~ 7.61g/L.Spore concentration is 7.23 × 10
3/ L ~ 7.23 × 10
4time between/L, the more aggravation that pH value declines along with the rising of spore concentration.When spore concentration continues to raise, final pH change not obvious (pH3.15 ~ pH3.30).Aspergillus niger all can form mycelium pellet (Fig. 2) within the scope of the spore concentration of this experiment.
Embodiment 2
Be inoculated into by aspergillus oryzae on the czapek's solution containing 2% agar, in 22 DEG C of-32 DEG C of incubators, cultivate 24-72h, filamentous fungus forms spore.
Utilize phosphoric acid buffer to wash filamentous fungus spore, by the spore suspension that obtains after microscopic counting, be dispensed in the tubule after sterilizing, be stored in 2-6 DEG C, stand-by.
Be inoculated in czapek's solution by the spore suspension after preparation, inoculating spores concentration is 7.23 × 10
8/ L.Temperature 30 DEG C, mixing speed 200rpm, after 72h cultivates.Initial pH is shown in Fig. 3 to the growing state of aspergillus niger in czapek's solution.Between initial pH3.5 ~ pH5.6, biomass raises gradually along with the increase of initial pH value.And initial pH higher than 5.6 time, biomass slightly declines, and illustrates that initial pH has faint suppression more than 5.6 cell growth.Initial pH is higher, and the amplitude that pH declines is larger.During initial pH3.5 ~ pH6.5, in czapek's solution, the content of residual glucose fluctuates very little (8.79g/L ~ 9.73g/L).As initial pH7.5, residual glucose is elevated to 11.31g/L.This result declined with biomass conforms to.Fig. 3 be aspergillus niger under the condition of the initial pH of difference, through the mycelium pellet that the cultivation of 72h is formed.Aspergillus niger can form mycelium pellet between initial pH3.5 ~ pH7.5 as seen from Figure 4.
Embodiment 3
Be inoculated into by Mortierella isabellina on YPD solid medium, in 22 DEG C of-32 DEG C of incubators, cultivate 168-240h, filamentous fungus forms spore.
Utilize sterilized water to wash filamentous fungus spore, by the spore suspension that obtains after microscopic counting, be dispensed in the tubule after sterilizing, be stored in 2-6 DEG C, stand-by.
Be inoculated into by spore suspension after preparation in the supernatant of soybean wastewater first time process, inoculating spores concentration is 7.23 × 10
8/ L.Culture condition is: pH5.5, temperature 30 DEG C, mixing speed 200rpm, and after 72h cultivates, filamentous fungus forms mycelium pellet.Glucose content is shown in Fig. 5 to aspergillus niger growing state in czapek's solution.Along with the rising of glucose content, biomass and remaining table sugar increase.When glucose content is 0.1g/L, pH does not decline.When glucose content is increased to 5.0g/L by 0.1g/L, pH fall strengthens gradually.Glucose content is higher than after 5.0g/L, and final pH keeps constant (about 3.70).Fig. 5 is the mycelium pellet that aspergillus niger is formed when different glucose concn.Fig. 6 shows that aspergillus niger can form mycelium pellet in 0g/L ~ 30g/L glucose concn, and nutrient solution also keeps clarification.
Claims (5)
1. control the method that Aspergillus filamentous fungus forms mycelium pellet, it is characterized in that, comprise the following steps:
The cultivation of the first step, spore
Be inoculated on solid medium by Aspergillus filamentous fungus, in 22 DEG C of-32 DEG C of incubators, cultivate 48-240h, Aspergillus filamentous fungus forms spore;
The preparation of second step, spore suspension
Utilize sterilized water or phosphoric acid buffer washing spore, obtain spore suspension, be dispensed in the tubule after sterilizing, preserve stand-by at 2-6 DEG C;
3rd step, control Aspergillus filamentous fungus form mycelium pellet
Be inoculated into by spore suspension in czapek's solution, inoculating spores concentration is 7.23 × 10
5/ L ~ 8.68 × 10
8/ L; Then, be placed in rotary shaker and cultivate, culture condition is: pH3.0 ~ 8.0, temperature 22-35 DEG C, shaking speed 100-200rpm; Cultivate 48h in czapek's solution after, Aspergillus filamentous fungus forms mycelium pellet.
2. control Aspergillus filamentous fungus according to claim 1 forms the method for mycelium pellet, and it is characterized in that, described Aspergillus filamentous fungus comprises aspergillus niger, aspergillus oryzae or Aspergillus nidulans.
3. control Aspergillus filamentous fungus according to claim 1 forms the method for mycelium pellet, it is characterized in that, described solid medium comprises PDA nutrient agar, containing the czapek's solution of 2% agar or YPD solid medium.
4. control Aspergillus filamentous fungus according to claim 1 forms the method for mycelium pellet, and it is characterized in that, the incubation time of the 3rd step in czapek's solution is 48-168h.
5. control Aspergillus filamentous fungus according to claim 1 forms the method for mycelium pellet, and it is characterized in that, the making method of described czapek's solution is as follows: get glucose25.0g, NaNO
33.0g, K
2hPO
41.0g, KCl0.5g, MgSO
47H
2o0.5g and FeSO
47H
2o0.01g, adds deionized water to 1L; PH is regulated to be 5.5 with 1mol/LHCl and 1mol/LNaOH again.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105733599A (en) * | 2016-01-28 | 2016-07-06 | 中南大学 | Cadmium-contaminated soil remediation fixing agent based on microorganism assembly synthesis as well as preparation and application method thereof |
| CN109609391A (en) * | 2019-02-15 | 2019-04-12 | 福州大学 | A kind of quantitative inoculation method of mycelium pellet |
| CN115181677A (en) * | 2022-07-13 | 2022-10-14 | 河北建筑工程学院 | Preparation method of composite biomass material for treating phenolic wastewater |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212484A (en) * | 2011-05-05 | 2011-10-12 | 南京工业大学 | Method for controlling growthform of filamentous fungi during fermentation process |
-
2014
- 2014-10-20 CN CN201410558748.3A patent/CN105505781A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212484A (en) * | 2011-05-05 | 2011-10-12 | 南京工业大学 | Method for controlling growthform of filamentous fungi during fermentation process |
Non-Patent Citations (1)
| Title |
|---|
| 黄勋娟: "优化黑曲霉菌丝球形成条件提高豆制品废水处理效率", 《工业微生物》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105733599A (en) * | 2016-01-28 | 2016-07-06 | 中南大学 | Cadmium-contaminated soil remediation fixing agent based on microorganism assembly synthesis as well as preparation and application method thereof |
| CN105733599B (en) * | 2016-01-28 | 2019-04-23 | 中南大学 | A kind of cadmium pollution soil reparation fixative and its methods for making and using same based on microorganism assembling synthesis |
| CN109609391A (en) * | 2019-02-15 | 2019-04-12 | 福州大学 | A kind of quantitative inoculation method of mycelium pellet |
| CN115181677A (en) * | 2022-07-13 | 2022-10-14 | 河北建筑工程学院 | Preparation method of composite biomass material for treating phenolic wastewater |
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Application publication date: 20160420 |
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