Method for com-culturing and improving hydrogen output by utilizing bacteria and chlamydomonas reinhardtii
A rhinestone and co-cultivation technology, applied in the field of biological hydrogen production, can solve the problems of easy pollution and easy pollution of other bacteria, and achieve the effect of increasing hydrogen production
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[0041] (a), preparation of bacterial genome: take 2 mL of logarithmic growth phase bacterial solution, put it into a sterile centrifuge tube, and centrifuge at 12,000 r / min for 5 min. Discard the supernatant, add 300 μL of 10 mg / mL lysozyme to the centrifuge tube containing the bacterial pellet, and incubate for 2 hours. Then, 15 μL of 10 mg / mL RNAase, 30 μL of 20 mg / mL proteinase K, and 100 μL of 10% SDS were sequentially added, mixed well, and incubated at 65° C. for 45 minutes until clear. Then 125 μL of 5M NaCl and 100 μL of 10% CTAB were added and incubated at 65° C. for 30 minutes to fully lyse cells and remove polysaccharides. Then, according to the routine steps of DNA extraction, the DNA was extracted with phenol, the DNA was precipitated with absolute ethanol, and finally the DNA was dissolved in pure water for use.
[0042] (b), primer design: according to the literature, the general primers for bacterial 16S rDNA gene amplification were selected: 27F (5′-AGAGTTTGA...
Embodiment 1
[0060] Example 1 Cultivation of Chlamydomonas reinhardtii
[0061] Chlamydomonas reinhardtii is a representative species of photosynthetic hydrogen production by microalgae with fast growth rate, low culture cost and high hydrogenase activity. In order to make the experiment easy to operate, the present invention preferably prefers the cell wall-deficient Chlamydomonas reinhardtii sp. cc849.
[0062] ①Normal culture conditions of Chlamydomonas reinhardtii: According to "The Chlamydomonas Sourcebook: a comprehensive guide to biology and laboratory use. New York: Academic Press. 1989" edited by Harris, the optimal conditions are 25±1℃, and the illumination intensity of fluorescent light (100~200μmol photons) m -2 s -1 ); liquid culture is in 50~100ml Tris-Acetate-Phosphate (TAP) medium, initial pH 7.2, horizontal shaker speed 100~130rpm, 1% inoculation subculture every 5~6 days; solid TAP plate medium contains 1.5% agar powder, preservation and purification of algal species i...
Embodiment 2
[0065] Example 2 Isolation and identification of Chlamydomonas commensal bacteria
[0066] ①Isolation, purification and culture of Chlamydomonas commensal bacteria:
[0067] Take 1 mL of Chlamydomonas culture solution infected with bacteria at different growth stages and dilute it with sterile double distilled water for 10 -1 , 10 -2 , 10 -3 Three concentration gradients were applied to TAP medium, TAP+Escherichia coli medium LB (TAP medium in turn) [7] and E. coli LB medium [8] Mix in a 1:1 volume ratio) and YEM medium [9] On the solid plate of 25 ℃, 28-30 ℃ and 37 ℃ respectively, after the colony grows out, pick a single colony and subculture it in the corresponding liquid medium.
[0068] Using TAP+LB, YEM and TAP three medium plates, the bacteria commensal with Chlamydomonas reinhardtii cc849 were preliminarily isolated. As a result, two single colonies with different characteristics were isolated on the TAP+LB mixed medium, which were named L2 respectively. and L3; ...
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