A method for determining the harvest time of oil-producing microalgae by using the chlorophyll fluorescence parameter fv/fm

A chlorophyll fluorescence, oil-producing microalgae technology, applied in the field of microalgae biology and bioenergy, can solve the problems of high time and reagent cost, accuracy interference, interference Raman spectral characteristics, etc., to reduce time and reagent cost, The effect of less interference factors and simple detection operation

Active Publication Date: 2017-02-15
INST OF AQUATIC LIFE ACAD SINICA
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Problems solved by technology

To assess the level of intracellular oil or neutral lipid accumulation and thus determine the harvest time of microalgae, the traditional gravimetric method (Bligh and Dyer, 1959, Canadian Journal of Biochemistry and Physiology, 37:911-917) involves cell disruption and oil extraction , takes a long time and requires at least 10-15mg of cells with a wet weight
Thin-layer chromatography (Reiser and Somerville, 1997, Journal of Bacteriology, 179:2969–2975) and gas chromatography-mass spectrometry (Gouveia et al., 2009, Journal of Industrial Microbiology & Biotechnology, 36:821-826) can detect different types of algae. Fatty acid composition, time-domain nuclear magnetic resonance system (Todt et al., 2006, Food Chemistry, 96:436-440) can conveniently quantify oil, but the above method also needs to break the cells for oil extraction before analysis, which takes time and reagent costs higher
In order to avoid the cell disruption process and non-invasively analyze the oil in algae cells, people use neutral lipid fluorescent probes such as BODIPY505 / 515 or Nile Red (Chen et al., 2009, Journal of Microbiological Methods, 77:41-47; Cooper et al., 2010, Journal of Bioscience and Bioengineering, 109:198-201) performed vital staining to detect intracellular neutral lipid levels, but due to the diversity of microalgae, the permeability of fluorescent probes to different microalgal cells There is a large difference, and it cannot stain the dead cells of microalgae, and its accuracy will be interfered by many factors
Raman spectroscopy is also an interesting method for assessing lipid levels in microalgal cells (Heraud et al., 2007, Fems Microbiology Letters, 275:24-30), but Raman spectroscopy in photosynthetic organisms is not compatible with the strong autofluorescence of pigments Concurrency interferes with the properties of Raman spectroscopy, limiting its applications

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  • A method for determining the harvest time of oil-producing microalgae by using the chlorophyll fluorescence parameter fv/fm
  • A method for determining the harvest time of oil-producing microalgae by using the chlorophyll fluorescence parameter fv/fm
  • A method for determining the harvest time of oil-producing microalgae by using the chlorophyll fluorescence parameter fv/fm

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Embodiment 1

[0031] A method for determining the harvest time of oleaginous microalgae using the chlorophyll fluorescence parameter Fv / Fm, with a 500 mL culture scale of Chlorella sp.C2 (Chen et al., 2014, Plant & Cell Physiology, 55:634-644) as For example, the steps are:

[0032] 1. Autotrophic culture of oleaginous microalgae (Chlorella) and nitrogen deficiency stress treatment: add sterile BG11 liquid medium into a 1L sterile Erlenmeyer flask, the volume of the medium is 500mL. Then inoculate the purified Chlorella sp.C2 preserved in the laboratory and liquid-activated in BG11 liquid medium, and the inoculation density is OD 700 =0.05, at the same time at the temperature of 25℃ and the light intensity of 70μmol m -2 the s -1 Under the conditions of continuous light and aeration culture;

[0033] With 700nm as the scanning wavelength, establish a standard curve between OD value and biomass, and calculate the concentration of biomass in the liquid medium by measuring the OD value of t...

