CN109752351B - Feedback control-based regulation method for microalgae nitrogen nutrition stress culture process - Google Patents

Feedback control-based regulation method for microalgae nitrogen nutrition stress culture process Download PDF

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CN109752351B
CN109752351B CN201711068393.XA CN201711068393A CN109752351B CN 109752351 B CN109752351 B CN 109752351B CN 201711068393 A CN201711068393 A CN 201711068393A CN 109752351 B CN109752351 B CN 109752351B
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薛松
刘娇
曹旭鹏
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Dalian Institute of Chemical Physics of CAS
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Abstract

The invention provides a method for adjusting a microalgae nitrogen nutrition stress culture process based on feedback control. The method defines a Nitrogen Stress Index (NSI) for quantifying the Nitrogen Stress degree of the microalgae cells, provides a control Index for accurately controlling the Nitrogen Stress formation degree in the microalgae culture process, realizes feedback control of the Nitrogen Stress degree of the microalgae cells based on chlorophyll fluorescence parameter guidance by establishing an in-situ online characterization method of the Nitrogen Stress degree based on the chlorophyll fluorescence parameter, and ensures that the microalgae cells are properly stressed.

Description

Feedback control-based regulation method for microalgae nitrogen nutrition stress culture process
Technical Field
The invention relates to a technology for regulating mild microalgae nutrition stress based on feedback control, in particular to a technology for regulating mild microalgae nutrition stress by using a microalgae chlorophyll fluorescence parameter (F)v/FmOr Δ F/Fm') real-time characterization of nitrogen stress degree of microalgae cells, feedback guidance of addition of nutrient elements in the culture process, and control of microalgae in proper stress state.
Background
Microalgae have the advantages of high photosynthetic efficiency, no land competition with agriculture and the like, and are highly valued in strategic research of renewable biomass energy sources at home and abroad.Under the condition of sufficient nutrition, microalgae can fix CO by cells2And the absorbed nutrient substances are mainly used for the growth and the propagation of the cells. When the cells are stressed (such as nutritional stress, environmental stress, etc.), the microalgae can fix energy and CO through photosynthesis2The flow direction is changed, wherein nitrogen stress is the most common stress means for microalgae culture at present. For oil-producing microalgae, under nitrogen stress conditions, microalgae cells cannot grow and divide normally, and redundant reducing power in the cells is used for oil synthesis. Therefore, cell growth and lipid accumulation are contradictory, and the highest lipid yield stage in the microalgae culture process is an unsteady state process. Therefore, it is necessary to control the stress level to which the cells are subjected during the culture process so that it is in an appropriate range. Within the stress range, the photosynthetic efficiency of the cells can be maintained at a certain level for the growth of the cells, and the cells can be stimulated to accumulate enough grease, so that the cells are maintained at the highest grease yield stage. Because the global change of cells caused by nutrition stress (such as nitrogen stress) of microalgae is extremely complex and the oil accumulation mechanism is not clear, the feasibility of establishing an oil-producing microalgae high-efficiency culture process guided by the oil accumulation mechanism theory is low, the current continuous high-efficiency controllable culture technology of oil-producing microalgae is not broken through, the development of the microalgae industry is limited, and the method becomes one of the hotspots and difficulties of microalgae research.
Currently, nitrogen supplementation is the most commonly used method for controlling the degree of cellular stress (AREMU A O et al. journal of physiology, 2015,51(4): 659-; in addition, there are studies using the cell N/C value to achieve control of the degree of stress (BONNEFOND H et al Biotechnology for Biofuels,2017,10(1): 25). However, due to technical limitations, real-time online monitoring of intracellular nitrogen content in the culture process cannot be realized, so that the accurate control of the nitrogen stress degree of cells in the culture process is limited. Patent CN10437475813 of Wangqiang et al uses Fv/FmThe relation with the oil content of the oil-producing microalgae under nitrogen stress guides the culture and harvest time. However, the technology can not realize the control of the nitrogen stress degree of the microalgae cells in the culture process, guide the supplement of nitrogen nutrient elements in the culture process and limit the supplement of the nitrogen nutrient elements in the culture processUse in different culture modes, in particular in fed batch, semi-continuous or continuous culture mode. Therefore, if a new parameter capable of representing the degree of the nitrogen stress of the cells on line can be used to realize feedback control of the cells to maintain moderate nutrition stress, the method has important significance for nitrogen limitation microalgae culture, and is particularly beneficial to realizing stable control of outdoor large-scale culture.
