CN102559505A - Microalgae cultivation method and device for controlling nutritive salt replenishment on basis of pH feedback - Google Patents

Microalgae cultivation method and device for controlling nutritive salt replenishment on basis of pH feedback Download PDF

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CN102559505A
CN102559505A CN2012100172460A CN201210017246A CN102559505A CN 102559505 A CN102559505 A CN 102559505A CN 2012100172460 A CN2012100172460 A CN 2012100172460A CN 201210017246 A CN201210017246 A CN 201210017246A CN 102559505 A CN102559505 A CN 102559505A
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salt
culture system
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nutritive salt
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CN102559505B (en
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丛威
鲍亦璐
张东梅
温树梅
刘明
吴霞
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Abstract

The present invention relates to field of microalgae cultivation, in particular it relates to a kind of method and device for adding nutritive salt culture microalgae based on pH feedback control. The method adds CO2 by pH feedback, and the pH value of cultivating system is made to maintain setting value, measures the CO2 additional amount of cultivating system, and using the relationship between CO2 additional amount and nutritive salt consumption, controls adding for nutritive salt. The additive amount of nutritive salt stream liquid feeding is calculated in the method by public formula (I),
Figure DDA0000132262460000011
Wherein, conversion coefficient of the α between nutritive salt consumption and CO2 additional amount, Δ CO2 are the CO2 additional amount of cultivating system. It is compared with the traditional method, the nutrient concentration of cultivating system can be controlled and be fluctuated in smaller range by method of the invention, can be improved the utilization rate of nutritive salt in incubation, save the cost.

Description

A kind of method and device of adding the nutritive salt culturing microalgae based on the pH feedback control
Technical field
The present invention relates to little algae and cultivate the field, particularly, the present invention relates to a kind of method and device of adding the nutritive salt culturing microalgae based on the pH feedback control.
Background technology
Little algae can utilize CO 2, light, water and nutritive salt carries out organic substances such as photosynthesis synthetic protein, glucide, lipid and pigment, be widely used at food, feed, medicine, fine chemistry industry and dye field.In recent years, along with the exhaustion day by day of fossil energies such as oil, coal, the new energy technology that utilizes little algae to produce hydrogen, system oil, refining alkene has caused people's great attention.
In little algae was cultivated, the proportion that the nutritive salt cost accounts for raw materials cost was very big, takes suitable nutritive salt control strategy, can when improving the nutritive salt utilization ratio, improve the output of cell or purpose product, effectively reduced little algae and cultivated cost.In little algae culturing process, the height of nutritive salt (being mainly nitrogenous source and phosphorus source) concentration is the important factor that influences micro algae growth and cellular component.The too much nutritive salt of disposable adding can produce the growth of little algae and suppress and toxic action (Effects of increased atmospheric CO in the substratum 2And N supply on photosynthesis, growth and cell composition of the cyanobacterium Spirulina platensis (Arthrospira) .J.Appl.Phycol., 1998,10:461～469; Review of nitrogen and phosphorus metabolism in seagrasses.J.Exp.Mar.Biol.Ecol.2000,250:133～167); And some nutritive salt disappearance will limit the growth of little algae; Change the cellular component of little algae simultaneously; As algae nitrogenous source, phosphorus source lack accumulation (the Effect of nitrogen that can promote the total fat of frustule, total carbohydrates or Serlabo slightly for certain; Salt; And iron content in the growth medium and light intensity on lipid production by microalgae isolated from freshwater sources in Thailand.Bioresource Technol., 2011,102:3043～3040; Nutrient limitation as a strategy for increasing starch accumulation in microalgae.Appl.Energ., 2011,88:3331～3335).Therefore, in little algae culturing process, the nutrient concentration of control culture system is very necessary.
At present, little algae culture process is also more extensive, one slightly algae manufacturing enterprise adopted some automatic control equipments; Can realize the monitoring or the control of conventional parameters such as pH, temperature; But aspect the control of nutritive salt, more employings is to add nutritive salt by artificial experience in the production, and perhaps the nutrient concentration in the determined off-line substratum is added again; Complex operation, lag.
Simultaneously; Because the polytropy of little algae culturing process; Difference to some extent between cultivating batch; And nitrogenous source (nitrate salt, urea and ammonium salt) commonly used, phosphorus source (phosphoric acid and hydrophosphate), magnesium etc. are difficult to the direct-on-line monitoring, and the actual change of nutrient concentration in the culturing process that is difficult to know for sure in real time is so adopt the concentration of direct feedback control nutritive salt to be difficult to realize.
Aspect the nutrient concentration control of little algae culture system; More existing flow feedings technology (as constant speed stream add, index stream adds, variable flow adds etc.) (Effects of carbon dioxide feeding rate and light intensity on thefed-batch pulse-feeding cultivation of Spirulina platensis in helical photobioreactor.Biochem.Eng.J.; 2008,39 (2): 369～375; Continuous and pulse feeding of urea as a nitrogen source in fed-batch cultivation of Spirulina platensis.Aquacult.Eng.; 2004,31:237～245) be used for the nutrient concentration control of little algae culture system.Though these flow feeding technology can be controlled the concentration of the nutritive salt of culture system to a certain extent; Improve purpose product output; But these flow feeding technology all are to be the feed forward control feed supplement of foundation with the incubation time; Program according to prior setting is operated, and can not truly reflect the variation of nutrient concentration in the culturing process.
On the other hand, the growth of little algae will consume CO 2, in nutrient solution, add proper C O 2Be the effective means that improves culture density, and in little algae is cultivated, keep normal growth and the metabolism that certain pH value helps little algae.Too high or cross when low when the pH of little algae culture system value; Little algae can stop growing even dead (Influence of temperature and pH on biomass production and protein biosynthesis in a putative Spirulina sp.Bioresource Technol.; 2007,98:2207～2211).Because the pH value and the CO of little algae culture system 2Interpolation closely related, existing patent is added CO through the pH feedback 2(CN200410009360.4 CN200510126465.2), still can not realize the real-time monitoring and the interpolation of nutritive salt in the pH value regulation and control of realization culture system in the time of as the carbon source culturing microalgae.
Summary of the invention
The objective of the invention is to, overcome existing little algae cultivate in nutritive salt add the defective that has blindness, influence production control and yield poorly, add CO existing the feedback based on pH 2On the method basis of culturing microalgae, a kind of method of adding the nutritive salt culturing microalgae based on the pH feedback control is provided: add CO through the pH feedback 2The pH value of culture system is maintained near the certain value (set(ting)value), the CO of metering culture system 2Additional amount utilizes CO 2Adding of additional amount control nutritive salt, thus production technique simplified, realize culture system pH value, carbon source, nutritive salt control simultaneously, improve algae biomass or product (total fat, albumen, polysaccharide etc.) content.
A purpose more of the present invention is in order to overcome the problems referred to above a kind of device of adding the nutritive salt culturing microalgae based on the pH feedback control to be provided.
Add the method for nutritive salt culturing microalgae according to according to the invention based on the pH feedback control, this method is added CO through the pH feedback 2, the pH value of culture system is maintained near the set(ting)value, near the fluctuating range of pH value set(ting)value of general culture system can be ± 0.1～± 0.5, the CO of metering culture system 2Additional amount, and utilize CO 2Relation between additional amount and the nutritive salt consumption is controlled adding of nutritive salt.
As a kind of preferred implementation of the present invention, pass through the addition that formula (I) calculates nutrition salt flowage liquid feeding in the said method,
Nutrition salt flowage liquid feeding addition (L)=flow (L/min) * stream adds the time (min)
Figure BDA0000132262440000031
Wherein, α is nutritive salt consumption and CO 2Reduction factor between the additional amount, Δ CO 2CO for culture system 2Additional amount,
Preestablish culture system and intend the pH value of keeping (pH set(ting)value); This pH set(ting)value is in the optimum pH scope of micro algae growth usually; Different and different according to the algae kind, be 9.0-10.0 like the optimum pH scope of growth of spirulina platensis, the optimum pH scope of chlorella growth is 7.0-8.0.Add CO through the pH feedback control 2The pH value of culture system is maintained near the pH set(ting)value, the CO of metering culture system 2Additional amount.Preestablish culture system nutritive salt feed supplement CO at interval 2Additional amount (set(ting)value) is as the CO of culture system 2When additional amount reached set(ting)value, addition and the enforcement of adopting formula (I) to calculate nutrition salt flowage liquid feeding were added; Perhaps, preestablish culture system nutritive salt feed supplement pitch time, the CO of culture system in the metering plant interval 2Additional amount is according to the CO in pitch time 2Additional amount adopts formula (I) to calculate the addition of nutrition salt flowage liquid feeding and implements to add.
Add the method for nutritive salt culturing microalgae according to of the present invention based on the pH feedback control, adopt conventional cultural method that little algae is cultivated, measure the CO of culture system in little algae culturing process 2The relation curve of additional amount and other nutritive salt consumption obtains nutritive salt consumption and CO 2Reduction factor α between the additional amount.
