Bacillus subtilis strain and application thereof
A technology of Bacillus subtilis and strains, applied in Bacillus subtilis strains and its application field, can solve the problems of narrow application area, water environment pollution, water resource shortage, etc., and achieve the effects of improving water quality, reducing COD, and simple cultivation conditions
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Embodiment 1
[0026] The isolation and screening of embodiment 1 spore
[0027] 1. Collection of sludge
[0028] Collect 7 parts of sludge and water samples from the breeding pond, weigh 10g of sludge respectively (10mL of water sample is pipetted) into a conical flask filled with 90mL of sterilized normal saline, shake in a shaker at 150r / min for 0.5h, Take it out and put it in an 80°C water bath for 30 minutes, and dilute the sample solution by a certain number of times using the ten-fold dilution method, and set it aside.
[0029] Take 0.1 mL of diluted sample solution and spread it on solid LB medium, culture it at 30°C for 48 hours, select the colonies with good growth, similar colony shape and the largest number, separate and purify until the single bacteria with the same colony shape, and obtain 12 strains of spores.
[0030] 2. Screening of Enzyme-producing Bacillus
[0031] (1) Cellulase detection:
[0032] Carboxymethylcellulose sodium plate medium (g / L): CMC-Na 15.0, NaCl 5.0,...
Embodiment 2
[0045] The identification of embodiment 2 spore CM-A12
[0046] (1) Morphological characteristics
[0047] After the strain CM-A12 was cultured on the solid LB growth medium for 24 hours, the colony was in the shape of transparent water droplets, with a raised surface, moist and shiny; after 48 hours of culture, the surface of the colony was dry and off-white. Under the microscope, it was observed that the strain cells were Brevibacteria, such as figure 1 shown.
[0048](2) Physiological and biochemical identification (as shown in Table 2)
[0049] Table 2
[0050]
[0051]
[0052] Note: "+": the reaction is positive; "-" the reaction is negative.
[0053] (3) 16S rDNA determination
[0054] The 16S rDNA gene sequence of the strain could not be determined by observing the morphological characteristics of the strain, and the identification at the molecular level was carried out.
[0055] PCR amplification of 16S rDNA of strain a
[0056] ①PCR amplification system:...
Embodiment 3
[0067] Embodiment 3 Bacillus subtilis (Bacillus subtilis) CM-A12 growth condition optimization
[0068] Strain culture medium: peptone 10g / L, beef extract 5g / L, NaCl 5g / L, pH 7-7.2.
[0069] (1) Optimum growth pH of the inventive bacterial strain
[0070] The initial pH of the culture solution was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 with 1.0 mol / L HCl and NaOH, respectively, and the CM-A12 was inserted into 1% (v / v) inoculum, Cultivate at 30°C and 150r / min. After culturing for 20h and 40h respectively, measure the absorbance at 600nm with an ultraviolet-visible spectrophotometer, and use the culture solution without inoculation as a blank control.
[0071] Depend on image 3 It is known that CM-A12 grows well when the initial pH is between 4 and 9, and the pH range is wide; when the initial pH value is greater than 9.0, the strain CM-A12 can hardly grow.
[0072] From an economic point of view, when CM-A12 is mass-produced and fermented, the medium should b...
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