Disclosure of Invention
The invention aims to provide a feedback control-based microalgae moderate nitrogen stress regulation technology, which aims to improve the controllability of microalgae culture and solve the problem that the stress degree cannot be accurately controlled by monitoring the stress degree on line in the existing microalgae nitrogen stress culture process.
A method for adjusting a microalgae nitrogen nutrition stress culture process based on feedback control is characterized in that in the microalgae culture process, a microalgae photosynthetic chlorophyll fluorescence parameter is used as an online characterization parameter of nitrogen stress degree, the nitrogen nutrition stress degree of cells is monitored in real time, and nitrogen nutrient elements are fed back and guided to be added in a culture system, so that the accurate control of the nitrogen stress of the microalgae cells in the culture process is realized.
In particular, the present invention relates to a method for producing,
(1) the nitrogen stress refers to the stress on the microalgae cells in the growth process when the culture system does not contain nitrogen nutrient elements in the culture environment in which the microalgae cells normally grow;
(2) characterizing the extent of nutritional Stress experienced by a cell based on the above defined Nitrogen Stress Index (NSI), NSI being according to formula NSIi=(Nmax-Ni)/(Nmax-Nmin) Calculating; wherein N ismaxRefers to the maximum nitrogen mass content, N, in microalgae cells under the condition of sufficient nutrition in the culture environment for normal growth of microalgae cellsminRefers to the lowest intracellular nitrogen mass content, N, at which cells remain viable under nitrogen stress conditionsiRefers to the intracellular nitrogen mass content at a certain moment under the condition of nitrogen stress culture;
(3) establishing a corresponding relation curve or function of microalgae photo-chlorophyll fluorescence parameters and NSI; specifically, in a stage from the time when the nitrogen nutrient element in a culture system is 0mg/L to the time when the cells reach a stationary phase, sampling and measuring the intracellular nitrogen content of the cells at no less than 3 culture times at equal time intervals, calculating an NSI value and a microalgae photo-chlorophyll fluorescence parameter at the corresponding time, and establishing a corresponding relation curve or function of the microalgae photo-chlorophyll fluorescence parameter and the NSI through correlation analysis;
(4) in the culture process, monitoring the change of the fluorescence parameters of the photosynthetic chlorophyll of the microalgae after nitrogen stress occurs, namely the nitrogen nutrient elements in the culture system are 0mg/L, calculating the current NSI by using the established association curve or function in the step (2), and supplementing the nitrogen nutrient elements into the culture system until the fluorescence parameters of the photosynthetic chlorophyll of the microalgae reach the set value again when the NSI is at the same level (the deviation range is within NSI +/-0.3);
in the process, the adding amount of the nitrogen nutrient elements of the unit (volume or mass) culture system is determined according to the cell dry weight increasing amount A required to be obtained by the unit culture system and the intracellular nitrogen content B corresponding to the set NSI level, namely A x B.
The culture environment for normal growth of the microalgae cells in the step (1) comprises temperature, light intensity, pH and nutrient elements of a culture system which meet the requirements of normal growth and proliferation processes of the microalgae cells;
the nutrient elements comprise nitrogen, phosphorus, sulfur, iron, copper, molybdenum, zinc, silicon, manganese and cobalt, and microorganisms and biotin can meet the normal growth of microalgae cells;
in the culture process, continuous illumination can be carried out for 24 hours, or illumination can be carried out in a certain light-dark cycle, and the light-dark cycle ratio ranges from 8 hours to 16 hours to 8 hours (light: dark);
nitrogen stress refers to stress to which microalgae cells are subjected during growth when the culture system contains no nitrogen nutrient elements.
In the whole culture process, other nutrient elements except nitrogen in the culture system, such as phosphorus, sulfur, iron, copper, molybdenum, zinc, silicon, manganese, cobalt and the like, as well as microorganisms, biotin and the like can meet the requirements of the growth of microalgae cells;
if the illumination is performed in the light-dark cycle mode, the sampling time should be selected at least 1h after the illumination.