Described predefined culture system nutritive salt feed supplement CO at interval 2Additional amount (Δ CO 2, mol) can be 0.001-1mol/m 2* culture area (m 2), or, 0.003-3mol/m 3* volume of culture (m 3); Described predefined nutritive salt feed supplement can be 0.1～24 hour pitch time, depends on the CO of algae kind, volume of culture, culture system 2The proportionlity of additional amount and nutritive salt consumption and the fluctuation range of the nutrient concentration that will control.
This culturing process proceeds to frustule concentration and reaches preset value, or the regular hour, or other can stop the culture condition appearance.
The pH set(ting)value of described culture system depend on cultivate the algae specific character, culture process requires and the measuring accuracy and the time of response of control instruments and pH transmitter, near the fluctuating range of pH value set(ting)value of culture system can be set at ± 0.1～± 0.5.
Nutritive salt in the described nutrition salt flowage liquid feeding comprises one or several in the algae culture desired nutritional salt such as nitrogenous source, phosphorus source, magnesium source, potassium source, source of iron, calcium source.Described nitrogenous source includes but not limited to one or more in nitrate salt, ammonium salt, the urea; Described phosphorus source includes but not limited to one or more in hydrophosphate, the phosphoric acid; Magnesium salts includes but not limited to sal epsom or magnesium chloride; Sylvite includes but not limited to vitriolate of tartar or Repone K; Molysite includes but not limited to the EDTA complex compound of ferrous sulfate; Calcium salt includes but not limited to calcium chloride or calcium sulfate.
The concentration of the nitrogenous source in the described nutrition salt flowage liquid feeding is 0.01～5mol/L, and the concentration in phosphorus source is 0.001～0.5mol/L, and the concentration of magnesium, potassium, iron, calcium salt is 0.0005～0.1mol/L.
Describedly add CO through the pH feedback control 2Near the implementation that the pH value of culture system is maintained the pH set(ting)value can be an on-off control, use this kind mode then preset control parameters CO is arranged 2Flow, pH value fluctuating range, time of lag etc.; Or, many grades of flow controls, use this mode then preset control parameters each grade CO is arranged 2Flow and corresponding pH value fluctuating range thereof; Or PID control, use this kind mode then parameter preset proportional (P), integration (I), derivative constant (D).
Being contemplated that of the method for adding the nutritive salt culturing microalgae based on the pH feedback control provided by the invention: little algae utilizes CO 2Carry out photosynthesis synthesis of organic substance matter as carbon source, and the pH value and the CO of culture system 2Concentration is interrelated, along with CO 2Consumption, the pH value of culture system constantly rises.Through adding CO to system 2, the CO that adds 2The CO that has consumed before having replenished 2Thereby, the pH value of culture system is maintained certain limit, in little algae culturing process, CO 2There is certain relation in the consumption of additional amount and other nutritive salt (nitrogenous source, phosphorus source etc.), through the CO of culture system in the metering culturing process 2Additional amount can estimate the consumption of other nutritive salt, adds nutritive salt according to the consumption of nutritive salt and can the nutrient concentration of culture system be controlled within the specific limits.
What the present invention proposed adds the method for nutritive salt culturing microalgae based on the pH feedback control; Comprise and use ordinary method to prepare substratum and kind liquid; Substratum is joined little algae incubator; Select for use according to algae kind characteristics, substratum characteristics or culturing purposes and to go out/degerming method, and implement to go out/degerming, add the algae kind then and carry out little algae and cultivate; According to the mixing solutions of the proportionlity preparation nutritive salt between various nutritive salt (nitrogen, phosphorus, potassium, the magnesium etc.) consumption, i.e. nutrition salt flowage liquid feeding; When culturing microalgae, metering CO 2Additional amount is according to the CO of culture system 2Proportionlity between additional amount and the nutritive salt consumption adds nutrition salt flowage liquid feeding to culture system, and the various nutritive salt that the compensation culture system consumes make it neither excessive, also do not constitute restriction.CO in the culturing process 2Proportionlity between additional amount and the various nutritive salt consumption and the proportionlity between the various nutritive salt consumption can adopt conventional batch formula to cultivate, batch feeding is cultivated or semicontinuous culture experiment obtains.
Little algae algae kind of the present invention comprises the tenaculat Habenaria (Spirulina platensis) of Cyanophyta; The chlorella of Chlorophyta (Chlorella sp.), grid algae (Scenedesmus sp.), Haematocoocus Pluvialls (Haematococcus Pluvialis), salt algae (Dunaliella sp.); The chrysophyceae of Chrysophyta (Isochrysis sphacrica); Bacillariophyta Phaeodactylum tricornutum (Phaeodactylum tricornutum), the Porphyridium cruentum of rhodophyta (Prophyridium Cruentum) etc.
Described substratum depends on the characteristic of cultivating the algae kind, can be the substratum that is fit to micro algae growth arbitrarily well known in the art, like Zarrouk substratum, BG-11 substratum, f/2 substratum, Provasoli substratum, BBM substratum etc.
The implementation of adding the method for nutritive salt culturing microalgae based on the pH feedback control that the present invention proposes is: in little algae culturing process, and along with the growth of frustule, CO in the culture system 2Reduce, the pH value raises, and nutrient concentration constantly reduces.In little algae culturing process, with the pH value of manual detection or online pH sensor detecting culture system, according to the pH value of culture system and the difference between the pH set(ting)value, artificial or by the pH sensor signal automatically feedback add CO 2The pH value of culture system is fluctuateed, labor measurement or near the pH set(ting)value by CO 2Flow metering mechanism measures the CO of culture system in the culturing process 2Additional amount is according to CO 2Additional amount obtains the nutritive salt consumption, adds the nutritive salt of equivalent then according to the nutritive salt consumption.Add nutritive salt and realize that through adding nutrition salt flowage liquid feeding the addition calculation formula of nutrition salt flowage liquid feeding is suc as formula (I).
The interpolation control of nutrition salt flowage liquid feeding can be taked one of following dual mode: preestablish culture system nutritive salt feed supplement CO at interval 2Additional amount (set(ting)value) continues metering (with manual work or use CO 2Flow metering mechanism) CO of culture system 2Additional amount is as the CO of culture system 2When additional amount reaches set(ting)value, adopt formula (I) to calculate the addition of nutrition salt flowage liquid feeding, start the interpolation of implementing one time of nutrition salt flowage liquid feeding, simultaneously with the CO of culture system 2After this additional amount zero clearing measures the CO of culture system again 2Additional amount; Or, preestablishing nutritive salt feed supplement pitch time of culture system, regularly metering is (with manual work or use CO 2Flow metering mechanism) CO of culture system in pitch time 2Additional amount is according to the CO that records 2Additional amount adopts formula (I) to calculate the addition of nutrition salt flowage liquid feeding and implements the interpolation of nutrition salt flowage liquid feeding, simultaneously with the CO of culture system 2After this additional amount zero clearing measures the CO of culture system again 2Additional amount.
The interpolation of nutrition salt flowage liquid feeding can be added according to required addition by manual work, also can quantitatively be added by member driving execution mechanism (adding pump like stream).Wherein, by member driving execution mechanism (adding pump) like stream quantitatively interpolation following mode can be arranged: the stream rate of acceleration that stream adds pump is set in (1), adds the time according to the addition of nutrition salt flowage liquid feeding by formula (I) calculated flow and controls the addition of nutrition salt flowage liquid feeding; (2) set the opening time that stream adds pump, the stream rate of acceleration that adds pump by formula (I) calculated flow according to the addition of nutrition salt flowage liquid feeding is controlled the addition of nutrition salt flowage liquid feeding; (3) use self can integrated flow pump, when the additional amount of pump reaches the addition of required nutrition salt flowage liquid feeding, stop automatically adding.
