CN109752351A - A kind of adjusting method of the microalgae nitrogen nutrition coercing cultivation process based on feedback control - Google Patents
A kind of adjusting method of the microalgae nitrogen nutrition coercing cultivation process based on feedback control Download PDFInfo
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Abstract
The adjusting method for the microalgae nitrogen nutrition coercing cultivation process based on feedback control that the present invention provides a kind of.Invention defines N stress index (the Nitrogen Stress Index for being used to N stress degree suffered by quantitative microalgae cell, NSI), Con trolling index is provided at degree for control N stress accurate during microdisk electrode, and the online characterizing method in original position by establishing the N stress degree based on chlorophyll fluorescence parameters, the feedback control microalgae cell N stress degree based on chlorophyll fluorescence parameters guidance is realized, guarantees that microalgae cell is suitably coerced.
Description
Technical field
The present invention relates to a kind of microalgae appropriateness Nutrient Stress regulation technology based on feedback control, specifically using micro-
Algae chlorophyll fluorescence parameters (Fv/FmOr Δ F/Fm') the N stress degree that is subject to of real-time characterization microalgae cell, feedback guidance culture
The addition of nutrient in the process, so that controlling microalgae is in appropriate stress state.
Background technique
Microalgae because it is high with photosynthetic efficiency, not with the advantages such as agricultural competition soil, at home and abroad recyclable organism mass-energy
Great attention is received in the strategic research of source.Microalgae is under conditions of nutritional sufficiency, the fixed CO of cell2And absorb
Nutriment is mainly used to cell itself growth and breeding.(such as Nutrient Stress, environment-stress), microalgae when cell is forced
The energy and CO fixed by photosynthesis2Flow direction changes, wherein N stress is the most common side of body of current microdisk electrode
Compel means.For oil-producing microalgae, under the conditions of N stress, microalgae cell can not carry out normal growth and division, and cell
Interior extra reducing power is just used for the synthesis of grease.Therefore cell growth and oil and fat accumulation contradict, microdisk electrode process
The middle lipid-producing acme is a kind of transient.Therefore need to control stress journey suffered by cell during the cultivation process
Degree, is at proper range.It coerces in range herein, both can guarantee that the photosynthetic efficiency of cell maintained certain level to thin
Intracellular growth, and the enough greases of Cellular Accumulation can be stimulated, so that cell be maintained to be in the lipid-producing acme.Due to microalgae by
Cell global change brought by Nutrient Stress (such as nitrogen stress) is extremely complex, and oil and fat accumulation mechanism is still unintelligible, therefore
Establishing with oil and fat accumulation mechanism theory be that the oil-producing microalgae high-efficient culture technique thinking feasibility that instructs is low, at present oil-producing microalgae
Breakthrough is not yet received always in the controllable culture technique of continuous high-efficient, limits the development of microalgae industry, becomes the hot spot of microalgae research
One of with difficult point.
Currently, adding for control nitrogen is most common method (the AREMU A O et for controlling Cellular stress degree
al.Journal of Phycology,2015,51(4):659-669)；In addition, also there is research to realize using cell N/C value
Control (BONNEFOND H et al.Biotechnology for Biofuels, 2017,10 (1): 25) of stress level.So
And due to technical restriction, the real time on-line monitoring of nitrogen content intracellular in incubation can not be still realized, to limit culture
The accurate control of cell N stress degree in the process.The patent CN10437475813 of the inventions such as Wang Qiang utilizes Fv/FmIt is micro- with oil-producing
The relationship guidance culture harvest time of oil content under algae N stress.However the technology can not achieve microalgae in control incubation
The N stress degree of cell instructs adding for nitrogen nutrition element in incubation, limits its answering in different training modes
With especially fed-batch, semicontinuous or continuous training mode.Therefore, if cell N stress can be characterized online using new
Extent index realizes that feedback control cell maintains appropriate Nutrient Stress, will limit microdisk electrode to nitrogen and be of great significance, especially
Contribute to realize the stability contorting of outdoor large-scale culture.
Summary of the invention
The object of the present invention is to provide a kind of microalgae appropriateness N stress regulation technology based on feedback control, it is micro- in order to be promoted
Algae culture controllability, solve stress level in current microalgae N stress incubation can not monitor on-line cannot achieve microalgae the side of body
The problem of urgent degree accurately controls.