Step (4), the supplement amount of the nitrogen nutrient elements of the unit (volume or mass) culture system is determined according to the cell dry weight increase A required to be obtained by the unit culture system and the intracellular nitrogen content B corresponding to the set NSI level, and the supplement amount is A x B; if the increase of the dry weight of the cells required to be obtained by culture is 1.0g/L, and the intracellular nitrogen content corresponding to NSI is set to be 4.0% wt, the nitrogen content required to be supplemented is (1.0 multiplied by 4.0%) g/L, namely 40 mg/L;
it should be noted that the increase of the dry weight of the cells to be obtained is consistent with the practical situation of microalgae culture and is not higher than the maximum dry weight of the cells obtained when the microalgae cells in the culture system reach the stationary phase.
Wherein the content of N can be determined by an element analyzer or Kjeldahl method, but is not limited to the method;
the microalgae photosynthetic chlorophyll fluorescence parameters comprise the maximum light quantum yield F for representing the microalgae photosynthetic system IIv/FmOr for characterizing the actual photon yield Δ F/F of the microalgae photosynthetic System IIm' Iso chlorophyll fluorescence parameters. Wherein the chlorophyll fluorescence parameter can be measured by a Water-PAM chlorophyll fluorescence apparatus (Walz, Germany), but is not limited to the use of the above apparatus.
The adding mode of the nitrogen nutrient in the culture medium of the culture system comprises continuous addition or batch intermittent addition;
in addition, the addition of other nutrient elements except nitrogen in the culture medium of the culture system needs to be controlled, and the addition amount of the nutrient elements and the addition time of the nutrient elements are included, so that the requirement of the growth of microalgae cells can be met when the other nutrient elements except nitrogen are sufficient in the culture process. 8. The method of adjusting of claim 1, wherein: the accurate control of nitrogen stress on the microalgae cells refers to that the cells reach the set NSI (non-specific cell differentiation) by feeding back and adjusting nitrogen supplement through real-time monitoring of chlorophyll fluorescence parameters, namely the set nitrogen stress degree is reached.
In order to accurately quantify the Nitrogen stress degree of cells, the Nitrogen Stress Index (NSI) of the cells is defined firstly. NSI according to formula NSIi=(Nmax-Ni)/(Nmax-Nmin) And (4) calculating.Wherein N ismaxRefers to the maximum intracellular nitrogen content, N, under conditions of adequate nutritionminRefers to the lowest intracellular nitrogen content, N, at which cells remain viable under nitrogen stress conditionsiRefers to the intracellular nitrogen content at a certain time under nitrogen stress conditions. Therefore, the NSI has a variation range of 0-1, and the larger the value of the NSI is, the stronger the stress degree of the cell is. In the case of nannochloropsis, the maximum intracellular nitrogen content under normal culture conditions is 8.10% wt, the minimum intracellular nitrogen content that can survive under nitrogen stress is 2.10% wt, and when the intracellular nitrogen content is 3.76% wt, the NSI is 0.72.
Based on the NSI defined above, the NSI and microalgae chlorophyll fluorescence parameter (F) are analyzed and established in the nitrogen stress culture processv/FmOr Δ F/Fm') correlation using microalgae chlorophyll fluorescence parameter (F)v/FmOr Δ F/Fm') real-time characterization of the nitrogen stress degree of the microalgae cells. The microalgae chlorophyll fluorescence parameters capable of being monitored in situ on line are used for replacing intracellular nitrogen content which cannot be measured on line in real time to indirectly represent the nitrogen stress degree of cells. Finally, the NSI and the microalgae chlorophyll fluorescence parameter (F) are utilizedv/FmOr Δ F/Fm') correspondence using microalgae chlorophyll fluorescence parameters (F)v/FmOr Δ F/Fm') as a nitrogen stress degree characterization parameter in the nitrogen stress culture process, and feeding back and guiding the addition of nutrient elements in the culture process to realize the moderate nutrient stress regulation of the microalgae. Specifically, when the NSI level to be controlled is set, the corresponding microalgae chlorophyll fluorescence parameter (F) is determinedv/FmOr Δ F/Fm') monitoring point. And when the chlorophyll fluorescence of the cells reaches a monitoring point, supplementing nitrogen nutrient elements to the culture system according to different culture modes and requirements. Chlorophyll fluorescence (F) of microalgae after nitrogen supplementationv/FmOr Δ F/Fm') Back-up, based on the preset nitrogen-supplemented chlorophyll fluorescence (F)v/FmOr Δ F/Fm') upper limit, real-time monitoring of microalgae chlorophyll fluorescence (F)v/FmOr Δ F/Fm') to achieve a predetermined range of lift-back by controlling the nitrogen addition to achieve a constant repetitive control of NSI. The specific flow diagram is shown in figure 1.