The present invention also provides a kind of and has added the device of nutritive salt culturing microalgae based on the pH feedback control, and said device comprises little algae incubator 5, nutrition salt flowage liquid feeding basin 6 and CO 2Basin 7, said device also comprises pH transmitter 1, CO 2 Flow metering mechanism 2, member 3 and topworks 4, wherein,
Said pH transmitter 1 and CO 2 Flow metering mechanism 2 all connects with member 3, and the signal output of member 3 connects with the signal input of topworks 4, said CO 2Basin 7 is through CO 2 Flow metering mechanism 2 is communicated with little algae incubator 5, and said nutrition salt flowage liquid feeding basin 6 is communicated with little algae incubator 5 through topworks 4;
Said member 3 detects the pH value of culture system through pH transmitter 1, and passes through CO 2The 2 control CO of flow metering mechanism 2Break-make or flow size, and obtain the CO of culture system 2Additional amount, and calculate the addition of nutrition salt flowage liquid feeding according to formula (I), implement to add through topworks 4 then;
Nutrition salt flowage liquid feeding addition (L)=flow (L/min) * stream adds the time (min)
Figure BDA0000132262440000061
As a kind of concrete implementation of such scheme, said pH transmitter 1 and CO 2Between flow metering mechanism 2 and the member 3 through cable or wireless connections, the pH value and the CO of the culture system that respectively pH transmitter 1 is obtained 2The CO that flow metering mechanism 2 obtains 2Flux values sends member 3 to;
The signal output of member 3 and CO 2The signal input of flow metering mechanism 2 and topworks 4 is through cable or wireless connections, CO 2The delivery port of basin 7 is through pipeline and CO 2The input aperture of flow metering mechanism 2 is communicated with, CO 2The delivery port of flow metering mechanism 2 is communicated with the input aperture of little algae incubator 5 through pipeline, and nutritive salt basin 6 is communicated with through pipeline with the input aperture of topworks 4, and the delivery port of topworks 4 is communicated with through pipeline with the input aperture of little algae incubator 5;
Member 3 is according to the pH value of the culture system of pH transmitter 1 transmission, to CO 2 Flow metering mechanism 2 sends instruction, control CO 2Break-make or flow size, CO 2The actual CO that flow metering mechanism 2 will measure 2Flow signal sends back member 3, the CO of member 3 accumulative total culture systems 2Additional amount; And calculate the addition of nutrition salt flowage liquid feeding according to formula (I); Member 3 sends instruction according to the addition of the nutrition salt flowage liquid feeding that calculates gained to topworks 4, and command adapted thereto is received by topworks 4 after nutrition salt flowage liquid feeding basin 6 carries out the interpolation of nutrition salt flowage liquid feeding.
As a kind of improvement of such scheme, described little algae incubator 5 can be open cultivation pool, like runway pond, circular shallow pond; Or the closed bioreactor, like duct type bioreactor, flat bioreactor and cylinder shape bioreactor etc.In open cultivation pool, add CO 2Mode can be for directly with CO 2Feed open cultivation pool or add CO through outside cultivation pool, setting up the gas-liquid crosspoint 2(like document Productivity, CO 2/ O 2Exchange and hydraulics in outdoor open high density microalgal (Chlorella sp.) photobioreactors operated in a Middle and Southern European climate.J.Appl.Phycol.; 2006; 18:811-826 is said), preferably adopt the described carbon compensator that is used for large-scale culturing micro-algae of patent CN200510126465.2 to add CO 2In the closed bioreactor, can add CO through gas distributor or other gas-liquid switch of reactor drum 2
The present invention is based on the pH feedback and add CO 2Make the pH value of culture system maintain certain value, according to CO 2The relation of additional amount and nutritive salt consumption is quantitatively added nutritive salt keeping the stable of nutrient concentration in the culturing process to culture system, thus realize microalgae mass cultivate in nutritive salt automatic control, add automatically.Compared with prior art, advantage of the present invention is:
1) utilizes pH signal feedback control CO 2Add and then realize adding automatically of nutritive salt; Realize the automatic adjusting of the pH value of culture system, inorganic carbon source, nutrient concentration; Cultivating for little algae provides culture environment secular, stable, that optimize, helps improving the output of frustule in the culturing process or product (total fat, albumen, polysaccharide etc.);
2) compare with traditional flow feeding method (constant speed feed supplement, index feed supplement and speed change feed supplement etc.), can the nutrient concentration of culture system be controlled at interior fluctuation, can improve the utilization ratio of nutritive salt in the culturing process, practice thrift and cultivate cost;
3) compare with the mode that the nutrient concentration feedback control nutritive salt that directly utilizes ion specific electrode to detect culture system adds; Can avoid in the culture system numerous zwitterions to the interference of ion specific electrode; Control error can be reduced, equipment cost can be reduced again.
4) simple to operate, it is convenient to implement, and is suitable for suitability for industrialized production.
Description of drawings
Fig. 1 is an apparatus structure synoptic diagram of adding the nutritive salt culturing microalgae based on the pH feedback control of the present invention.
The accompanying drawing sign
1, pH transmitter 2, CO 2 Flow metering mechanism 3, member
4, topworks 5, little algae incubator 6, nutrition salt flowage liquid feeding basin
7, CO 2Basin
Embodiment
Below in conjunction with accompanying drawing device of the present invention is further explained, as shown in Figure 1 according to an embodiment of the present invention, omitted valve, strainer and other utility appliance among the figure, comprising:
1, pH transmitter: detect the pH signal;
2, CO 2Flow metering mechanism: can detect CO 2Flow signal also sends member to, and can accept switch, analog or digital signal that member sends, carries out CO 2Regulating is as being CO 2The combination of mass flow controller, under meter and SV or other can be measured and the device of pilot-gas flow;
3, member: can receive pH transmitter and CO 2The analog or digital signal that flow metering mechanism sends, and give CO 2Flow metering mechanism and topworks send the instrument or the computingmachine of switch, analog or digital steering order;
4, topworks: can carry out the instruction, adjusting of member 3 and the addition of metering nutrition salt flowage liquid feeding, as can or regulating volume pump, peristaltic pump or other fluid delivery system of aperture continuously for two, multidigit;
5, little algae incubator: comprise closed bioreactor or open cultivation pool, stirring that is used for mixed-culture medium or breather and the illumination apparatus or the natural light of source of artificial light are provided.
6, nutrition salt flowage liquid feeding basin;
7, CO 2Basin.
PH transmitter 1 and CO in the device of the present invention 2Pass through cable or wireless connections between flow metering mechanism 2 and the member 3, with the pH measured value and the CO of pH transmitter 1 acquisition 2The CO that flow metering mechanism 2 obtains 2The flow measurement value sends member 3 to; The signal input of member 3 signal outputs and topworks 4 executes instruction to transmit through cable or wireless connections; Nutritive salt basin 6 is communicated with through pipeline with the input aperture of topworks 4; The delivery port of topworks 4 is communicated with through pipeline with little algae incubator 5 input apertures, and valve, strainer and under meter further are housed on the connecting tube.
Said device comprises little algae incubator 5, nutrition salt flowage liquid feeding basin 6 and CO 2Basin 7, said device also comprises pH transmitter 1, CO 2 Flow metering mechanism 2, member 3 and topworks 4, wherein,
Through cable or wireless connections, the pH value of the culture system that pH transmitter 1 is obtained sends member 3 between said pH transmitter 1 and the member 3;
CO 2Pass through cable or wireless connections between flow metering mechanism 2 and the member 3, with CO 2The CO that flow metering mechanism 2 obtains 2Flux values sends member 3 to;
The signal output of member 3 and CO 2The signal input of the signal input of flow metering mechanism 2 and topworks 4 is through cable or wireless connections; Nutrition salt flowage liquid feeding basin 6 is communicated with through pipeline with the input aperture of topworks 4, and the delivery port of topworks 4 is communicated with through pipeline with the input aperture of little algae incubator 5;
Member 3 is according to the pH value of the culture system of pH transmitter 1 transmission, to CO 2 Flow metering mechanism 2 sends instruction, control CO 2Break-make or flow size, CO 2The actual CO that flow metering mechanism 2 will measure 2Flow signal transfers to member 3, the CO of member 3 accumulative total culture systems 2Additional amount; And calculate the addition of nutrition salt flowage liquid feeding according to formula (I); Member 3 sends instruction according to the addition of the nutrition salt flowage liquid feeding that calculates gained to topworks 4, and command adapted thereto is received by topworks 4 after nutritive salt basin 6 carries out nutrition salt flowage liquid feeding
Comparative example 1
Little algae algae kind of using is carried out constant speed pulse batch feeding with above-mentioned algae kind and is cultivated as spirulina plalensis (Spirulina platensis, 439, typical case's culture collection council of Chinese Academy of Sciences algae kind storehouse).
Consisting of of substratum: sodium hydrogencarbonate 5g/L, ammonium sulfate 0.15g/L, sodium-chlor 0.3g/L, Repone K 0.3g/L, sal epsom 0.06g/L, potassium hydrogenphosphate 0.15g/L, Presfersul 0.003g/L, EDTA disodium 0.024g/L, calcium chloride 0.012g/L.Used nutrition salt flowage liquid feeding is the ammonium sulfate solution of 0.75mol/L.
Little algae incubator adopts 1000m 2Open runway pond, nutrient solution liquid layer thickness are 15cm.
Above-mentioned substratum is without sterilization, and directly the preparation back adds the algae kind in the runway pond, and initial frustule concentration is 0.5g/L, and temperature is outdoor temperature (25～35 ℃), and intensity of illumination is outdoor natural lighting (575～1200 μ mol/m 2.s), adopt the impeller whipping appts to drive the mobile mixing of nutrient solution, the nutrient solution flow velocity is 15m/min.In the culturing process, adopt pH feed back control system feedback to add CO 2Make the pH value of culture system maintain 9.8 ± 0.1, add nutrition salt flowage liquid feeding every day once, add 100L at every turn, add 6 days continuously, the fluctuation range of the ammonium sulfate concentrations of culture system is 0.03～2.3mmol/L.Cultivate termination during to 7 days, the biomass concentration of frustule reaches 1.08g/L, and average yield per unit reaches 12.43g/m 2D, the utilization ratio of ammonium sulfate is 58.9%, and the utilization ratio of potassium hydrogenphosphate is 64.8%, and the protein of the tenaculat Habenaria of acquisition, polysaccharide, total fat and chlorophyllous contents on dry basis are respectively 62.31%, 12.87%, 4.74% and 10.87mg/g.