A kind of adjusting method of the microalgae nitrogen nutrition coercing cultivation process based on feedback control, is in microdisk electrode process
In, the online characterization parameter by using the photosynthetic chlorophyll fluorescence parameters of microalgae as N stress degree, real-time monitoring cell institute
By nitrogen nutrition stress level, the addition of nitrogen nutrition element in feedback guidance cultivating system is realized suffered by incubation microalgae cell
The accurate control of N stress.
Specifically,
(1) N stress refers in the culture environment of microalgae cell normal growth, controls not nitrogenous battalion in cultivating system
When supporting element, microalgae cell suffered stress during the growth process；
(2) cell institute is characterized based on above-mentioned defined N stress index (Nitrogen Stress Index, NSI)
By Nutrient Stress degree, NSI presses formula NSIi=(Nmax-Ni)/(Nmax-Nmin) calculate；Wherein NmaxRefer in microalgae cell normal growth
Culture environment in maximum nitrogen mass content intracellular, N under the conditions of nutritional sufficiencyminUnder the conditions of finger N stress when cell survival
Minimum nitrogen mass content intracellular, NiRefer to the nitrogen mass content intracellular at a certain moment under N stress condition of culture；
(3) the photosynthetic chlorophyll fluorescence parameters of microalgae and the corresponding relationship curve or function of NSI are established；Specifically, training
Nitrogen nutrition element is to play cell the 0mg/L moment to reach stage stationary phase in the system of supporting, and constant duration is measured by sampling no less than 3
The nitrogen content intracellular of a culture moment cell calculates NSI value and the photosynthetic chlorophyll fluorescence parameters of the microalgae at corresponding moment, passes through
Correlation analysis establishes the photosynthetic chlorophyll fluorescence parameters of microalgae and the corresponding relationship curve or function of NSI；
(4) during the cultivation process, it after there is N stress, i.e., after nitrogen nutrition element is 0mg/L in cultivating system, monitors micro-
Algae photosynthetic chlorophyll fluorescence parameters variation, utilizes (2) established invariance curve or function to calculate NSI at that time, when with set at NSI
When same level (deviation range is within NSI ± 0.3), nitrogen nutrition element is added into cultivating system until the photosynthetic leaf of microalgae
Green element fluorescence parameter reaches setting value again；
During this, the additional amount of unit (volume or quality) cultivating system nitrogen nutrition element is according to unit cultivating system need
Nitrogen content B intracellular corresponding to the dry cell weight incrementss A to be obtained and setting NSI is horizontal is determining, as A*B.
The culture environment of step (1) the microalgae cell normal growth includes meeting microalgae cell normal growth and being proliferated
Temperature, light intensity, pH and the cultivating system nutrient of journey；
Nutrient includes that nitrogen phosphate and sulfur, iron, copper, molybdenum, zinc, silicon, manganese, cobalt and microorganism, biotin can meet microalgae
Cell normal growth；
In incubation can continuous illumination for 24 hours, or can also with certain Dark-light cycle carry out illumination, Dark-light cycle ratio
Range 8h:16h~16h:8h (illumination: dark)；
N stress refers to that microalgae cell is suffered during the growth process when being only free of nitrogen nutrition element in above-mentioned cultivating system
The stress arrived.
Step (3) during the whole culture process, in cultivating system in addition to nitrogen nutrition other each nutrients include phosphorus,
Sulphur, iron, copper, molybdenum, zinc, silicon, manganese, cobalt etc. and microorganism, biotin etc. can meet needed for microalgae cell growth；
If illumination is carried out with Dark-light cycle mode, after sample time should be selected in illumination at least 1h.
The additional amount of step (4) unit (volume or quality) cultivating system nitrogen nutrition element is according to unit cultivating system needs
Nitrogen content B intracellular corresponding to the dry cell weight incrementss A and setting NSI level of acquisition is determining, as A*B；Such as cultivate needs
The dry cell weight incrementss 1.0g/L of acquisition sets the corresponding nitrogen content intracellular of NSI as 4.0%wt, then the nitrogen for needing to add contains
Amount is (1.0 × 4.0%) g/L, as 40mg/L；
It should be noted that needing the dry cell weight incrementss obtained that should meet microdisk electrode actual conditions, not higher than training
It can be obtained maximum cell dry weight when microalgae cell reaches stationary phase in the system of supporting.