The feedback control-based microalgae moderate nutrition stress regulation technology established by the invention uses microalgae chlorophyll fluorescence parameters (F) which can be monitored in situ on linev/FmOr Δ F/Fm') as a real-time characterization parameter of the nitrogen stress degree, solves the limitation that the intracellular nitrogen content can not be measured in real time for evaluating the nitrogen stress degree. Provides a new control strategy for microalgae controllable nitrogen stress culture.
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The invention is illustrated in fig. 5, wherein:
FIG. 1 utilization of microalgae chlorophyll fluorescence parameters (F) under nitrogen stress culturev/FmOr Δ F/Fm') is used as a nitrogen stress degree real-time characterization parameter to feed back and guide the nitrogen nutrient element supplement flow diagram.
FIG. 2 is a schematic diagram of a columnar photobioreactor microalgae culture system.
FIG. 3. delta. F/F of Nannochloropsis oculata in nitrogen-limited culture modem' and intracellular nitrogen content.
FIG. 4. delta.F/F of Nannochloropsis at an NSI of 0.81 under the culture conditions of example 1m' and intracellular nitrogen content.
FIG. 5. delta.F/F of Nannochloropsis at a NSI of 0.73 in a semi-continuous culture mode under the culture conditions of example 2m' and intracellular nitrogen content.
Detailed Description
The invention firstly provides a feedback control-based microalgae moderate nutrition stress adjusting technology, namely the stress degree of microalgae cells is adjusted by a feedback control means in the nitrogen limitation microalgae culture process, so as to realize moderate nutrition stress.
Firstly, culturing microalgae under the condition of sufficient nutrition, collecting the microalgae after the microalgae grow to a stable period, and measuring the intracellular nitrogen content of the microalgae dry powder by using an element analyzer, namely Nmax. The method for measuring the intracellular nitrogen content is not limited to the use of an element analyzer, and other methods capable of measuring the intracellular nitrogen content, such as a Kjeldahl method, are applicable.
Timed sampling for viability determination during nitrogen limitation cultureChlorophyll fluorescence parameter (F) of cellsv/FmOr Δ F/Fm') until the cell dies, determining the intracellular nitrogen content N at that timemin. Investigating the regression relationship between chlorophyll fluorescence parameter and intracellular nitrogen content, selecting F preferentiallyv/FmOr Δ F/Fm' As a characteristic parameter of the degree of nitrogen stress, i.e., one having a high correlation was selected. Wherein the chlorophyll fluorescence parameter can be measured by a Water-PAM chlorophyll fluorescence instrument (Walz, Germany) by the method used by YAO et al (YAO C et al. Bioresource Technology,2016,212: 26-34.). It should be noted that the measurement of the fluorescence parameters of chlorophyll in microalgae is not limited to the above-mentioned apparatus and method, and other apparatuses and methods for measuring fluorescence parameters of chlorophyll in microalgae are suitable.
And then, under the condition of nitrogen limitation culture, determining the degree of nutrient stress, determining a control range based on chlorophyll fluorescence parameter feedback control according to the established relation between the chlorophyll fluorescence parameter and the intracellular nitrogen content, and guiding the supplement of nutrient element nitrogen in the culture process.
The process and results of the present invention are illustrated by the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1:
taking the cultivation of nannochloropsis in a 500mL cylindrical glass bubble reactor (FIG. 2) as an example, the reaction conditions are respectively a cultivation temperature of 25 + -2 deg.C and an illumination intensity of 140 μmol m–2s–1CO of air2The ventilation rate is 100mL min–1Introduction of CO only during illumination2,CO2The air inlet amount of the light source is 2% of the air inlet amount, the light-dark ratio is 14h:10h, and the illumination time per day is 8: 00-20: 00. Seawater culture, using modified F/2 nutrient salts, wherein the final concentration of each nutrient salt in mother liquor is (per 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. The nitrogen nutrient concentration in the system is expressed as the final concentration of nitrogen element.