Embodiment 2

[0038] A method for determining the harvest time of oleaginous microalgae using the chlorophyll fluorescence parameter Fv / Fm, taking the 1L culture scale of Chlorella sorokiniana C3 (Zhang et al., 2013, PLoS ONE 8(7):e69225) as an example , whose steps are:

[0039] 1. Autotrophic cultivation of oleaginous microalgae (Chlorella) and treatment of nitrogen deficiency stress: add sterile BG11 liquid medium into a 2L sterile Erlenmeyer flask, and the volume of the medium is 1L. Then inoculate the purified Chlorellasorokiniana C3 preserved in the laboratory and liquid-activated in BG11 liquid medium at an inoculation density of OD 700 =0.05, at the same time at the temperature of 25℃ and the light intensity of 70μmol m -2 the s -1 Under the conditions of continuous light and aeration culture;

[0040] With 700nm as the scanning wavelength, establish a standard curve between OD value and biomass, and calculate the concentration of biomass in the liquid medium by measuring the OD ...

Embodiment 3

[0045] In order to verify the method and further highlight the superiority of the method, the applicant took Chlorellasp. The parameter Fv / Fm determines the harvesting time of oil-producing microalgae, and at the same time compares it with the method of determining the harvesting time of oil-producing microalgae by thin-layer chromatography analysis after oil extraction combined with image analysis software. A method using chlorophyll fluorescence parameter Fv / Fm to determine the production time The method for oil microalgae harvest time, its steps are:

[0046] 1. Chlorella autotrophic culture and nitrogen deficiency stress treatment: add sterile BG11 liquid medium into a 2L sterile Erlenmeyer flask, the medium volume is 1L. Then inoculate the purified Chlorella sp.C2 preserved in the laboratory and liquid-activated in BG11 liquid medium, and the inoculation density is OD 700 =0.05, at the same time at the temperature of 25℃ and the light intensity of 70μmol m -2 the s -1 ...

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Abstract

The invention discloses a method for determining the oleaginous microalgae harvesting time by utilizing the chlorophyll fluorescence parameter Pv / Fm. The method comprises the following steps: (1) carrying out conventional culture and stress induction on oleaginous microalgae, namely carrying out conventional autotrophic culture on the oleaginous microalgae; culturing the oleaginous microalgae to mid-log phase, and inducing accumulation of neutral fat under a stress condition; (2) detecting the chlorophyll fluorescence parameter Fv / Fm, namely sampling in an oil-production inducing process, and detecting Fv / Fm; (3) detecting the content of neutral fat, namely extracting algae oil by chloroform / methanol, and detecting the content of the neutral fat by means of a thin layer chromatography method and image analysis software; (4) determining the harvesting time, namely harvesting microalgae after the neutral fat in cells starts to be remarkably accumulated for 1-2 days, determining the Fv / Fm range after the neutral fat starts to be remarkably accumulated for 1-2 days to be the Fv / Fm harvesting range if the detected Fv / Fm and the neutral fat content are remarkably related, and determining the harvesting time when the algae solution Fv / Fm reaches the harvesting range in induction of neutral fat production under the same stress induction. The method is simple and quick in the chlorophyll fluorescence parameter Pv / Fm detection operation, has few interference factors, and is accurate and stable.

Description

technical field [0001] The invention belongs to the technical field of microalgae biology and bioenergy, and more specifically relates to a method for determining the harvest time of oil-producing microalgae by using the chlorophyll fluorescence parameter Fv / Fm, which is applicable to the production of microalgae bioenergy. Background technique [0002] Microalgae are prokaryotic or eukaryotic single-celled photosynthetic microorganisms. They are very efficient solar energy converters. They are distributed in fresh or salt water and absorb light energy, water and CO transmitted by the water environment. 2 Accumulated biomass can convert light energy into chemical energy and store it in the cell in the form of organic matter such as oil or starch. As the oldest lower photosynthetic organisms, some microalgae can directly use sunlight, CO 2 And simple nutrients such as N and P grow rapidly and synthesize a large amount of oil (mainly neutral fat triglyceride) in the cells, th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
Inventor 王强陈辉何晨柳乔亚琴
Owner INST OF AQUATIC LIFE ACAD SINICA
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