Wherein N content, which measures, can be used elemental analyser or the measurement of triumphant formula nitriding, but be not limited to the above method；
The photosynthetic chlorophyll fluorescence parameters of the microalgae include producing for characterizing maximum amount of microalgae photosynthetical system II
Rate Fv/FmOr the practical quantum yield Δ F/F for characterizing microalgae photosynthetical system IIm' etc. chlorophyll fluorescence parameters.Its middle period
Green element fluorescence parameter can be measured with Water-PAM chlorophyll fluorescence instrument (Walz, Germany), but be not limited to using
Above-mentioned instrument.
The adding manner of nitrogen nutrition substance includes to be continuously added to or intermittently add in batches in the culture medium of the cultivating system
Enter；
In addition, except being denitrogenated in the culture medium of cultivating system other nutrients addition need control include nutrition member
The additional amount of element and the addition time of nutrient, it is ensured that other each nutrients are sufficient in addition to nitrogen nutrition in incubation
It can meet needed for microalgae cell growth.8. adjusting method according to claim 1, it is characterised in that: the microalgae cell
The accurate control of suffered N stress, which refers to, reaches cell by the adding for feedback regulation nitrogen of real time monitoring of chlorophyll fluorescence parameters
The NSI of setting reaches the N stress degree of setting.
The present invention is the N stress degree of precise quantification cell, first defines the N stress index (Nitrogen of cell
Stress index, NSI).NSI presses formula NSIi=(Nmax-Ni)/(Nmax-Nmin) calculate.Wherein NmaxRefer in nutritional sufficiency condition
Under maximum nitrogen content intracellular, NminNitrogen content intracellular minimum when cell survival, N under the conditions of finger N stressiRefer to N stress item
The nitrogen content intracellular at a certain moment under part.Therefore, the variation range of NSI is 0~1, and value shows that more greatly cell is suffered and coerces journey
It spends stronger.By taking micro- quasi- ball algae as an example, its maximum nitrogen content intracellular is 8.10%wt under normal cultivation conditions, maintains to deposit under N stress
Minimum nitrogen content intracellular living is 2.10%wt, when nitrogen content intracellular is 3.76%wt, NSI 0.72.
NSI based on above-mentioned definition, in N stress incubation, NSI and microalgae chlorophyll fluorescence parameters are established in analysis
(Fv/FmOr Δ F/Fm') correlation, utilize microalgae chlorophyll fluorescence parameters (Fv/FmOr Δ F/Fm') real-time characterization microalgae cell
The N stress degree being subject to.Using can the microalgae chlorophyll fluorescence parameters of online in-situ monitoring replace can not real-time online measurement
Nitrogen content intracellular characterizes cell N stress degree indirectly.Finally utilize the NSI of above-mentioned foundation and microalgae chlorophyll fluorescence parameters
(Fv/FmOr Δ F/Fm') corresponding relationship, use microalgae chlorophyll fluorescence parameters (Fv/FmOr Δ F/Fm') it is used as N stress culture
The microalgae appropriateness nutrition side of body is realized in N stress degree characterization parameter in journey, the addition of nutrient in feedback guidance incubation
Compel to adjust.Specifically, corresponding microalgae chlorophyll fluorescence parameters have been determined after the NSI that setting needs to control is horizontal
(Fv/FmOr Δ F/Fm') monitoring point.After cell chlorophyll fluorescence reaches monitoring point, according to different training method and require to
Cultivating system is adding nitrogen nutrition element.After mending nitrogen, microalgae chlorophyll fluorescence (Fv/FmOr Δ F/Fm') go up, according to setting in advance
Chlorophyll fluorescence (the F of fixed nitrogen coveringv/FmOr Δ F/Fm') upper limit, real-time monitoring microalgae chlorophyll fluorescence (Fv/FmOr Δ F/
Fm') variation, added by control nitrogen, reach preset rise range, to realize the continuous Repetitive controller of NSI.
Idiographic flow schematic diagram such as Fig. 1.