Embodiment 1
Little algae algae kind of using is spirulina plalensis (Spirulina platensis, 439, typical case's culture collection council of Chinese Academy of Sciences algae kind storehouse), carries out adding based on the pH feedback control batch feeding cultivation of nutritive salt with above-mentioned algae kind.
The composition of substratum and nutrition salt flowage liquid feeding is with comparative example 1.
Little algae incubator adopts 1000m 2Open runway pond adopts the impeller whipping appts to drive the mobile mixing of nutrient solution, and the nutrient solution liquid layer thickness is 15cm.The nutritive salt gear is referring to Fig. 1, and wherein, the pH transmitter is commercially available online pH transmitter (405-DPAS-SC-S8, a plum Teller-Tuo benefit Instr Ltd.), CO 2Flow metering mechanism is commercially available CO 2Mass flow controller (D07-11C, Beijing Qixing Huachuang Electronics Co., Ltd), member is an a.c..In culturing process, adopt the on-off control mode to feed CO 2The pH value of culture system is maintained near the pH set(ting)value, promptly on the pH value of culture system reaches the pH value of setting, prescribe a time limit CO 2Mass flow controller leaves, according to the CO that sets 2Flow is added CO 2And metering CO 2Additional amount, the pH value that drops to setting when the pH of culture system value is prescribed a time limit CO down 2Mass flow controller closes.CO when actual culture system 2Additional amount surpasses the CO that sets 2During additional amount, this a.c. is with the CO of plug-in according to actual culture system 2Additional amount calculates nutrition salt flowage liquid feeding addition according to formula (I), and driving execution mechanism, and topworks is multidigit adjustable diaphragm formula volume pump (GM80, the high pump industry of power).
Above-mentioned substratum is without sterilization, and directly the preparation back adds the algae kind in the runway pond, and initial frustule concentration is 0.5g/L, temperature natural (22～37 ℃), and illumination is natural light, intensity of illumination is 575～1200 μ mol/m 2.s, the nutrient solution flow velocity is 15m/min.In the culturing process, the pH value with culture system on the instrument of member is set in 9.8 ± 0.1, CO 2Flow set is 10L/min (CO 2Mass flow controller leaves) or 0 (CO 2Mass flow controller closes), feed supplement CO at interval 2Additional amount (Δ CO 2) to be set at 100mol (be 0.1mol/m 2* 1000m 2=100mol), the flow set of volume pump is 25L/min (pump leaves) or 0L/min (pump pass).When the pH of culture system value reaches 9.9, CO 2Mass flow controller is opened automatically, feeds CO 2And carry out CO 2The metering of additional amount, when the pH of culture system value drops to 9.7, CO 2Mass flow controller cuts out automatically, stops CO 2The metering of additional amount; CO when actual culture system 2When additional amount reached 100mol, member calculated nutrition salt flowage liquid feeding addition according to formula (I), wherein, calculates used parameter alpha=0.103mol ammonium sulfate/mol CO 2, the consumption and the CO of the ammonium sulfate of cultivating by the constant speed pulse batch feeding of comparative example 1 2The ratio of additional amount obtains.The computation process of parameter alpha is: the ammonium sulfate total addition level is 81.6kg (being 618mol) in the culturing process, and the ammonium sulfate total amount of cultivating culture system when stopping is 13.3kg (101mol), and the consumption that can calculate ammonium sulfate thus is 517mol, and passes through CO 2The mass flow controller metering obtains the CO in the culturing process 2Additional amount is 220.7kg (being 5016mol), and α is ammonium sulfate consumption (517mol) and CO 2The ratio of additional amount (5016mol), then α=0.103mol ammonium sulfate/mol CO 2Member calculates according to formula (I) and opens the stream that volume pump carries out nutrition salt flowage liquid feeding behind the nutrition salt flowage liquid feeding addition automatically and add; Add up nutrition salt flowage liquid feeding addition simultaneously; When the accumulative total addition reached required nutrition salt flowage liquid feeding addition, volume pump was closed automatically.When volume pump is opened, the CO in the member 2The additional amount zero clearing.Along with the growth of frustule, CO in the culture system 2Reduce, the pH value raises, and constantly carries out the pH feedback and adds CO 2And metering CO 2The operation of additional amount, the CO of a moment culture system instantly 2When additional amount reached 100mol, member repeated aforesaid operations, adds the stream that stops nutrition salt flowage liquid feeding after 6 days and adds.In the culturing process, the ammonium sulfate concentrations of culture system maintains 0.3～11mmol/L.Cultivate termination in the time of 7 days, the biomass concentration of frustule reaches 1.15g/L, and yield per unit reaches 13.93g/m 2D, the utilization ratio of ammonium sulfate is 84.7%.The protein of the tenaculat Habenaria that obtains, polysaccharide, total fat and chlorophyllous contents on dry basis are respectively 63.12%, 10.31%, 5.11% and 11.56mg/g.Compare with comparative example 1, yield per unit has improved 12.1.0%, and the ammonium sulfate utilization ratio has improved 43.8%.
Comparative example 2
Little algae algae kind of using is carried out batch culture as spirulina plalensis (Spirulina platensis, 439, typical case's culture collection council of Chinese Academy of Sciences algae kind storehouse) with above-mentioned algae kind.
Sodium hydrogencarbonate 8.4g/L, SODIUMNITRATE 1.25g/L, phosphoric acid 0.1g/L, sodium-chlor 1.0g/L, Repone K 1.0g/L, sal epsom 0.1g/L, Presfersul 0.01g/L, EDTA disodium 0.08g/L, calcium chloride 0.04g/L.
Little algae incubator adopts 314m 2Circular shallow pond adopts whipping appts to mix.The nutrient solution liquid layer thickness is 10cm.The shallow pond of circle layer directly adds above-mentioned substratum and algae kind, and initial frustule concentration is 0.15g/L, temperature natural (22～37 ℃), and illumination is natural light, intensity of illumination is 575～1200 μ mol/m 2.s.Cultivate initially, the sodium hydroxide of employing 10mol/L is 9.8 with the pH regulator of culture system.In the culturing process, the pH value of labor measurement culture system, and add CO according to the pH value manual feedback of culture system 2The pH value of culture system is maintained 9.8 ± 0.2.Cultivate termination in the time of 7 days, the biomass concentration of frustule reaches 0.79g/L, and average yield per unit reaches 9.14g/m 2D, the utilization ratio of SODIUMNITRATE is 50.3%, the utilization ratio of phosphoric acid is 59.1%.
Embodiment 2
Little algae of using is spirulina plalensis (Spirulina platensis, 439, typical case's culture collection council of Chinese Academy of Sciences algae kind storehouse).Carry out adding the batch feeding cultivation of nutritive salt based on the pH feedback control with above-mentioned algae kind.
Consisting of of substratum: sodium hydrogencarbonate 8.4g/L, SODIUMNITRATE 0.5g/L, phosphoric acid 0.1g/L, sodium-chlor 1.0g/L, Repone K 1.0g/L, sal epsom 0.1g/L, Presfersul 0.01g/L, EDTA disodium 0.08g/L, calcium chloride 0.04g/L.Nutrition salt flowage liquid feeding is the mixed aqueous solution of SODIUMNITRATE, phosphoric acid and sal epsom; Wherein the concentration of SODIUMNITRATE is 5mol/L; Concentration of phosphoric acid is 0.216mol/L, and the concentration of sal epsom is 0.017mol/L (the concentration ratio of SODIUMNITRATE, phosphoric acid, sal epsom is confirmed by the ratio that the three in the batch culture of comparative example 2 consumes).