In this example, microalgae cells were cultured under 4 culture conditions. In the 4 culture processes, other nutrient elements except nitrogen nutrition in the system can meet the requirements of microalgae cell growth, including phosphorus, sulfur, iron, copper, molybdenum, zinc, silicon, manganese, cobalt and the like, as well as microorganisms, biotin and the like; the light intensity, temperature and ventilation were maintained constant. At the initial inoculation under each culture condition, the dry weight of cells in the culture system was 0.18 g/L.
Culturing condition 1, wherein the nutrition is sufficient during the culturing process, the normal growth of microalgae cells can be met, the culturing is carried out until the stable period, and an element analyzer is utilized to determine the maximum intracellular nitrogen content NmaxIt was 8.10% wt. The specific nutrient elements are supplemented as follows: adding F/2 mother liquor into the culture medium during inoculation, and adding 5mL of F/2 nutrient salt mother liquor into each liter of culture system when the nitrogen element level in the system is 63mg L-1(expressed as a 5 XF/2 level); monitoring nitrogen concentration in the system during culture, and consuming nitrogen in the system to 10mg L by 3 days-1At this time (i.e., 1 XF/2 level), 4mL of F/2 nutrient salt stock solution was supplemented to the medium until the nitrogen concentration reached 60mg L-1(reaching about 5 XF/2 level again), when the culture is carried out for 6 days, 4mL of F/2 nutrient salt mother liquor is supplemented again, the culture is continued for 10 days, and the microalgae is harvested to measure the content of the intracellular nitrogen.
Culturing under nitrogen stress condition 2, adding no nitrogen nutrient element in the system during initial inoculation, adding other nutrient elements under the same condition as 1, culturing for 10 days, harvesting microalgae, determining intracellular nitrogen content, and determining the lowest intracellular nitrogen content N for maintaining cell survivalminIt was 2.10% wt.
Culturing under nitrogen limitation condition 3, adding NaNO in F/2 nutrient salt mother liquor during initial inoculation3The nitrogen concentration in the system is 24.0mg/L, the addition of other nutrient elements except nitrogen is the same as the culture condition 1, and the nitrogen nutrient elements are not supplemented in the culture process. Sampling at 15:00 pm every day, and determining chlorophyll fluorescence parameter delta F/Fm' and intracellular nitrogen content, the specific changes are shown in FIG. 3. Culturing for 3 days, wherein the nitrogen concentration in the system is 0mg L-1The cells are in a nitrogen stressed state. Under nitrogen stress,. DELTA.F/Fm' has a high correlation with NSI, and the regression curve is-1.4945 x +1.5228, R20.9713(y is NSI, x is DeltaF/F)m'). Determination of use of Δ F/F by correlation analysism' as an on-line characterization parameter for the degree of nitrogen stress.
Culture conditions 4 were used to achieve NSI control. When NSI was controlled to 0.81 (intracellular nitrogen content 3.23% wt), Δ F/F of the cells at this timemThe' value 0.470 is the online characterization parameter value of the current nitrogen stress degree. Specifically, microalgae were cultured under culture 3 conditions, and after 5 days of culture, cells were cultured at Δ F/Fm' reached 0.460, at which time the microalgal cell NSI was 0.81, reaching the control point for NSI control. And then supplementing nitrogen into the culture system until the nitrogen concentration of the system is 24.5mg/L (when the increase of the cell dry weight required to be obtained by culture is 0.75g/L and the intracellular nitrogen content is 3.23% wt, the nitrogen concentration in the system is required to be supplemented to be 24mg/L), supplementing the rest nutrient elements to the 5 xF/2 level, recording the nitrogen content again as 0 day, and not supplementing the nutrient elements in the culture process. Culture Process monitoring Δ F/Fm', intracellular Nitrogen content and Δ F/Fm' variations are as in FIG. 4. As can be seen from the figure, after supplementing nitrogen, the cells were cultured for 5 days at a ratio of. DELTA.F/Fm' to 0.477, at which the intracellular nitrogen content is 3.27% wt and the NSI is 0.81, stable control of the nitrogen stress level is achieved.
