CN104531584A - 15 N stable isotope marked blue-green algae and biological culture method thereof - Google Patents

Abstract

The invention discloses 15 N stable isotope marked blue-green algae and a biological culture method thereof. Nitrogen atoms in the blue-green algae are replaced with 15 N stable isotopes. The method comprises the following steps of blue-green algae enrichment culturing and 15 N-blue-green algae culturing. The culturing technology and a formula are suitable for 15 N stable isotope marked synechocystis growing. The abundance of obtained algal products is high, and the production cost is low.

Description

15the blue-green algae of N cold labeling and biological culturing method thereof

Technical field

The present invention relates to one ¹⁵the blue-green algae of N cold labeling and biological culturing method thereof, belong to algae and cultivate field.

Background technology

Blue-green algae is that a class can carry out the photosynthetic prokaryotic organism of plant type product oxygen, and its strong adaptability, has some characteristics of plant and bacterium concurrently, and be therefore otherwise known as cyanobacteria.Blue-green algae is extensively distributed in tellurian all types of water body and some terrestial enviornments, is the most important originally producer in water body.Blue-green algae decapacitation is carried out outside photosynthetic autotrophs growth, and about have the blue-green algae of half that exogenous organic matter can be utilized under illumination condition to carry out the growth of mixotrophism type, minority blue-green algae can also utilize exogenous organic matter to carry out chmosynthetic heterotrophs growth under complete no light condition.

Cytoalgae (Synechocystis sp.) is the unicellular non-Azotica of a class, lives in fresh water, can carry out vegetal photosynthesis ^[1].Because its genetic operating system is ripe, genetic background is clear, be therefore widely used in photosynthesis research as a kind of modular system, it is also expression system conventional in the important object of algae molecular biology research and algal gene engineering simultaneously.

Along with the develop rapidly of stable isotopic tracer technology, increasing scholar starts to utilize ¹⁵n stable isotope research algae ^[2-5]. ¹⁵n stable isotope technology has the advantages such as highly sensitive, the algae absorption test time is short, the critical process of nitrogen absorption is clearer.

Summary of the invention

Technical problem to be solved by this invention is: provide a kind of ¹⁵the blue-green algae of N cold labeling and biological culturing method thereof.

For solving the problems of the technologies described above, the technical solution used in the present invention is:

The invention provides one ¹⁵the blue-green algae of N cold labeling, the nitrogen-atoms in its body is by stable isotope ¹⁵n replaces.

Meanwhile, the present invention also provides a kind of cultivation ¹⁵the method of N cold labeling blue-green algae, comprises the following steps:

(1) blue-green algae enrichment culture

Get blue algae liquid, with the centrifugation 5min of 3 200r/min, wash with nitrogen-free agar after removing supernatant liquor, recentrifuge removes supernatant liquor, so repeats 2-5 time, is inoculated in and is equipped with in the triangular flask of nutrient solution,

Incubator light source is fluorescent lamp, illumination 2 000lux, and light dark period is 12h/12h, and temperature is 25 DEG C, 250mL triangular flask, and the volume of every bottle of nutrient solution is 150mL, and pH value is 7.0, the inoculation of the algae in vegetative period of taking the logarithm, every day hand bottle 3 times;

(2) ¹⁵the cultivation of N-blue-green algae

The blue-green algae of enrichment is transferred to ¹⁵combined hardening model is carried out in the blue-green algae substratum of N cold labeling, add 5.2g/L glucose in the medium, cultivate and carry out in closed photo bioreactor, reactor comprises three 30W daylight type luminescent lamps, incident intensity 7 000 lux, culture temperature 25 DEG C, stirring velocity 400 revs/min, air flow quantity is 2.0L/min, continuous illumination, cultivates 1-2 week.

Preferably, in step (2), incubation time is 10 days.

Blue-green algae of the present invention is cytoalgae.

Beneficial effect of the present invention:

Culture process of the present invention and formula, suitable ¹⁵n cold labeling cytoalgae grows.The algae kind product abundance obtained is high, and production cost is low.

Accompanying drawing explanation

Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.

Before Fig. 1 cultivates, cytoalgae stable isotope mass spectrum m/z 28 [ ¹⁴n ¹⁴n] and m/z 29 [ ¹⁴n ¹⁵n] mass spectrum.

