CN103852440A - Method for measuring microalgae biomass components through Fourier transform infrared spectroscopy (FTIR) - Google Patents
Method for measuring microalgae biomass components through Fourier transform infrared spectroscopy (FTIR) Download PDFInfo
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- CN103852440A CN103852440A CN201210517281.9A CN201210517281A CN103852440A CN 103852440 A CN103852440 A CN 103852440A CN 201210517281 A CN201210517281 A CN 201210517281A CN 103852440 A CN103852440 A CN 103852440A
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- polysaccharide
- protein
- acid amides
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- transform infrared
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Abstract
The invention relates to a method for measuring microalgae biomass components through Fourier transform infrared spectroscopy (FTIR). The relative contents of oil, proteins and polysaccharides in microalgae biomass are important indicators of evaluating the quality of the microalgae biomass, and the oil, proteins and polysaccharides are needed to be respectively measured one by one in a traditional chemical method. The microalgae biomass is measured by utilizing the FTIR, protein amide I band serves as an internal reference peak, and the relative content of the oil and polysaccharides is calculated. According to the method, the relative contents of the oil, proteins and polysaccharides in the microalgae cells are rapidly and simultaneously measured, the method is simple and high in repeatability, the quantity demand of the sample is low, and the defects that the chemical measurement method is complicated and time-consuming and the like are overcome.
Description
Technical field
The invention belongs to marine biotechnology field, be specially and utilize Fourier transform infrared spectroscopy the relative content of grease and polysaccharide in micro-algae to be carried out the method for rapid evaluation simultaneously
Background technology
Micro-algae has that kind is many, photosynthetic efficiency is high, biological yield is high, growth cycle is short and self fat content advantages of higher, is considered to one of biomass energy raw material of preparing biodiesel the best.In microalgae cell, the mensuration of full constituent content is cultivated micro-algae and regulates and controls to have directive significance, generally adopts each component concentration in chemical gauging microalgae cell.In micro-algae, fat content is the important indicator that determines micro-algae biodiesel cost, and conventional weight method is measured microalgae grease content needs algae powder amount large, and leaching process is loaded down with trivial details, and a large amount of uses of organic reagent also can bring the problem such as environment and health in addition; Nile red decoration method can Fast Measurement cell in neutral fats content, be applied to screening and the incubation neutral fats assay of the strain of rich oil algae, but condition of culture, algae kind, dyeing temperature and dyeing time etc. all can affect fluorescence intensity, be unfavorable for comparing between laboratory interchange.The mensuration of polysaccharide and protein content generally adopts the method for colour developing to measure, and sample need, through broken, extract the steps such as colour developing.In chemical gauging cell, the assay process of three large components is more loaded down with trivial details, consuming time longer.So traditional chemical method is unsuitable for micro-algae endogenous substance content to do rapid evaluation.
Fourier transform infrared spectroscopy (FTIR) belongs to molecular spectrum, is the powerful of determining material composition, quick with it, and harmless, the advantage of microdetermination is used widely in identification.Macromolecular substances all has absorption at FTIR middle infrared as protein, lipid, nucleic acid, polysaccharide etc. in cell, this region spectrum can provide each material special absorption of vibrations spectrum simultaneously, and makes it to carry out rapid evaluation to the content of grease, protein, polysaccharide in micro-algal biomass simultaneously.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing Fourier transform infrared spectroscopy fast micro-algal biomass to be assessed.
To achieve these goals, the technical solution used in the present invention content is:
(1) sample preparation
Algae powder to be measured 1-5mg and spectroscopic pure potassium bromide are carried out to ground and mixed by 1.0-3.0:100 mass ratio, get 40-80mg biased sample, infrared grinding tool carries out compressing tablet.
(2) sample determination
The Fourier transform infrared spectroscopy condition of scanning: instrumental resolution: 4cm
-1; Scanning wave number: 4000-700cm
-1; Cumulative scan number of times is 16-64 time.With pressing potassium bromide troche scanning spectrogram spectrogram as a setting, prepare Sample Scan, obtain sample infrared spectrum.