The microalgae appropriateness Nutrient Stress regulation technology based on feedback control that the present invention establishes, using can online in-situ monitoring
Microalgae chlorophyll fluorescence parameters (Fv/FmOr Δ F/Fm') real-time characterization parameter as N stress degree, solve nitrogen intracellular
Content is unable to the limitation that real-time measurement is used to assess N stress degree.New control plan is provided for the controllable N stress culture of microalgae
Slightly.
Detailed description of the invention
5 width of attached drawing of the present invention, in which:
Under Fig. 1 N stress culture, microalgae chlorophyll fluorescence parameters (F is utilizedv/FmOr Δ F/Fm') real as N stress degree
When characterization parameter feedback guidance nitrogen nutrition element add flow diagram.
Fig. 2 column bioreactor microalgae culture system.
Fig. 3 limits micro- quasi- ball algae Δ F/F under nitrogen training modem' and nitrogen content intracellular variation.
Under 1 condition of culture of Fig. 4 embodiment, control NSI micro- quasi- ball algae Δ F/F when being 0.81m' and nitrogen content intracellular variation.
Under 2 condition of culture of Fig. 5 embodiment, Semi-continuous cultivation scheme control NSI micro- quasi- ball algae Δ F/F when being 0.73m' and born of the same parents
Interior nitrogen content variation.
Specific embodiment
Present invention firstly provides a kind of microalgae appropriateness Nutrient Stress regulation technology based on feedback control limits micro- in nitrogen
By stress level suffered by feedback control means adjustable micro-algal cell in algae incubation, appropriate Nutrient Stress is realized.
Microdisk electrode is carried out under the conditions of nutritional sufficiency first, after micro algae growth to stationary phase, microalgae is collected, uses element
The nitrogen content intracellular of analysis-e/or determining microalgae dry powder, i.e. Nmax.The method for wherein measuring nitrogen content intracellular is not limited to using element
Analyzer, other can measure for example triumphant examination nitriding of method of nitrogen content intracellular etc. and be applicable in.
In nitrogen limitation incubation, timing sampling measures living cells chlorophyll fluorescence parameters (Fv/FmOr Δ F/Fm'), directly
To cell death, nitrogen content N intracellular at this time is determinedmin.The regression relation for investigating chlorophyll fluorescence parameters and nitrogen content intracellular, is selected
It is preferred that selecting Fv/FmOr Δ F/Fm' characterization parameter as N stress degree, i.e. high one of selection correlation.Its Determination of Chlorophyll is glimmering
Optical parameter can be measured with Water-PAM chlorophyll fluorescence instrument (Walz, Germany), and measuring method, which can refer to YAO etc., to be made
Method (YAO C et al.Bioresource Technology, 2016,212:26-34.).It may be noted that microalgae leaf
The measurement of green element fluorescence parameter is not limited to using above-mentioned instrument and method, other can measure the instrument of microalgae chlorophyll fluorescence parameters
Device and method are applicable in.
Then, it in the case where nitrogen limits condition of culture, determines the degree of Nutrient Stress, is joined by the chlorophyll fluorescence of above-mentioned foundation
Several relationships with nitrogen content intracellular determine the control range based on chlorophyll fluorescence parameters feedback control, instruct to seek in incubation
Support adding for elemental nitrogen.
The method of the present invention and result are illustrated below by specific embodiment.It should be understood that these embodiments are only used for
Illustrate the present invention rather than limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, this
Field technical staff can make various changes or modification to the present invention, and such equivalent forms equally fall within right appended by the application and want
Seek book limited range.
Embodiment 1:
By taking quasi- ball algae culture micro- in 500mL column glass bubbling reactor (Fig. 2) as an example, reaction condition is respectively to cultivate temperature
25 ± 2 DEG C of degree, 140 μm of ol m of intensity of illumination–2s–1, the CO of air2Ventilatory capacity is 100mL min–1, only CO is passed through in illumination2,
CO2Intake be passed through air 2%, Light To Dark Ratio 14h:10h, daily light application time is 8:00~20:00.Cultivation in sea water,
Using the F/2 nutritive salt of improvement, each nutritive salt of mother liquor is final concentration of (every liter): 75.00g NaNO3, 5.00g NaH2PO4,
3.15g FeCl3·6H2O, 4.36g Na2EDTA, 9.80mg CuSO4·5H2O, 6.30mg Na2MoO4·2H2O, 22.00mg
ZnSO4·7H2O, 10.00mg CoCl2·6H2O, 0.18g MnCl2·4H2O, 1.00mg vitamin B12,0.20g vitamin
B1,1.10mg biotin.Nitrogen nutrition concentration is indicated in system with nitrogen final concentration.