After Fig. 2 cultivates, do not adjust before receiving cup resistance, cytoalgae stable isotope mass spectrum m/z 28 [ ¹⁴n ¹⁴n] and m/z 29 [ ¹⁴n ¹⁵n] mass spectrum.

After Fig. 3 cultivates, after adjustment receives cup resistance, cytoalgae stable isotope mass spectrum m/z 28 [ ¹⁴n ¹⁴n] and m/z 29 [ ¹⁴n ¹⁵n] mass spectrum.

Embodiment

Embodiment 1 present embodiments provides a kind of cultivation ¹⁵the method of N cold labeling cytoalgae, comprises the following steps:

(1) cytoalgae enrichment culture

Get cytoalgae algae liquid, with the centrifugation 5min of 3 200r/min, wash with nitrogen-free agar after removing supernatant liquor, recentrifuge removes supernatant liquor, so repeats 2-5 time, is inoculated in and is equipped with in the triangular flask of nutrient solution.

Incubator light source is fluorescent lamp, illumination 2 000lux, and light dark period is 12h/12h, and temperature is 25 DEG C, 250mL triangular flask, and the volume of every bottle of nutrient solution is 150mL, and pH value is 7.0, the inoculation of the algae in vegetative period of taking the logarithm, every day hand bottle 3 times.

(2) ¹⁵the cultivation of N-cytoalgae

The blue-green algae of enrichment is transferred to ¹⁵carry out Combined hardening model in the blue-green algae substratum of N cold labeling, add 5.2g/L glucose in the medium.Cultivate and carry out in closed photo bioreactor, reactor comprises three 30W daylight type luminescent lamps, incident intensity 7 000lux, culture temperature 25 DEG C, stirring velocity 400 revs/min, and air flow quantity is 2.0L/min, continuous illumination, cultivates 10 days.

The cytoalgae that aforesaid method is cultivated is analyzed.

(1) index test part

1 instrument, reagent and material

DELTA V-type isotope ratio mass spectrometer is manufactured by Thermo Fisher Scientific company of the U.S., and Flash 2000 HT type elemental analyser and main frame DELTA V are online, accessory current following device ConFlo IV, formation EA-IRMS system on-line determination solid sample ¹⁵n, ¹⁴n isotopics.

Reference material used is USGS40 (δ ¹⁵n=-4.42+0.06 ‰, nitrogen element content is 9.52%).

2 empirical theory bases

In an atmosphere, ¹⁵n exists as a kind of more stable isotropic substance, and its abundance is stable and uniform, and average abundance 0.3663%, is considered to ¹⁵n standard isotopic abundance ^[11].At occurring in nature, in animals and plants and environment ¹⁵atom N abundance and 0.3663% difference very little, be convenient to compare and study to amplify difference, usually with in sample ¹⁵relative ratio (the δ of N ¹⁵n) its abundance is represented.Its calculation formula is:

${\mathrm{\δ}}^{15}N=\frac{R_{\mathrm{smpl}}-R_{\mathrm{st}}}{R_{\mathrm{st}}}\×1000$

Wherein, R is isotopic absolute ratio, is typically expressed as the ratio of heavy isotope and the abundance of light isotope, i.e. the ratio of mole number content:

$R=\frac{\mathrm{atom}{\%}^{15}N}{\mathrm{atom}{\%}^{14}\text{N}}$

It is according to occurring in nature δ that the general-purpose of DELTA V equipment receives cup resistance acquiescence ¹⁵the range of normal value of N is arranged, during test nitrogen stable isotope, receive m/z 28 [ ¹⁴n ¹⁴n] and m/z 29 [ ¹⁴n ¹⁵n] the signal amplification factor of reception cup be about 100 times and 10000 times respectively.Through the cytoalgae that context of methods is cultivated, ¹⁵n abundance greatly improves.Carry out nitrogen stable isotope again after needing adjustment to receive cup resistance, and be not suitable for using δ ¹⁵n represents ¹⁵the abundance of N, directly can use atom% ¹⁵n represents its abundance.

2 experimental techniques

Closely wrapped up by sample tin can, send in 960 DEG C of Reaktionsofens, oxygenolysis in the oxygen and helium flow of high density by automatic sampler, reaction generates mixed gas, is wherein reduced into N containing nitrogen oxide through going back native copper ₂, the mixed gas of formation, under the delivery of high-purity helium (99.999%), is separated through chromatographic column, enters mass spectrum successively, measure its nitrogen isotope relative ratio by the rectification of Confio IV device, isotropic substance spends absolutely ratio and nitrogen element content.The reception cup resistance (RC-KOMBINAT) of m/z28 and m/z 29 should be replaced by 30G/5PF and 300M/470PF respectively before measuring the cytoalgae after cultivating.

