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