The microalgae cell culture of 4 kinds of condition of culture is carried out in the present embodiment.In 4 kinds of incubations in system except nitrogen nutrition it
Other outer each nutrients include phosphorus, sulphur, iron, copper, molybdenum, zinc, silicon, manganese, cobalt etc. and microorganism, biotin etc. can meet it is micro-
Needed for frustule growth；Light intensity, temperature and ventilatory capacity remain unchanged.Under every kind of condition of culture when initial inoculation, in cultivating system
Dry cell weight is 0.18g/L.
Condition of culture 1, nutritional sufficiency in incubation are able to satisfy microalgae cell normal growth, and culture to stationary phase utilizes
Elemental analyser measures its maximum nitrogen content N intracellularmaxFor 8.10%wt.Specific nutrient is added as follows: inoculation Shi Xiangpei
It supports and F/2 mother liquor is added in base, 5mL F/2 nutritive salt mother liquor is added in every liter of cultivating system, and nitrogen level exists in system at this time
63mg L-1(being represented by 5 × F/2 level)；Nitrogen concentration in monitoring system in incubation, when culture was to the 3rd day, nitrogen in system
It is consumed to 10mg L-1When (1 × F/2 horizontal), it is dense to nitrogen that 4mL F/2 nutritive salt mother liquor is added into culture medium at this time
Degree reaches 60mg L-1(reach again about 5 × F/2 level) adds 4mL F/2 nutritive salt mother liquor when culture was to the 6th day again, after
To 10 days, harvesting microalgae measured nitrogen content intracellular for continuous culture.
Condition of culture 2 does not add nitrogen nutrition element in system when initial inoculation under N stress condition of culture, other battalion
The addition of element is supported with condition of culture 1, after culture 10 days, harvesting microalgae measures nitrogen content intracellular, determines and maintains cell survival
Minimum nitrogen content N intracellularminFor 2.10%wt.
Condition of culture 3 is cultivated in the case where nitrogen limits condition of culture, and when initial inoculation is individually added into F/2 nutritive salt into system
NaNO in mother liquor3, make nitrogen concentration 24.0mg/L in system, denitrogenate the addition of other outer nutrients with condition of culture 1, culture
Nitrogen nutrition element is no longer mended in the process.15:00 every afternoon sampling, measures chlorophyll fluorescence parameters Δ F/Fm' and nitrogen intracellular
Content, specific variation are as shown in Figure 3.Culture was to 3 days, and nitrogen concentration is 0mg L in system-1, cell is in N stress state.The nitrogen side of body
Under compeling, Δ F/Fm' with NSI have high correlation, regression curve y=-1.4945x+1.5228, R2=0.9713 (y is
NSI, x are Δ F/Fm').It is determined by correlation analysis and uses Δ F/Fm' online characterization parameter as N stress degree.
Control of the condition of culture 4 to realize NSI.When controlling NSI 0.81 (nitrogen content intracellular is 3.23%wt),
The Δ F/F of cell at this timem' value 0.470 be current N stress degree online characterization parameter value.Specifically, in culture 3
Microdisk electrode is carried out under part, after culture 5 days, cell Δ F/Fm' reach for 0.460, microalgae cell NSI is 0.81 at this time, is reached
Control the control point NSI.Then to added in cultivating system nitrogen to system nitrogen concentration be 24.5mg/L (when culture need obtain it is thin
Born of the same parents' dry weight incrementss are 0.75g/L, and when nitrogen content intracellular is 3.23%wt, needing to add to nitrogen concentration in system is 24mg/L),
Remaining nutrient is supplemented to 5 × F/2 level, is denoted as again at this time 0 day, no longer supplement the nutrients element in incubation.Culture
Process monitoring Δ F/Fm', nitrogen content and Δ F/F intracellularm' variation such as Fig. 4.It can be seen that being further cultured for after mending nitrogen 5 days, Δ F/
Fm' reaching 0.477, nitrogen content intracellular is 3.27%wt at this time, NSI 0.81 realizes the stability contorting of N stress degree.