3. ¹⁵n cytoalgae test data

Get the cytoalgae parallel testing δ before cultivation ¹⁵n and atom% ¹⁵n, then get use ¹⁵n substitutes the cytoalgae six parallel testing atom% after nitrogenous source cultivation ¹⁵n, data are as table 1:

Front and back δ cultivated by table 1 ¹⁵n and atom% ¹⁵n value

As can be seen from measured value, the cytoalgae after cultivation, ¹⁵n stable isotope substitution rate is very high, atom% ¹⁵n value brings up to more than 97% from 0.374%.From stable isotope mass spectrum m/z 28 [ ¹⁴n ¹⁴n] and m/z 29 [ ¹⁴n ¹⁵n] can find out in mass spectrum, do not adjust before receiving cup resistance, almost can't see m/z 28 [ ¹⁴n ¹⁵n] peak.After the reception cup resistance (RC-KOMBINAT) of m/z 28 and m/z 29 is replaced by 30G/5PF and 300M/470PF respectively, m/z 29 [ ¹⁴n ¹⁵n] peak is obvious, algae kind ¹⁵n stable isotope substitution rate is high, and abundance is high.

The above is only preferred embodiment of the present invention, and be not restriction the present invention being made to other form, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the Equivalent embodiments of equivalent variations.But everyly do not depart from technical solution of the present invention content, any simple modification, equivalent variations and the remodeling done above embodiment according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.

Claims (5)

1. ¹⁵the blue-green algae of N cold labeling, is characterized in that, the nitrogen-atoms in its body is by stable isotope ¹⁵n replaces.

2. ¹⁵the substratum of the blue-green algae of N cold labeling, is characterized in that, culture medium prescription is as follows:

1、NaNO ₃, 1.5g/L；

2、K ₂HPO ₃·3H ₂O 0.052g/L；

3、MgSO ₄·7H ₂O 0.075g/L；

4、CaCl ₂·2H ₂O 0.036g/L；

5, citric acid 6.0mg/L;

6 、EDTANa ₂1.0mg/L；

7、NaCO ₃0.02g/L；

8、H ₃BO ₃2.86mg/L；

9、MnCl ₂·4H ₂O 1.86mg/L；

10、ZnSO ₄·7H ₂O 0.22mg/L；

11、Na ₂MoO ₄·2H ₂O 0.39mg/L；

12、CuSO ₄·5H ₂O 0.08mg/L；

Nitrogenous source is NaNO ₃, the nitrogen-atoms of nitrogenous source adopts ¹⁵n marks.

3. a cultivation ¹⁵the method of N cold labeling blue-green algae, is characterized in that, comprise the following steps:

(1) blue-green algae enrichment culture

Get blue algae liquid, with centrifugation 5 min of 3 200 r/min, wash with nitrogen-free agar after removing supernatant liquor, recentrifuge removes supernatant liquor, so repeats 2-5 time, is inoculated in and is equipped with in the triangular flask of nutrient solution;

Incubator light source is fluorescent lamp, illumination 2 000 lux, and light dark period is 12 h/ 12h, and temperature is 25 DEG C, 250 mL triangular flasks, and the volume of every bottle of nutrient solution is 150 mL, and pH value is 7.0, the inoculation of the algae in vegetative period of taking the logarithm, every day hand bottle 3 times;

(2) ¹⁵the cultivation of N-blue-green algae

The blue-green algae of enrichment is transferred to ¹⁵combined hardening model is carried out in the blue-green algae substratum of N cold labeling, add 5.2g/L glucose in the medium, cultivate and carry out in closed photo bioreactor, reactor comprises three 30W daylight type luminescent lamps, incident intensity 7 000 lux, culture temperature 25 DEG C, stirring velocity 400 revs/min, air flow quantity is 2.0L/min, continuous illumination, cultivates 1-2 week.

4. cultivation according to claim 3 ¹⁵the method of N cold labeling blue-green algae, is characterized in that, in step (2), incubation time is 10 days.

5. the blue-green algae described in claim 1 or 2 is cytoalgae.