Example 2:
in this example, microalgae cells were cultured under 4 kinds of culture conditions, and the other 3 kinds of culture conditions were the same as in example 1 except for the culture condition 4. The culture condition 4 adopts semi-continuous culture, namely when the degree of cell stress reaches a set value, diluting according to a set dilution rate, supplementing nutrient salts (the specific supplementing mode is the same as the culture condition 4 in the example 1), and starting a new cycle. Wherein the dilution rate is determined during the semi-continuous culture processIn (2), the volume of fresh medium added at the time of re-dilution is a proportion of the total culture volume. Specifically, in culture condition 4 of this example, NSI is controlled to 0.73 intracellular nitrogen content of 3.73% wt, and the parameter Δ F/F for online characterization of the current nitrogen stress level is presentmThe value of' is 0.503. FIG. 5 is a graph based on Δ F/Fm' semi-continuous culture results with feedback control of the degree of nitrogen stress of the cells, the operating dilution rate was 0.6. As can be seen from the figure,. DELTA.F/F after 4 days of culturem' reaching 0.503 with intracellular nitrogen content of 3.73 wt% and NSI of 0.73, re-diluting the culture system at 0.6 dilution rate and supplementing nitrogen nutrient elements to maintain the nitrogen content of the system at about 24.0mg/L, continuing culturing for 8 days, and culturing for Δ F/Fm' to 0.504, re-diluting and re-culturing according to the above steps; at 12 days of culture,. DELTA.F/FmWhen the nutrient content reaches 0.514, the nutrient elements can be continuously supplemented according to the steps and then cultured. The culture conditions of this example 4 were only cultured for 12 days. As can be seen from FIG. 5, in the present embodiment, the parameter Δ F/F is characterized online by monitoring the degree of nitrogen stressm'; when the cell is cultured for 4 days, 8 days and 12 days, the intracellular nitrogen content is respectively 3.73 percent wt, 3.56 percent wt and 3.67 percent wt, and the corresponding NSI is respectively 0.73, 0.75 and 0.74, thereby realizing the repeated stable control of NSI. This result indicates that the degree of nitrogen stress in cells can be controlled by using Δ F/Fm' As a cell nitrogen stress degree characterization parameter, the feedback control of the cell nitrogen stress degree is realized.

Claims (7)

1.一种基于反馈控制的微藻氮营养胁迫培养过程的调节方法,其特征在于:是在微藻培养过程中,通过使用微藻光合叶绿素荧光参数作为氮胁迫程度的在线表征参数,实时监测细胞所受氮营养胁迫程度,反馈指导培养体系内氮营养元素的加入,实现培养过程微藻细胞所受氮胁迫的精确控制;1. a kind of regulation method based on the microalgae nitrogen nutrient stress cultivation process of feedback control, it is characterized in that: be in the microalgae cultivation process, by using microalgae photosynthetic chlorophyll fluorescence parameter as the on-line characterization parameter of nitrogen stress degree, real-time monitoring The degree of nitrogen nutrient stress on cells is fed back to guide the addition of nitrogen nutrient elements in the culture system to achieve precise control of nitrogen stress on microalgae cells during the culture process; 所述调节方法,具体来说,The adjustment method, specifically, (1)所述氮胁迫指在微藻细胞正常生长的培养环境中,控制培养体系中不含氮营养元素时,微藻细胞在生长过程中所受到的胁迫;(1) The nitrogen stress refers to the stress that the microalgae cells are subjected to during the growth process when the control culture system does not contain nitrogen nutrients in the culture environment where the microalgae cells grow normally; (2)基于上述所定义的氮胁迫指数(Nitrogen Stress Index,NSI)表征细胞所受营养胁迫程度,NSI按式NSIi= (Nmax- Ni)/(Nmax- Nmin)计算;其中Nmax指在微藻细胞正常生长的培养环境中营养充足条件下胞内最大氮质量含量,Nmin指氮胁迫条件下细胞维持存活时最低的胞内氮质量含量,Ni指氮胁迫培养条件下某一时刻的胞内氮质量含量;(2) Based