Little algae incubator adopts 314m 2Circular shallow pond adopts whipping appts to mix.The nutrient solution liquid layer thickness is 10cm.The shallow pond of circle layer directly adds above-mentioned substratum and algae kind, and initial frustule concentration is 0.15g/L, temperature natural (22～37 ℃), and illumination is natural light, intensity of illumination is 575～1200 μ mol/m 2.s, will cultivate the pH of initial incubation system and be adjusted to 9.8 through adding 10mol/L sodium hydroxide.In the culturing process, the pH value of labor measurement culture system is also added CO according to the pH value of culture system feedback 2, the pH value stabilization that makes culture system is 9.8 ± 0.2; Human metering CO 2Additional amount, every day, 18:00 was regularly according to the CO of culture system 2Additional amount calculates the addition of nutrition salt flowage liquid feeding according to formula (I), and (addition of nutrition salt flowage liquid feeding is that benchmark calculates with SODIUMNITRATE, calculates used parameter alpha=0.182mol SODIUMNITRATE/mol CO 2, by the SODIUMNITRATE consumption and the CO of the batch culture of comparative example 2 2The ratio of additional amount obtains), and manual work quantitatively adds nutrition salt flowage liquid feeding, adds the stream that stops nutrition salt flowage liquid feeding after 6 days and adds.In the culturing process, the sodium nitrate concentration of culture system maintains 4.3～6.9mmol/L, and phosphoric acid concentration maintains 0.7～1.2mmol/L, and magnesium sulfate concentration maintains 0.004～0.006mol/L.Cultivate termination in the time of 7 days, the biomass concentration of frustule reaches 0.83g/L, and yield per unit reaches 9.71g/m 2D, the utilization ratio of SODIUMNITRATE can reach 93.6%, and the utilization ratio of phosphoric acid can reach 95.1%.Compare with comparative example 2, yield per unit has improved 6.2%, and the SODIUMNITRATE utilization ratio has improved 86.1%, and the utilization ratio of phosphoric acid has improved 60.9%.
Comparative example 3
Little algae algae kind of using is carried out batch culture as chlorella (Chlorella sp., 1298, typical case's culture collection council of Chinese Academy of Sciences algae kind storehouse) with above-mentioned algae kind.
Consisting of of substratum: SODIUMNITRATE 1.5g/L, potassium hydrogenphosphate 0.04g/L, bitter salt 0.075g/L, two hydration calcium chloride 0.02g/L, yellow soda ash 0.02g/L, Hydrocerol A 0.006g/L, ferric ammonium citrate 0.006g/L, EDTA disodium 0.001g/L.
Little algae incubator adopts the duct type bioreactor, and pipe diameter is 8cm, and length is 200m, and actual volume of culture is 1m 3Reactor drum adds above-mentioned substratum and algae kind after adding the hypochlorous acid sterilizing and washing, and initial frustule concentration is 0.2g/L, and temperature is controlled to be 22-30 ℃, and illumination is natural light, and intensity of illumination is 575～1200 μ mol/m 2.s, the nutrient solution flow velocity is 12m/min.Adopt pH feed back control system feedback to add CO 2Make the pH value of culture system maintain 7.0 ± 0.5.After the cultured continuously 12 days, the biomass concentration of frustule reaches 1.42g/L, and average unit volume productive rate is 101.7mg/L.d, and the butt total lipid content that obtains chlorella is 15.3%, and total fat productive rate is 15.6mg/L.d.
Embodiment 3
Little algae algae kind of using is chlorella (Chlorella sp., 1298, typical case's culture collection council of Chinese Academy of Sciences algae kind storehouse), carries out adding based on the pH feedback control batch feeding cultivation of nutritive salt with above-mentioned algae kind.
Consisting of of substratum: SODIUMNITRATE 0.1g/L, potassium hydrogenphosphate 0.04g/L, bitter salt 0.075g/L, two hydration calcium chloride 0.02g/L, yellow soda ash 0.02g/L, Hydrocerol A 0.006g/L, ferric ammonium citrate 0.006g/L, EDTA disodium 0.001g/L.Nutrition salt flowage liquid feeding 1 is a 0.25mol/L SODIUMNITRATE.
Little algae incubator adopts the duct type bioreactor, and pipe diameter is 8cm, and length is 200m, and actual volume of culture is 1m 3The nutritive salt system is referring to Fig. 1, and wherein the pH transmitter is commercially available online pH transmitter (GKF-4-a, Shen, Shanghai Dong Shenghuachuanganqichang).CO 2Flow metering mechanism is the combination of sales gas mass flowmeter (D07-7BM, Beijing Qixing Huachuang Electronics Co., Ltd) and solenoid valve (DT2B, Beijing Qixing Huachuang Electronics Co., Ltd), and member is a computingmachine.In culturing process, adopt the on-off control mode to feed CO 2The pH value of culture system is maintained near the pH set(ting)value.CO when actual culture system 2Additional amount surpasses the CO that sets 2During additional amount, this a.c. with plug-in according to actual CO 2Additional amount calculates the flow of nutrition salt flowage liquid feeding according to formula (I), and driving execution mechanism, and topworks is the adjustable peristaltic pump of multidigit (BT300-2J, a Baoding LanGe constant flow pump Co., Ltd).
Bioreactor adds above-mentioned substratum and algae kind after adding the hypochlorous acid sterilizing and washing, and initial frustule concentration is 0.2g/L, and temperature is controlled at 22-30 ℃, and intensity of illumination is 575～1200 μ mol/m 2.s, the nutrient solution flow velocity is 12m/min.In the culturing process, the pH value with culture system on the computingmachine of member is set in 7.0 ± 0.3, CO 2Flow set is 0.5L/min (CO 2Mass flow controller leaves) or 0 (CO 2Mass flow controller closes), feed supplement CO at interval 2Additional amount (Δ CO 2) to be set at 5mol (be 5mol/m 3* 1m 3=5mol), the opening time of peristaltic pump is set at 4min.Cultivate initially, when the pH of culture system value reaches 7.3, CO 2Mass flow controller is opened automatically, feeds CO 2And carry out CO 2The metering of additional amount, when the pH of culture system value drops to 6.7, CO 2Mass flow controller cuts out automatically, stops CO 2The metering of additional amount.CO when actual culture system 2When additional amount reached 5mol, the flow that member calculates nutrition salt flowage liquid feeding according to formula (I) (calculated used parameter alpha=0.093mol SODIUMNITRATE/mol CO 2, by the SODIUMNITRATE consumption and the CO of the batch culture of comparative example 3 2The ratio of additional amount obtains), and open the stream that peristaltic pump carries out nutrition salt flowage liquid feeding automatically and add, add up stream simultaneously and add the time, when accumulative total stream adds the time when reaching 4min, peristaltic pump is closed automatically.When peristaltic pump is opened, the CO in the member 2The additional amount zero clearing.Along with the growth of frustule, CO in the culture system 2Reduce, the pH value raises, and constantly carries out the pH feedback and adds CO 2And metering CO 2The operation of additional amount, the CO of a moment culture system instantly 2When additional amount reached 5mol, member repeated aforesaid operations, adds the stream that stops nutrition salt flowage liquid feeding after 9 days and adds, and the sodium nitrate concentration of culture system maintains 0.5～1.4mmol/L.Cultivate termination in the time of 12 days, the biomass concentration of frustule reaches 1.75g/L, and unit volume output reaches 129.2mg/Ld, and the utilization ratio of SODIUMNITRATE is 91.9%, and the butt total lipid content of the chlorella of acquisition is 20.1%, and total fat productive rate is 25.97mg/L.d.Compare with comparative example 3, chlorella unit volume productive rate has improved 27.0%, and total lipid content improves 31.4%, and total fat productive rate improves 66.5%.
Embodiment 4
Algae kind, little algae incubator and culture condition are with embodiment 3.The nutritive salt system is referring to Fig. 1, and wherein the pH transmitter is commercially available online pH transmitter (GKF-4-a, Shen, Shanghai Dong Shenghuachuanganqichang), CO 2Flow metering mechanism is commercially available CO 2Mass flow controller (D07-7B, Beijing Qixing Huachuang Electronics Co., Ltd), member is a computingmachine.In culturing process, adopt the on-off control mode to feed CO 2The pH value of culture system is maintained near the pH set(ting)value.When actual nutritive salt feed supplement surpassed set(ting)value pitch time, computingmachine was according to actual CO 2Additional amount calculates nutrition salt flowage liquid feeding stream dosage with plug-in according to formula (I), and driving execution mechanism, and topworks is the adjustable peristaltic pump of multidigit (BT300-2J, a Baoding LanGe constant flow pump Co., Ltd; BT50-1J, Baoding LanGe constant flow pump Co., Ltd).
Consisting of of substratum: SODIUMNITRATE 0.75g/L, potassium hydrogenphosphate 0.004g/L, bitter salt 0.075g/L, two hydration calcium chloride 0.02g/L, yellow soda ash 0.02g/L, Hydrocerol A 0.006g/L, ferric ammonium citrate 0.006g/L, EDTA disodium 0.001g/L.Nutrition salt flowage liquid feeding stream liquid feeding is divided into two kinds: nutrition salt flowage liquid feeding 1 is the mixed aqueous solution of SODIUMNITRATE, potassium hydrogenphosphate; Wherein the concentration of SODIUMNITRATE is 0.63mol/L, and the concentration of potassium hydrogenphosphate is 0.02mol/L (the concentration ratio of SODIUMNITRATE, potassium hydrogenphosphate is confirmed by the ratio of both consumption in the batch culture of comparative example 3); Nutrition salt flowage liquid feeding 2 is the ferric ammonium citrate of 1mmol/L.