Embodiment 2:
The microalgae cell culture that 4 kinds of condition of culture are carried out in the present embodiment, in addition to condition of culture 4, remaining 3 kinds cultures are the same as real
Apply example 1.Condition of culture 4 uses Semi-continuous cultivation, i.e., when Cellular stress degree reaches setting value, carries out by the dilution rate of setting
Dilution, adds nutritive salt (specific additional way is with condition of culture 4 in embodiment 1), starts new circulation.Wherein, dilution rate is
Refer to during Semi-continuous cultivation, the ratio for the total volume of culture of fresh culture volume Zhan added when diluting again.It is specific and
Speech, in the present embodiment condition of culture 4, NSI control is 3.73%wt in 0.73 nitrogen content intracellular, at this time current N stress degree
Online characterization parameter Δ F/Fm' value be 0.503.Fig. 5 is based on Δ F/Fm' feedback control cell N stress degree semicontinuous training
It supports as a result, operation dilution rate is 0.6.It can be seen that after culture 4 days, Δ F/Fm' reaching 0.503, nitrogen content intracellular is at this time
3.73%wt, NSI 0.73, diluting cultivating system again by 0.6 dilution rate at this time and adding nitrogen nutrition element makes nitrogen in system
Content maintains 24.0mg/L or so, continues to cultivate, after culture 8 days, Δ F/Fm' reach 0.504, it is diluted again by above-mentioned steps
It is further cultured for；When cultivating 12 days, Δ F/Fm' up to 0.514 when, can continue to add nutrient by above-mentioned steps being further cultured for.This implementation
Example condition of culture 4 is only cultivated 12 days.From figure 5 it can be seen that in the present embodiment, by the online characterization parameter Δ for monitoring N stress degree
F/Fm'；At culture 4 days, 8 days and 12 days, nitrogen content intracellular is respectively 3.73%wt, 3.56%wt and 3.67%wt, corresponding NSI
Respectively 0.73,0.75 and 0.74 realize the repetition stability contorting of NSI.This is the result shows that cell N stress degree can lead to
It crosses and uses Δ F/Fm' as cell N stress degree characterization parameter realize cell N stress degree feedback control.
Claims (8)
1. a kind of adjusting method of the microalgae nitrogen nutrition coercing cultivation process based on feedback control, it is characterised in that: be in microalgae
In incubation, online characterization parameter by using the photosynthetic chlorophyll fluorescence parameters of microalgae as N stress degree is supervised in real time
Nitrogen nutrition stress level suffered by cell is surveyed, incubation microalgae is realized in the addition of nitrogen nutrition element in feedback guidance cultivating system
The accurate control of N stress suffered by cell.
2. adjusting method according to claim 1, it is characterised in that: specifically,
(1) N stress refers in the culture environment of microalgae cell normal growth, controls in cultivating system without nitrogen nutrition member
When plain, microalgae cell suffered stress during the growth process；
(2) based on the above-mentioned defined suffered battalion of N stress index (Nitrogen Stress Index, NSI) characterization cell
Stress level is supported, NSI presses formula NSIi=(Nmax-Ni)/(Nmax-Nmin) calculate；Wherein NmaxRefer to the training in microalgae cell normal growth
Support in environment maximum nitrogen mass content intracellular, N under the conditions of nutritional sufficiencyminIt is minimum when cell survival under the conditions of finger N stress
Nitrogen mass content intracellular, NiRefer to the nitrogen mass content intracellular at a certain moment under N stress condition of culture；
(3) the photosynthetic chlorophyll fluorescence parameters of microalgae and the corresponding relationship curve or function of NSI are established；Specifically, in culture body
Nitrogen nutrition element is to play cell the 0mg/L moment to reach stage stationary phase in system, and no less than 3 trainings are measured by sampling in constant duration
The nitrogen content intracellular for supporting moment cell calculates NSI value and the photosynthetic chlorophyll fluorescence parameters of the microalgae at corresponding moment, passes through correlation
Property analysis establish the photosynthetic chlorophyll fluorescence parameters of microalgae and the corresponding relationship curve or function of NSI；
(4) during the cultivation process, after there is N stress, i.e., after nitrogen nutrition element is 0mg/L in cultivating system, microalgae light is monitored
It closes chlorophyll fluorescence parameters to change, (2) established invariance curve or function is utilized to calculate NSI at that time, be in together when with setting NSI
When one level, nitrogen nutrition element is added into cultivating system until the photosynthetic chlorophyll fluorescence parameters of microalgae reach setting value again；
During this, the additional amount of unit (volume or quality) cultivating system nitrogen nutrition element is obtained according to unit cultivating system needs
Nitrogen content B intracellular corresponding to the dry cell weight incrementss A and setting NSI level obtained is determining, as A*B.
