A high-temperature-resistant Bacillus bacterium njau-nd8 for accelerating compost maturity and its application
A technology of NJAU-ND8 and Bacillus genus, applied in the field of agricultural microorganisms, can solve the problems of weak promotion of composting effect and unstable composting strains
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Embodiment 1
[0035] Embodiment 1, isolation and identification of functional strains
[0036] Put the samples in the high temperature phase of composting into a 250ml Erlenmeyer flask with glass beads and 100ml of sterile water, shake at 25°C and 170r / min for 20min to form a soil suspension, and draw 10 -2 、10 -3 、10 -4 、10 -5Dilute 0.1ml of the fertilizer suspension, spread it on the lignocellulose culture plate, repeat each concentration 3 times, culture at 28°C for 5 days, pick and purify the growing colonies on the plate for more than 5 times, and select the colonies that can still grow Transfer to slant culture and store at 4°C for later use.
[0037] Through qualitative and quantitative screening, a bacterial strain named NJAU-ND8 was finally obtained. The colony of the bacterial strain NJAU-ND8 on the LB plate was large, pale white, and the edges were scattered. The bacteria were soft and sticky to a certain extent. provoke. The starch decomposition is positive and the tryptoph...
Embodiment 2
[0047] Embodiment 2, NJAU-ND8 produces the determination of various enzyme activities
[0048] Use LB medium as NJAU-ND8 enzyme production medium: 10g peptone, 5g yeast powder, 10g NaCl, 1000ml deionized water, pH 7.0, sterilized at 121°C for 20min. Use 3ml of sterile water to make the bacterial suspension into bacterial suspension, take 0.5ml and inoculate them in the enzyme-producing medium respectively, and culture on a shaking table at 170r / min at 50°C. After 24 hours, the bacterial solution was taken, and after adding the extract solution for extraction, the cell membrane was broken to form a stock solution by using an ultrasonic breaker (300W, ultrasonic for 3s, with an interval of 7s, for 3min).
[0049] Cellulase activity was determined according to the method of Albrecht et al. (2008). The stock solution was mixed with acetate buffer (50mM, pH 5) at a ratio of 1:5 (W / V), shaken on a horizontal shaker for 1h, and then centrifuged at 10000r / min at 4°C for 10min to obta...
Embodiment 3
[0056] Embodiment 3, the generation of liquid inoculum
[0057] Streak the NJAU-ND8 strains stored in glycerol tubes at -80°C on LB solid medium plates for activation, and cultivate them in a 37°C incubator for one hour. Pick a single colony of NJAU-ND8 and culture it in a 3ml liquid test tube at 37°C with shaking at 170r / min for 10 hours, and the bacterial solution is used as the seed solution. With 1% (v / v) inoculum size, a seed solution was transferred to LB liquid shake flask, and cultivated at 37°C and 170r / min until mid-logarithmic phase (OD 600 = 1.0), the cells were collected by centrifugation at 4°C, washed three times with distilled water, and resuspended in an equal volume of distilled water for later use.
[0058] The preparation method of the LB culture medium used is as follows, taking the preparation of 1L medium as an example: protein building 10g, yeast powder 5g, NaCl 10g agar 20g, constant volume to 1000ml, pH natural, sterilized at 121°C for 20min.
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