on the above-defined nitrogen stress index (Nitrogen Stress Index, NSI) to characterize the degree of nutrient stress to cells, NSI is calculated according to the formula NSI i = (N max - N i )/(N max - N min ); wherein N max refers to the maximum intracellular nitrogen mass content under nutrient sufficient conditions in the culture environment of normal growth of microalgae cells, N min refers to the lowest intracellular nitrogen mass content when the cells maintain survival under nitrogen stress conditions, and Ni refers to nitrogen stress culture conditions The mass content of intracellular nitrogen at the next moment; (3)建立微藻光合叶绿素荧光参数与NSI的对应关系曲线或函数;具体而言,在培养体系中氮营养元素为0 mg/L时刻起到细胞达到稳定期阶段,等时间间隔取样测定不少于3个培养时刻细胞的胞内氮含量计算NSI值以及相应时刻的微藻光合叶绿素荧光参数,通过相关性分析建立微藻光合叶绿素荧光参数与NSI的对应关系曲线或函数;(3) Establish the corresponding relationship curve or function between the photosynthetic chlorophyll fluorescence parameters of microalgae and NSI; specifically, when the nitrogen nutrient element in the culture system is 0 mg/L until the cells reach the stable stage, sampling and measuring at equal time intervals Calculate the NSI value of the intracellular nitrogen content of the cells at less than 3 cultivation times and the microalgae photosynthetic chlorophyll fluorescence parameters at the corresponding time, and establish the corresponding relationship curve or function between the microalgae photosynthetic chlorophyll fluorescence parameters and NSI through correlation analysis; (4)在培养过程中,当出现氮胁迫后,即培养体系中氮营养元素为0 mg/L后,监测微藻光合叶绿素荧光参数变化,利用(2)所建立关联曲线或函数计算当时NSI,当与设定NSI处于同一水平时,向培养体系内补加氮营养元素直至微藻光合叶绿素荧光参数再次达到设定值;(4) During the culture process, when nitrogen stress occurs, that is, after the nitrogen nutrient element in the culture system is 0 mg/L, monitor the changes of the photosynthetic chlorophyll fluorescence parameters of the microalgae, and use the correlation curve or function established in (2) to calculate the current NSI. , when it is at the same level as the set NSI, nitrogen nutrients are added to the culture system until the microalgae photosynthetic chlorophyll fluorescence parameter reaches the set value again; 此过程中,单位体积或质量培养体系氮营养元素的补加量根据单位培养体系需要获得的细胞干重增加量A与设定NSI水平所对应的胞内氮含量B确定,即为A*B。In this process, the supplementary amount of nitrogen nutrient elements per unit volume or mass culture system is determined according to the increase in dry cell weight A that needs to be obtained per unit culture system and the intracellular nitrogen content B corresponding to the set NSI level, that is, A*B . 2.根据权利要求1所述调节方法,其特征在于:步骤(1)所述微藻细胞正常生长的培养环境包括满足微藻细胞正常生长和增殖过程的温度、光强、pH以及培养体系营养元素;2 . The adjustment method according to claim 1 , wherein the culture environment for the normal growth of microalgae cells in step (1) includes temperature, light intensity, pH and nutrients in the culture system that satisfy the normal growth and proliferation process of microalgae cells. 3 . element; 营养元素包括氮、磷、硫、铁、铜、钼、锌、硅、锰、钴以及微生物、生物素;Nutrients include nitrogen, phosphorus, sulfur, iron, copper, molybdenum, zinc, silicon, manganese, cobalt, as well as microorganisms, biotin; 培养过程中24h连续光照,或以一定的光暗循环进行光照,光暗循环比的范围8h:16h~16h:8h;光暗指光照和黑暗;During the culturing process, continuous light for 24h, or light with a certain light-dark cycle, the range of light-dark cycle ratio is 8h:16h~16h:8h; light and dark refer to light and darkness; 氮胁迫指在上述培养体系中仅仅不含氮营养元素时,微藻细胞在生长过程中所受到的胁迫。Nitrogen stress refers to the stress that the microalgae cells are subjected to during the growth process when the above-mentioned culture system does not contain only nitrogen nutrients. 3.