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Hydrogen production chlamydomonas chloroplast separation method

A technology for chloroplasts and hydrochlamydomonas, applied in the biological field, can solve problems such as difficulty in separation, and achieve the effects of time saving, high yield and simple steps

Active Publication Date: 2015-01-14
INST OF BOTANY CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, some metabolic and structural changes have occurred in the hydrogen-producing Chlamydomonas cells, which makes it difficult for people to isolate the chloroplasts of the sulfur-deficient Chlamydomonas hydrogen-producing cells by the pressure method.

Method used

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  • Hydrogen production chlamydomonas chloroplast separation method
  • Hydrogen production chlamydomonas chloroplast separation method
  • Hydrogen production chlamydomonas chloroplast separation method

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Embodiment 1

[0056] Embodiment 1, the separation of Chlamydomonas chloroplast

[0057] 1. Culture of Chlamydomonas cells

[0058] Inoculate Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) CC400 into a 100ml Erlenmeyer flask containing 30ml of normal TAP culture medium at a temperature of 25°C and a light intensity of 200μE·m -2 ·s -1 Cultivate in a shaker at 60rpm under continuous light for 24-36h. Collect the pre-cultured Chlamydomonas cells and inoculate them into 900ml of normal TAP culture medium with an initial concentration of 4×10 4 pcs / ml (OD 750 about 0.02); at a temperature of 25°C, the light intensity is 120μE·m -2 ·s -1 Under the conditions, the synchronous culture of "12h light: 12h dark" was carried out, co-cultured for 3 photoperiods, and Chlamydomonas cells were collected at 4-6h of the fourth photoperiod.

[0059] 2. Sulfur deficiency treatment induces hydrogen production reaction

[0060] 1. Centrifuge at 2500 rpm for 4 minutes at 25°C to collect the Chlamydo...

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Abstract

The invention discloses a hydrogen production chlamydomonas chloroplast separation method which comprises the following steps: 1) hanging Chlamydomonas in a lysate, adding saponin until the final concentration is 0.25g / 100mL; 2) putting the step 1) solution on the ice for 1-2min for chlamydomonas cell lysis; 3) centrifuging the step 2) solution for 1min at 2300g at 4 DEG C; 4) using the lysate for rinsing steps 3) precipitate, centrifuging for 1min at 1000-2300g at 4 DEG C to obtain crude chloroplast extract; 5) hanging the crude chloroplast extract in a lysate; 6) adding 75% by volume of Percoll on the centrifugal pipe bottom, adding 45% by volume of Percoll into the upper layer, adding the step 5) solution into the uppermost layer to the form the gradient, centrifuging for 20min at 4600g at 4 DEG C to form dark green bands between two different volume fractions of Percoll; and 7) taking the dark green bands out, using the lysate to dilute, centrifuging for 2min at 890-1000g at 4 DEG C, and collecting precipitate to obtain chlamydomonas chloroplast. The experiment proves that the method has the advantages of simple operation, high chlamydomonas chloroplast yield and shorter time.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for isolating Chlamydomonas chloroplasts. Background technique [0002] Photosynthetic hydrogen production by algae is a biological process in which microalgae use solar energy to split water to release hydrogen gas. It has obvious advantages such as regeneration and clean hydrogen production process, and has potential application value. It is one of the important ways to realize the sustainable production of hydrogen energy. Chlamydomonas is a model alga species for photosynthetic hydrogen production, with high hydrogen production efficiency and easy cultivation. Chloroplast is the organelle for photosynthetic hydrogen production. It is very important to analyze the process and regulation of photosynthetic hydrogen production from isolated and purified chloroplasts for the research and development of artificial simulation systems for photosynthetic hydrogen production. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/12C12R1/89
CPCC12N1/12
Inventor 黄芳孙永乐张锦陈梅
Owner INST OF BOTANY CHINESE ACAD OF SCI
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