(3) grease, polysaccharide relative content calculate
Respectively to fat absorption bands of a spectrum (3002-2807cm
-1), protein acid amides I absorption band (1722-1585cm
-1) and polysaccharide absorption band (1200-953cm
-1) carry out area integral, calculate grease protein content relative to polysaccharide as interior reference peak using protein acid amides I band;
Grease relative content is: fat absorption peak area/acid amides I is with area; Polysaccharide relative content is fat absorption peak area/acid amides I band area.
Described micro-algal biomass refers to grease, protein and the polysaccharide (can measure as requested wherein two or more indexs) in micro-algae.
Advantage of the present invention is as follows:
1. full constituent is measured simultaneously: Fourier transform infrared spectroscopy scanning spectrogram can obtain the absorption peak of microalgae cell internal protein, polysaccharide, grease simultaneously, so can analyze three kinds of components simultaneously, measure one by one with traditional chemical method and determination step loaded down with trivial details compared with, save time.
2, mensuration process is simple: will gather in the crops algae mud dry, and grind evenly compressing tablet with potassium bromide.Fourier transform infrared spectrometer scans, and is about 30s sweep time.
3, required sample size is few: general chemical gauging grease needs 100mg left and right dry algae powder, and Fourier transform infrared spectroscopy full constituent is measured required sample size only 1-5mg.
4, measurement result accuracy is high: in rich oil Strain selection and regulation culture monitoring, full constituent measurement result is consistent with chemical measurement result simultaneously for Fourier transform infrared spectroscopy.
Attached caption
Table 1 is that micro-algae infrared spectrum bands of a spectrum distribute.
Fig. 1 is that Fourier transform infrared spectroscopy is measured screening algae strain grease relative content and polysaccharide relative content; Note: Fig. 1 a is with fat absorption peak (3002-2807cm
-1) area/acid amides I area A (Lipid/AmideI) calculates the relative content of total fat; Fig. 1 b is with polysaccharide absorption band (1200-953cm
-1) area/acid amides I area A (Carbo/Amide I) calculates the relative content of total reducing sugar; 1: purple ball algae; 2:H16; 3: sub-heart-shaped four slit bamboo or chopped wood algaes; 4: grid algae; 5:H56; 6: Chaetoceros; 7: chlorella pyrenoidosa.
Fig. 2 FT-IR measures the linear relationship of microalgae grease and polysaccharide relative content and chemical assay result.
Embodiment
Below by specific embodiment, method of the present invention and result are described.
The sub-heart type four slit bamboo or chopped wood algaes of algae strain used (Tetraselmis subcordiformis), grid algae (Scenedesmusobliquus), the each component characteristics of micro-algae sample infrared spectrum absorbs in table 1.3000-2800cm-1 scope is C-H stretching vibration, is mainly derived from lipid, and 1740cm-1 is the vibration absorption peak of C=O in fat and fatty acid Zhong Zhi functional group, and these two region absorption intensity sizes are corresponding to the content of grease in frustule.1650cm
-1and 1540cm
-1be respectively with protein in the flexural vibrations of C=O stretching vibration and N-H, absorption intensity size is corresponding to frustule internal protein content.1200-900cm
-1for the vibration absorption peak of carbohydrate C-O-C, polyoses content in the corresponding frustule of absorption intensity size.
By heart-shaped Asia four slit bamboo or chopped wood algaes or grid algae sample 3mg and potassium bromide (1:100 mass ratio) mixed grinding in proportion, get 60mg compressing tablet, utilize Fourier transform infrared spectrometer (Bruker TENSOR27, Germany) to carry out Sample Scan, the condition of scanning: spectral resolution: 4cm
-1, cumulative scan 32 times, sweep limit 4000-700cm
-1.