In the culturing process, the pH value with culture system on the computingmachine of member is set in 7.0 ± 0.3, CO 2Flow set is 0.5L/min (CO 2Mass flow controller leaves) or 0 (CO 2Mass flow controller closes), the nutritive salt feed supplement is set at 4h pitch time, and the stream of peristaltic pump 1 control nutrition salt flowage liquid feeding 1 adds, and flow set is 0.2L/min (pump leaves) or 0L/min (pump pass); The stream of peristaltic pump 2 control nutrition salt flowage liquid feedings 2 adds, and flow set is 1mL/min (pump leaves) or 0L/min (pump pass).Cultivate initially, when the pH of culture system value reaches 7.3, CO 2Mass flow controller is opened automatically, feeds CO 2And carry out CO 2The metering of additional amount, when the pH of culture system value drops to 6.7, CO 2Mass flow controller cuts out automatically, stops CO 2The metering of additional amount.When cultivating 4 hours, detect the CO of culture system 2Additional amount, member is according to CO 2Additional amount calculates nutrition salt flowage liquid feeding addition according to formula (I), and (addition of nutrition salt flowage liquid feeding 1 is that benchmark calculates with SODIUMNITRATE, calculates used parameter alpha with embodiment 3, and nutrition salt flowage liquid feeding 2 calculates used parameter alpha=7.3 * 10 -5Mol ferric ammonium citrate/mol CO 2, by the ferric ammonium citrate consumption and the CO of the batch culture of comparative example 3 2The ratio of additional amount obtains), and open peristaltic pump 1 and 2 automatically, the stream that carries out nutrition salt flowage liquid feeding 1 and 2 respectively adds, addition totally respectively simultaneously, when addition totally reached required nutrition salt flowage liquid feeding addition, peristaltic pump was closed automatically.When peristaltic pump is opened, the CO in the member 2The additional amount zero clearing.Along with the growth of frustule, CO in the culture system 2Reduce, the pH value raises, and constantly carries out the pH feedback and adds CO 2And metering CO 2The operation of additional amount, when cultivating 8 hours, member repeats aforesaid operations, adds the stream that stops nutrition salt flowage liquid feeding after 9 days and adds, and the sodium nitrate concentration in the culture system maintains 0.6～1.4mmol/L, and potassium hydrogenphosphate concentration maintains 0.02～0.03mmol/L.Cultivate termination in the time of 12 days, the biomass concentration of frustule reaches 1.86g/L, and unit volume output reaches 138.3mg/Ld, and the utilization ratio of SODIUMNITRATE is 93.7%, and the butt total lipid content of the chlorella of acquisition is 27.2%, and total fat productive rate is 37.6mg/L.d.Compare with comparative example 3, chlorella unit volume productive rate has improved 36.0%, and total lipid content improves 77.8%, and total fat productive rate improves 141.2%.
Comparative example 4
Little algae algae kind of using is two type grid algaes (Scenedesmus dimorphus, 1266, typical case's culture collection council of Chinese Academy of Sciences algae kind storehouses), carries out batch culture with above-mentioned algae kind.
Substratum consists of: SODIUMNITRATE 1.5g/L, potassium hydrogenphosphate 0.04g/L, bitter salt 0.075g/L, two hydration calcium chloride 0.02g/L, yellow soda ash 0.02g/L, Hydrocerol A 0.006g/L, ferric ammonium citrate 0.006g/L, EDTA disodium 0.001g/L.
Little algae incubator adopts CN201110197276.X (a kind of airlift photobioreactor of realizing little algae sparkle effect) the described 300L cylinder shape of patent airlift photobioreactor; The high 1125mm of tank body, internal diameter 600mm, the high 700mm of guide shell; Internal diameter 400mm, actual volume of culture 200L.Bioreactor with the Youxiaolin washing and sterilizing after, add above-mentioned substratum, the inoculation mouth through reactor drum inserts the algae kind, initial frustule concentration is 0.3g/L, temperature is 25 ± 1 ℃, average intensity of illumination is 120 μ mol/m 2.s.Air flow is 20L/min, adopts pH feed back control system feedback to add CO 2The pH value of culture system is maintained 7.5 ± 0.5.Cultivate termination in the time of 15 days, the biomass concentration of frustule reaches 1.37g/L, and the unit volume productive rate is 71.3mg/L.d, and the butt total lipid content that obtains two type grid algaes is 14.8%, and total fat productive rate is 10.6mg/L.d.
Embodiment 5
Little algae algae kind of using is two type grid algaes (Scenedesmus dimorphus, 1266, typical case's culture collection council of Chinese Academy of Sciences algae kind storehouses), carries out adding based on the pH feedback control batch feeding cultivation of nutritive salt with above-mentioned algae kind.
Substratum consists of: SODIUMNITRATE 0.05g/L, potassium hydrogenphosphate 0.04g/L, bitter salt 0.075g/L, two hydration calcium chloride 0.02g/L, yellow soda ash 0.02g/L, Hydrocerol A 0.006g/L, ferric ammonium citrate 0.006g/L, EDTA disodium 0.001g/L.Used nutrition salt flowage liquid feeding is the 1mol/L sodium nitrate solution.
Little algae incubator adopts CN201110197276.X (a kind of airlift photobioreactor of realizing little algae sparkle effect) the described 300L cylinder shape of patent airlift photobioreactor; The high 1125mm of tank body, internal diameter 600mm, the high 700mm of guide shell; Internal diameter 400mm, actual volume of culture 200L.The nutritive salt automatic control device is referring to Fig. 1, wherein the pH transmitter be commercially available online pH transmitter (405-60, EUTTCH-JAMES), CO 2Flow metering mechanism is commercially available CO 2Mass flowmeter (MFC3030V, Dalian elegance and talent sensing Science and Technology Ltd.), member is a computingmachine.In culturing process, adopt many grades of flow control modes to feed CO 2The pH value of culture system is maintained near the pH set(ting)value, promptly cultivating initial CO of initial setting 2Flow reaches when the pH of culture system value on the pH value of setting in limited time, and member sends and instructs to CO 2Mass flow controller adopts high-grade CO 2Flow is set the pH value down in limited time when the pH of culture system value drops to, and member sends and instructs to CO 2Mass flow controller adopts low-grade CO 2Flow.The CO of metering culture system in this process 2Additional amount, when actual nutritive salt feed supplement surpassed set(ting)value pitch time, computingmachine was according to the CO of metering 2Additional amount calculates nutrition salt flowage liquid feeding addition with plug-in according to formula (I), and driving execution mechanism, and topworks is multidigit adjustable dosing pump (JCM 2-15.1, Zhejiang Province Airyokpo Pump Co., Ltd).
Bioreactor with the Youxiaolin washing and sterilizing after, add above-mentioned substratum, the inoculation mouth through reactor drum inserts the algae kind, initial frustule concentration is 0.3g/L, temperature is 25 ± 1 ℃, average intensity of illumination is 120 μ mol/m 2.s, Ventilation Rate is 20L/min, initial CO 2Flow is 0.5L/min.In the culturing process, the pH value with culture system on the computingmachine of member is set in 7.5 ± 0.5, low-grade CO 2Flow set is 0.5L/min, high-grade CO 2Flow set is 2.5L/min, and the nutritive salt feed supplement is set at 24h pitch time, and the flow set of volume pump is 0.1L/min (pump leaves) or 0L/min (pump pass).Cultivate initially CO 2Flow is 0.5L/min, and when the pH of culture system value reached 8.0, member sent and instructs to CO 2Mass flow controller is with CO 2Flow is adjusted into 2.5L/min, and when the pH of culture system value dropped to 7.0, member sent and instructs to CO 2Mass flow controller is with CO 2Flow is adjusted into 0.5L/min, in this process, constantly measures the CO of culture system 2Additional amount.When cultivating 24 hours, detect the CO of culture system 2Additional amount, member is according to the CO of culture system 2Additional amount calculates nutrition salt flowage liquid feeding addition according to formula (I) and (calculates used parameter alpha=0.086mol SODIUMNITRATE/mol CO 2, by the SODIUMNITRATE consumption and the CO of the batch culture of comparative example 4 2The ratio of additional amount obtains), and open volume pump automatically, the stream that carries out nutrition salt flowage liquid feeding adds, and adds up addition simultaneously, and when the accumulative total addition reached required nutrition salt flowage liquid feeding addition, volume pump was closed automatically.When volume pump is opened, the CO in the member 2The additional amount zero clearing.Along with the growth of frustule, CO in the culture system 2Reduce, the pH value raises, and constantly carries out the pH feedback and adds CO 2And metering CO 2The operation of additional amount, when cultivating 48 hours, member repeats aforesaid operations, adds the stream that stops nutrition salt flowage liquid feeding after 12 days and adds, and the sodium nitrate concentration of culture system maintains 0.3～1.0mmol/L.Cultivate termination in the time of 15 days, the biomass concentration of frustule reaches 1.56g/L, and unit volume output reaches 84.0mg/Ld, and the utilization ratio of SODIUMNITRATE is 94.3%, and the butt total lipid content of two type grid algaes of acquisition is 24.5%, and total fat productive rate is 20.6mg/L.d.Compare with comparative example 4, two type grid algae unit volume productive rates have improved 17.8%, and total lipid content improves 65.5%, and total fat productive rate improves 94.3%.