根据权利要求1所述调节方法,其特征在于:3. The adjustment method according to claim 1, wherein: 步骤(3)在整个培养过程中,培养体系中除氮营养之外其他各营养元素包括磷、硫、铁、铜、钼、锌、硅、锰、钴以及微生物、生物素;Step (3) During the whole culture process, in addition to nitrogen nutrition, other nutrient elements in the culture system include phosphorus, sulfur, iron, copper, molybdenum, zinc, silicon, manganese, cobalt, as well as microorganisms and biotin; 若以光暗循环模式进行光照,则取样时间应选在光照至少1h后。If the light is illuminated in a light-dark cycle mode, the sampling time should be selected at least 1 hour after the light is illuminated. 4.根据权利要求1所述调节方法,其特征在于:4. The adjustment method according to claim 1, wherein: 步骤(4)单位体积或质量培养体系氮营养元素的补加量根据单位培养体系需要获得的细胞干重增加量A与设定NSI水平所对应的胞内氮含量B确定,即为A*B;如培养需要获得的细胞干重增加量1.0 g/L,设定NSI对应的胞内氮含量为4.0 %wt,则需要补加的氮含量为(1.0×4.0 %) g/L,即为40 mg/L;Step (4) The supplementary amount of nitrogen nutrient elements per unit volume or mass culture system is determined according to the increase amount A of cell dry weight that needs to be obtained by the unit culture system and the intracellular nitrogen content B corresponding to the set NSI level, that is, A*B ; If the increase in dry weight of cells to be obtained for culture is 1.0 g/L, and the intracellular nitrogen content corresponding to NSI is set to be 4.0 %wt, the nitrogen content that needs to be supplemented is (1.0×4.0 %) g/L, which is 40 mg/L; 需要注意的是,需要获得的细胞干重增加量应符合微藻培养实际情况,不高于培养体系中微藻细胞达到稳定期时所能获得的最大细胞干重。It should be noted that the increase in dry cell weight to be obtained should be in line with the actual situation of microalgae culture, and not higher than the maximum dry cell weight that can be obtained when the microalgal cells in the culture system reach a stable phase. 5.根据权利要求1所述调节方法,其特征在于:其中N含量测定可使用元素分析仪或凯式定氮法测定,但不限定于上述方法;5. The adjustment method according to claim 1, characterized in that: wherein the N content can be measured using an elemental analyzer or Kjeldahl method, but is not limited to the above-mentioned method; 所述的微藻光合叶绿素荧光参数包括用于表征微藻光合系统II的最大光量子产率F v / F m 或用于表征微藻光合系统II的实际光量子产率ΔF/F m 叶绿素荧光参数,其中叶绿素荧光参数用Water-PAM叶绿素荧光仪(Walz, Germany)进行测定,但不限定于使用上述仪器。The microalgal photosynthetic chlorophyll fluorescence parameters include the maximum light quantum yield Fv / Fm used to characterize the microalgal photosynthetic system II or the actual light quantum yield ΔF/Fm ' chlorophyll fluorescence parameter used to characterize the microalgal photosynthetic system II , wherein the chlorophyll fluorescence parameter is measured with a Water-PAM chlorophyll fluorescence instrument (Walz, Germany), but is not limited to the use of the above-mentioned instrument. 6.根据权利要求1所述调节方法,其特征在于:所述培养体系的培养基内氮营养物质的加入方式包括以连续加入或分批间歇加入;6. The regulating method according to claim 1, wherein the method of adding nitrogen nutrients in the culture medium of the culture system comprises adding continuously or intermittently in batches; 此外,培养体系的培养基内除氮之外其他营养元素的加入需要控制的包括营养元素的加入量与营养元素的加入时间,确保培养过程中除氮营养之外其他各营养元素充足均可满足微藻细胞生长所需。In addition, the addition of other nutrient elements except nitrogen in the medium of the culture system needs to be controlled, including the addition amount of nutrient elements and the addition time of nutrient elements, to ensure that the nutrient elements other than nitrogen nutrition are sufficient during the cultivation process. Required for microalgal cell growth. 7.根据权利要求1所述调节方法,其特征在于:所述的微藻细胞所受氮胁迫的精确控制指通过叶绿素荧光参数的实时监控反馈调节氮的补加使细胞达到设定的NSI,即达到设定的氮胁迫程度。7. The adjustment method according to claim 1, wherein the precise control of nitrogen stress on the microalgae cells refers to adjusting the addition of nitrogen through real-time monitoring of chlorophyll fluorescence parameters to make the cells reach the set NSI, That is, the set nitrogen stress level is reached.
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