Spectrogram process software OPUS 6.5(Bruker, Germany) to sample infrared spectrum grease (3002-2807cm
-1), protein acid amides I (1722-1585cm
-1) and polysaccharide (1200-953cm
-1) absorption band carries out area integral, with acid amides I(1722-1585cm
-1) bands of a spectrum are as interior reference peak, with fat absorption peak (3002-2807cm
-1) area/acid amides I area A (Lipid/Amide I) calculates the relative content of grease, with polysaccharide absorption band (1200-953cm
-1) area/acid amides I area A (Carbo/Amide I) calculates the relative content of polysaccharide.Sub-heart-shaped four slit bamboo or chopped wood algaes and grid algae grease relative content are respectively 0.568,0.708; Polysaccharide relative content is respectively 3.07,1.03.Be this grid algae fat content of infrared spectrum measurement higher than and sub-heart-shaped four slit bamboo or chopped wood polysaccharides content higher than grid algae.
Experiment algae used strain: sub-heart type four slit bamboo or chopped wood algaes (Tetraselmis subcordiformis), Chaetoceros (Chaetoceros), chlorella pyrenoidosa (Chlorella pyrenoidosa), grid algae rm (Scenedesmusobliquus), Chlamydomonas reinhardtii (Chlamydomonas reinhardtii CC 124), purple ball algae (Porphyridium cruentum), H56, H16(do not determine the strain of title algae)
The each component characteristics of micro-algae sample infrared spectrum absorbs in table 1.3000-2800cm
-1scope is C-H stretching vibration, is mainly derived from lipid, and 1740cm-1 is the vibration absorption peak of C=O in fat and fatty acid Zhong Zhi functional group, and these two region absorption intensity sizes are corresponding to the content of grease in frustule.1650cm
-1and 1540cm
-1be respectively with protein in the flexural vibrations of C=O stretching vibration and N-H, absorption intensity size is corresponding to frustule internal protein content.1200-900cm
-1for the vibration absorption peak of carbohydrate C-O-C, polyoses content in the corresponding frustule of absorption intensity size.
To be with screening algae powder 3mg sample and potassium bromide (1:100 mass ratio) mixed grinding in proportion, get 60mg compressing tablet, and utilize Fourier transform infrared spectrometer (Bruker TENSOR27, Germany) to carry out Sample Scan, the condition of scanning: spectral resolution: 4cm
-1, sweep limit 4000-700cm
-1, cumulative scan 32 times.
Spectrogram process software OPUS 6.5(Bruker, Germany) to sample infrared spectrum grease (3002-2807cm
-1), protein acid amides I (1722-1585cm
-1) and polysaccharide (1200-953cm
-1) absorption band carries out area integral, with acid amides I(1722-1585cm
-1) bands of a spectrum are as interior reference peak, with fat absorption peak (3002-2807cm
-1) area/acid amides I area A (Lipid/Amide I) calculates the relative content of grease, with polysaccharide absorption band (1200-953cm
-1) area/acid amides I area A (Carbo/Amide I) calculates the relative content (Fig. 1) of polysaccharide.Chemical method (Bligh, E.G., and Dyer, W.J. (1959) A rapid method of total lipid extraction and purification, Canadian journal of biochemistry and physiology.37,911-917.; Bradford, M.M., (1976) .A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal.Biochem.72,248254.; Somani, B.L., Khanade, J., and Sinha, R. (1987) A modified anthrone sulfuric-acid method for the determination of fructose in the presence of certain proteins, Analytical biochemistry 167,327-330.) grease, protein, the polyoses content of above algae strain sample are measured to (table 2), to verify the accuracy of Fourier transform infrared spectroscopy scanning result.Fig. 1 a shows that measuring relative fat content A (Lipid/Amide I) value of H56 by FTIR is that 1.32 to be significantly higher than other algae kinds higher.H16 (0.75), grid algae (0.71), Chaetoceros (0.66), chlorella pyrenoidosa (0.59), sub-heart-shaped four slit bamboo or chopped wood algaes (0.57), fat content is more or less the same relatively, and fat content is lower relatively for purple ball algae (0.18).Traditional chemical method is measured (table 2) to screening algae strain grease, polysaccharide, protein content.Result show fat content higher algae strain be H56, H16 account for respectively algae powder 31.0% and 28.5%, traditional chemical method is measured other screening algae strain fat content grid algaes, Chaetoceros, chlorella pyrenoidosa, sub-heart-shaped four slit bamboo or chopped wood algae content are more or less the same at 10-13%; Purple ball algae fat content is minimum is 7.2%.Fig. 1 b shows that measuring total reducing sugar relative content A (Carbo/Amide I) by FTIR is up to sub-heart-shaped four slit bamboo or chopped wood algaes (3.07), other algae strain content are slightly low, be followed successively by Chaetoceros (6.78), H16 (11.7), H56 (4.27), chlorella pyrenoidosa (2.16), purple ball algae (4.58), grid algae (3.12).Table 2 show total sugar content the highest be sub-heart-shaped four slit bamboo or chopped wood algaes, total sugar content is 47.4%, what content was slightly low is respectively 21.5% and 18.4% for H16, Chaetoceros content, other algae strain total sugar contents of chemical gauging are followed successively by H56, chlorella pyrenoidosa, purple ball algae, grid algae.To survey algae strain polyoses content consistent with FTIR measurement result.
Measure grease and polysaccharide relative content and chemical gauging result with Fourier transform infrared spectroscopy and carry out linear regression (Fig. 2), relatively the linear relationship of fat content and chemical method result: Y=0.02837X+0.26204R
2=0.91798; The linear relationship of polyoses content and chemical assay result: Y=0.05354X+0.4885R relatively
2=0.9716.Further prove Fourier transform infrared spectroscopy, can be used as a kind of method quickly and accurately and be applied to the Fast Measurement of micro-algal biomass.
Table 1
The total fat of table 2 chemical gauging screening algae strain, polysaccharide, protein content.
Claims (4)
1. a Fourier transform infrared spectroscopy is measured the method that micro-algal biomass forms, it is characterized in that: utilize Fourier transform infrared spectroscopy to scan micro-algal biomass sample, using protein acid amides I bands of a spectrum as interior reference peak, calculate the relative content of grease and polysaccharide.
2. it is characterized in that in accordance with the method for claim 1:
Concrete steps are as follows:
(1) preparation of samples:
Take and treat micrometer algae powder 1-5mg, according to 1.0-3.0:100 mass ratio and KBr mixed grinding, get 40-80mg biased sample compressing tablet;
(2) Fourier transform infrared spectroscopy is measured:
Fourier transform infrared spectrometer is carried out Sample Scan, the condition of scanning: spectral resolution is 4cm
-1, sweep limit 4000-700cm
-1, cumulative scan 16-64 time;
Spectrogram 1722-1585cm
-1absorption peak is protein acid amides I absorption band, 3002-2807cm
-1absorption peak is fat absorption bands of a spectrum, 1200-953cm
-1absorption peak is polysaccharide absorption band;
(3) relative content calculates
Respectively fat absorption bands of a spectrum, protein acid amides I absorption band and polysaccharide absorption band are carried out to area integral, calculate grease protein content relative to polysaccharide as interior reference peak using protein acid amides I band;
Grease relative content is: fat absorption peak area/acid amides I is with area; Polysaccharide relative content is fat absorption peak area/acid amides I band area.
3. it is characterized in that in accordance with the method for claim 1: described micro-algal biomass refers to grease, protein and the polysaccharide in micro-algae.
4. in accordance with the method for claim 1, it is characterized in that: described protein acid amides I bands of a spectrum are the characteristic absorption peak of protein C=O key in infrared spectrum, and wave-number range is 1722-1585cm
-1.
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Application publication date: 20140611 |