Comparative example 5
Little algae algae kind of using is Phaeodactylum tricornutum (Phaeodactylum tricornutum, FACHB-863, typical case's culture collection council of Chinese Academy of Sciences algae kind storehouse), the batch culture of carrying out constant speed pulse feed supplement with above-mentioned algae kind.
Substratum is with the f/2 substratum of seawater configuration, consists of: ammonium chloride 0.05g/L, sodium-chlor 24.5g/L, sodium sulfate 4.09g/L; Repone K 0.7g/L, sodium hydrogencarbonate 0.20g/L, Potassium Bromide 0.10g/L, boric acid 0.003g/L; Sodium Fluoride 0.003g/L, magnesium chloride hexahydrate 11.10g/L, calcium chloride 1.54g/L, Strontium dichloride hexahydrate 0.017g/L; SODIUM PHOSPHATE, MONOBASIC 0.005g/L, nine hydrated sodium silicate 0.03g/L, EDTA disodium 0.004g/L, six Ferric Chloride Hydrated 0.003g/L.
Little algae incubator adopts the flat gas lift bioreactor of 80L, and length * wide * height is of a size of 800mm * 100mm * 1000mm, actual volume of culture 70L.After bioreactor adds above-mentioned substratum, feed steam and make substratum boiling sterilization in 20 minutes, the algae kind is inserted through the inoculation mouth of reactor drum in the cooling back, and initial frustule concentration is 0.2g/L, and temperature is 20 ± 1 ℃, and average intensity of illumination is 100 μ mol/m 2.s, air flow is 10L/min.Adopt pH feed back control system feedback to add CO 2The pH value of culture system is maintained 7.7 ± 0.3.In the culturing process, add 0.05g/L ammonium chloride every day once, add 8 days continuously.Cultivate in the time of 10 days and stop; The biomass concentration of frustule reaches 0.77g/L; The unit volume productive rate is 57mg/L.d; The butt protein, glucide, the total lipid content that obtain Phaeodactylum tricornutum are respectively 38.4%, 14.9% and 18.2%, and timnodonic acid (EPA) accounts for 12.8% of total fatty acid content.
Embodiment 6
Little algae algae kind of using is Phaeodactylum tricornutum (Phaeodactylum tricornutum, FACHB-863, typical case's culture collection council of Chinese Academy of Sciences algae kind storehouse), carries out adding based on the pH feedback control batch feeding cultivation of nutritive salt with above-mentioned algae kind.
The composition of substratum is with comparative example 5, and nutrition salt flowage liquid feeding is the sodium nitrate solution of 0.1mol/L.
Little algae incubator adopts the flat gas lift bioreactor of 80L, and length * wide * height is of a size of 800 * 100 * 1000mm, actual volume of culture 70L.The nutritive salt automatic control device sees also Fig. 1, and wherein the pH transmitter is commercially available online pH transmitter (GP211, the new new sensor in a Shanghai ltd), CO 2Flow metering mechanism is commercially available CO 2Mass flow controller (SFC4000 contains and thinks sharp ltd), member is a computingmachine.In culturing process, adopt the PID mode to feed CO 2The pH value of culture system is maintained near the pH set(ting)value, promptly calculate pid value according to the pH value of the culture system of surveying and the difference of pH set(ting)value in the culturing process, regulate CO according to pid value 2The size of flow, and in this process the CO of automatic gauge culture system 2Additional amount is as the CO of culture system 2Additional amount surpasses the CO that sets 2During additional amount, computingmachine calls the CO of plug-in according to metering 2Additional amount is according to the flow of formula (I) calculating nutrition salt flowage liquid feeding, and driving execution mechanism, and topworks is the adjustable peristaltic pump of multidigit (BT50-1J, a Baoding LanGe constant flow pump Co., Ltd).
After bioreactor adds above-mentioned substratum, feed steam and make substratum boiling sterilization in 20 minutes, the algae kind is inserted through the inoculation mouth of reactor drum in the cooling back, and initial frustule concentration is 0.2g/L, and temperature is 20 ± 1 ℃, and average intensity of illumination is 100 μ mol/m 2.s, air flow is 10L/min.
In the culturing process, the pH value with culture system on the computingmachine of member is set in 7.5, initial CO 2Flow set is 1L/min, rate constant (P)=3, and integration constant (I)=1, derivative constant (D)=1, the nutritive salt feed supplement is set at 12h pitch time, and the stream of peristaltic pump adds the time and is set at 5min.In the culturing process, adopt PID control passing through CO 2Mass flow controller adjustment CO 2Flow makes the pH value of culture system maintain 7.7 ± 0.3, in this process, constantly measures the CO of culture system 2Additional amount.When cultivating 12 hours, detect the CO of culture system 2Additional amount, member is according to the CO of culture system 2The flow that additional amount calculates nutrition salt flowage liquid feeding according to formula (I) (calculates used parameter alpha=0.091mol ammonium chloride/mol CO 2, the ammonium chloride consumption and the CO that cultivate by the batch feeding of comparative example 5 2The ratio of additional amount obtains), and open peristaltic pump automatically, the stream that carries out nutrition salt flowage liquid feeding adds, and adds up stream simultaneously and adds the time, and when accumulative total stream adds the time when reaching 5min, peristaltic pump is closed automatically.When peristaltic pump is opened, the CO in the member 2The additional amount zero clearing.Along with the growth of frustule, CO in the culture system 2Reduce, the pH value raises, and constantly carries out the pH feedback and adds CO 2And metering CO 2The operation of additional amount, when cultivating 24 hours, member repeats aforesaid operations, adds the stream that stops nutrition salt flowage liquid feeding after 8 days and adds, and the sodium nitrate concentration of culture system maintains 0.4～1.0mmol/L.Cultivate termination in the time of 10 days, the biomass concentration of frustule reaches 0.86g/L, and unit volume output reaches 66.0mg/Ld.Butt albumen, glucide and the total lipid content of the Phaeodactylum tricornutum that obtains are respectively 35.5%, 8.5% and 25.3%, and total fat productive rate is 20.6mg/L.d, and timnodonic acid (EPA) accounts for 19.1% of total fatty acid content.Compare with comparative example 5, Phaeodactylum tricornutum unit volume productive rate has improved 13.6%, and total lipid content improves 39.0%, and timnodonic acid (EPA) accounts for total fatty acid content and improves 33.0%.
Comparative example 6
Little algae algae kind of using is carried out batch culture as Porphyridium cruentum (Prophyridium Cruentum, 981, typical case's culture collection council of Chinese Academy of Sciences algae kind storehouse) with above-mentioned algae kind.
Substratum is with the configuration of sterilization seawater, and it consists of: urea 0.5g/L, sodium hydrogencarbonate 3.0g/L, potassium primary phosphate 0.03g/L, VB125 μ g/L, VB10.9mg/L, Fe-EDTA 0.11mg/L.
Little algae incubator adopts 70L cylinder shape software airlift photobioreactor (Dalian Huixin Titanium Equipment Development Co., Ltd), and the high 1800mm of urceolus, internal diameter are 220mm, and the high 1500mm of guide shell, internal diameter are 160mm, actual volume of culture 65L.After bioreactor adds above-mentioned substratum, feed steam and make substratum boiling sterilization in 20 minutes, the algae kind is inserted through the inoculation mouth of reactor drum in the cooling back, and initial frustule concentration is 0.2g/L, and temperature is 29 ± 1 ℃, and average intensity of illumination is 100 μ mol/m 2.s, air flow is 6.5L/min.Adopt pH feed back control system feedback to add CO 2(CO 2Air flow be 250mL/min) make the pH value of culture system maintain 7.5 ± 0.5.Cultivate termination in the time of 10 days, the biomass concentration of frustule reaches 1.08g/L, and unit volume output reaches 88mg/Ld, and the B-phycoerythrin content of the Porphyridium cruentum of acquisition can reach 6.12%, and the concentration of the exocellular polysaccharide in the nutrient solution can reach 258mg/L.
Embodiment 7
Little algae algae kind of using is Porphyridium cruentum (Prophyridium Cruentum, 981, typical case's culture collection council of Chinese Academy of Sciences algae kind storehouse), carries out adding based on the pH feedback control batch feeding cultivation of nutritive salt with above-mentioned algae kind.
Substratum is with the configuration of sterilization seawater, and it consists of: urea 0.12g/L, sodium hydrogencarbonate 3.0g/L, potassium primary phosphate 0.03g/L, VB125 μ g/L, VB10.9mg/L, Fe-EDTA 0.11mg/L.Used nutritive salt feed supplement liquid is the urea soln of 0.1mol/L.