3. adjusting method according to claim 2, it is characterised in that: the culture of step (1) the microalgae cell normal growth
Environment includes the temperature for meeting microalgae cell normal growth and breeding, light intensity, pH and cultivating system nutrient；
Nutrient includes that nitrogen phosphate and sulfur, iron, copper, molybdenum, zinc, silicon, manganese, cobalt and microorganism, biotin can meet microalgae cell
Normal growth；
In incubation can continuous illumination for 24 hours, or can also with certain Dark-light cycle carry out illumination, the model of Dark-light cycle ratio
Enclose 8h:16h~16h:8h (illumination: dark)；
When N stress is referred in above-mentioned cultivating system only without nitrogen nutrition element, microalgae cell is suffered during the growth process
Stress.
4. adjusting method according to claim 2, it is characterised in that:
Step (3) during the whole culture process, in cultivating system in addition to nitrogen nutrition other each nutrients include phosphorus, sulphur, iron,
Copper, molybdenum, zinc, silicon, manganese, cobalt etc. and microorganism, biotin etc. can meet needed for microalgae cell growth；
If illumination is carried out with Dark-light cycle mode, after sample time should be selected in illumination at least 1h.
5. adjusting method according to claim 2, it is characterised in that:
The additional amount of step (4) unit (volume or quality) cultivating system nitrogen nutrition element needs to obtain according to unit cultivating system
Dry cell weight incrementss A and setting NSI level corresponding to nitrogen content B intracellular determine, as A*B；Such as culture needs to obtain
Dry cell weight incrementss 1.0g/L, set the corresponding nitrogen content intracellular of NSI as 4.0%wt, then the nitrogen content for needing to add is
(1.0 × 4.0%) g/L, as 40mg/L；
It should be noted that needing the dry cell weight incrementss obtained that should meet microdisk electrode actual conditions, not higher than culture body
It can be obtained maximum cell dry weight when microalgae cell reaches stationary phase in system.
6. adjusting method according to claim 1, it is characterised in that: wherein N content, which measures, can be used elemental analyser or triumphant
The measurement of formula nitriding, but it is not limited to the above method；
The photosynthetic chlorophyll fluorescence parameters of the microalgae include for characterizing the sub- yield F of the maximum amount of microalgae photosynthetical system IIv/
FmOr the practical quantum yield Δ F/F for characterizing microalgae photosynthetical system IIm' etc. chlorophyll fluorescence parameters.Its Determination of Chlorophyll is glimmering
Optical parameter can be measured with Water-PAM chlorophyll fluorescence instrument (Walz, Germany), but be not limited to using above-mentioned instrument
Device.
7. adjusting method according to claim 2, it is characterised in that: nitrogen nutrition substance in the culture medium of the cultivating system
Adding manner includes to be continuously added to or be intermittently added in batches；
In addition, what the addition of other nutrients needed to control except denitrogenating in the culture medium of cultivating system includes nutrient
The addition time of additional amount and nutrient, it is ensured that other each nutrient abundances can expire in addition to nitrogen nutrition in incubation
Needed for sufficient microalgae cell growth.
8. adjusting method according to claim 1, it is characterised in that: the accurate control of N stress suffered by the microalgae cell
Refer to the real time monitoring feedback regulation nitrogen for passing through chlorophyll fluorescence parameters adds the NSI for making cell reach setting, that is, reaches setting
N stress degree.
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