Little algae incubator adopts 70L cylinder shape software airlift photobioreactor (Dalian Huixin Titanium Equipment Development Co., Ltd), and the high 1800mm of urceolus, internal diameter are 220mm, and the high 1500mm of guide shell, internal diameter are 160mm, actual volume of culture 65L.The nutritive salt automatic control device is referring to Fig. 1, and wherein the pH transmitter is commercially available online pH transmitter ((405-DPAS-SC-S8, plum Teller-Tuo benefit Instr Ltd.), CO 2Flow metering mechanism is commercially available CO 2Mass flow controller (D07-11C, Beijing Qixing Huachuang Electronics Co., Ltd), member is an a.c..In culturing process, adopt the on-off control mode to feed CO 2The pH value of culture system is maintained near the pH set(ting)value.CO when actual culture system 2Additional amount surpasses the CO that sets 2During additional amount, this a.c. with plug-in according to actual CO 2Additional amount calculates nutrition salt flowage liquid feeding addition according to formula (I), and driving execution mechanism, and topworks is multidigit adjustable dosing pump (JCM 2-9.5, Zhejiang Province Airyokpo Pump Co., Ltd).
After bio-reactor added above-mentioned substratum, through the inoculation mouth access algae kind of reactor drum, initial frustule concentration was 0.2g/L, and temperature is 23 ± 1 ℃, and average intensity of illumination is 100 μ mol/m 2.s, air flow is 6.5L/min.
In the culturing process, the pH value with culture system on the instrument of member is set in 7.5 ± 0.5, CO 2Flow set is 0.65L/min (CO 2Mass flow controller leaves) or 0 (CO 2Mass flow controller closes), feed supplement CO at interval 2Additional amount (Δ CO 2) to be set at 0.65mol (be 10mol/m 3* 0.065m 3=0.65mol), the flow set of volume pump is 0.05L/min (pump leaves) or 0L/min (pump pass).Cultivate initially, when the pH of culture system value reaches 8.0, CO 2Mass flow controller is opened automatically, feeds CO 2And the CO of metering culture system 2Additional amount, when the pH of culture system value reaches 7.0, CO 2Mass flow controller cuts out automatically, stops CO 2The metering of additional amount.CO when culture system 2When additional amount reached 0.65mol, member calculated nutrition salt flowage liquid feeding addition according to formula (I) and (calculates used parameter alpha=0.045mol urea/mol CO 2The urea consumption and the CO that cultivate by the batch feeding of comparative example 6 2The ratio of additional amount obtains), and open the stream that volume pump carries out nutrition salt flowage liquid feeding automatically and add, add up addition simultaneously, when the accumulative total addition reached required nutrition salt flowage liquid feeding addition, volume pump was closed automatically.When volume pump is opened, the CO in the member 2The additional amount zero clearing.Along with the growth of frustule, CO in the culture system 2Reduce, the pH value raises, and constantly carries out the pH feedback and adds CO 2And metering CO 2The operation of additional amount, the CO of a moment culture system instantly 2When additional amount reached 0.2mol, member repeated aforesaid operations, adds the stream that stops nutrition salt flowage liquid feeding after 6 days and adds.In the culturing process, the urea concentration of culture system maintains 0.9～1.8mmol/L.Cultivate termination in the time of 10 days, the biomass concentration of frustule reaches 1.21g/L, and unit volume output reaches 101.0mg/Ld, and the B-phycobiliprotein phycoerythrin content of the Porphyridium cruentum of acquisition can reach 8.07%, and the concentration of the exocellular polysaccharide in the nutrient solution can reach 425mg/L.Compare with comparative example 6, frond unit volume productive rate has improved 14.8%, and B-phycobiliprotein content improves 31.9%, and the exocellular polysaccharide concentration in the nutrient solution improves 64.7%.
It should be noted last that above embodiment is only unrestricted in order to technical scheme of the present invention to be described.Although the present invention is specified with reference to embodiment; Those of ordinary skill in the art is to be understood that; Technical scheme of the present invention is made amendment or is equal to replacement, do not break away from the spirit and the scope of technical scheme of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.

Claims (11)

1. add the method for nutritive salt culturing microalgae based on the pH feedback control for one kind, this method is added CO through the pH feedback 2, make the pH value of culture system maintain set(ting)value, the CO of metering culture system 2Additional amount, and utilize CO 2Relation between additional amount and the nutritive salt consumption is controlled adding of nutritive salt.
2. according to claim 1ly add the method for nutritive salt culturing microalgae, it is characterized in that, calculate the addition of nutrition salt flowage liquid feeding in the said method through formula (I) based on the pH feedback control,
Figure FDA0000132262430000011
Wherein, α is nutritive salt consumption and CO 2Reduction factor between the additional amount, Δ CO 2CO for culture system 2Additional amount,
Preestablish culture system nutritive salt feed supplement CO at interval 2Additional amount is as the CO of culture system 2When additional amount reached set(ting)value, addition and the enforcement of adopting formula (I) to calculate nutrition salt flowage liquid feeding were added; Perhaps
Preestablish culture system nutritive salt feed supplement pitch time, the CO of culture system in the metering plant interval 2Additional amount is according to the CO in pitch time 2Additional amount adopts formula (I) to calculate the addition of nutrition salt flowage liquid feeding and implements to add.
3. according to claim 2ly add the method for nutritive salt culturing microalgae, it is characterized in that, adopt conventional cultural method that little algae is cultivated, measure the CO of culture system in little algae culturing process based on the pH feedback control 2The relation curve of additional amount and nutritive salt consumption obtains nutritive salt consumption and CO 2Reduction factor α between the additional amount.
4. according to claim 2ly add the method for nutritive salt culturing microalgae, it is characterized in that the said culture system nutritive salt feed supplement CO at interval that preestablishes based on the pH feedback control 2Additional amount is 0.001-1mol/m 2* culture area m 2Or 0.003-3mol/m 3* volume of culture m 3, or
Described predefined nutritive salt feed supplement pitch time is 0.1～24 hour.
5. the method for adding the nutritive salt culturing microalgae based on the pH feedback control according to claim 2; It is characterized in that; The Porphyridium cruentum of chrysophyceae, Bacillariophyta Phaeodactylum tricornutum or the rhodophyta of the tenaculat Habenaria that said little algae is a Cyanophyta, the chlorella of Chlorophyta, grid algae, Haematocoocus Pluvialls, salt algae, Chrysophyta
The growth medium of said little algae is Zarrouk substratum, BG-11 substratum, f/2 substratum, Provasoli substratum or BBM substratum;
Nutritive salt in the described nutrition salt flowage liquid feeding comprises one or several in nitrogenous source, phosphorus source, magnesium source, potassium source, source of iron and the calcium source.
6. according to claim 5ly add the method for nutritive salt culturing microalgae, it is characterized in that described nitrogenous source comprises one or more in nitrate salt, ammonium salt and the urea based on the pH feedback control;
Described phosphorus source comprises hydrophosphate and/or phosphoric acid; Magnesium salts comprises sal epsom and/or magnesium chloride; Sylvite comprises vitriolate of tartar and/or Repone K; Molysite comprises the EDTA complex compound of ferrous sulfate; Calcium salt comprises calcium chloride and/or calcium sulfate;
The concentration of nitrogenous source is 0.01～5mol/L in the described nutrition salt flowage liquid feeding, and the concentration in phosphorus source is 0.001～0.5mol/L, and the concentration of magnesium, potassium, iron, calcium salt is 0.0005～0.1mol/L.
7. realize that one of claim 1 to 6 is said and add the device of nutritive salt culturing microalgae method that said device comprises little algae incubator (5), nutrition salt flowage liquid feeding basin (6) and CO for one kind based on the pH feedback control 2Basin (7) is characterized in that, said device also comprises pH transmitter (1), CO 2Flow metering mechanism (2), member (3) and topworks (4), wherein,
Said pH transmitter (1) and CO 2Flow metering mechanism (2) all connects with member (3), and the signal output of member (3) connects said CO with the signal input of topworks (4) 2Basin (7) is through CO 2Flow metering mechanism (2) is communicated with little algae incubator (5), and said nutrition salt flowage liquid feeding basin (6) is communicated with little algae incubator (5) through topworks (4);
Said member (3) is through the pH value of pH transmitter (1) detection culture system, through CO 2The control CO of flow metering mechanism (2) 2Break-make or flow size, and obtain the CO of culture system 2Additional amount according to the addition of formula (I) calculating nutrition salt flowage liquid feeding, is implemented to add through topworks (4) then at last;
Figure FDA0000132262430000021
8. according to claim 7ly add the device of nutritive salt culturing microalgae, it is characterized in that said pH transmitter (1) and CO based on the pH feedback control 2Flow metering mechanism (2) connects with member (3), the pH value and the CO of the culture system that respectively pH transmitter (1) is obtained 2The CO that flow metering mechanism (2) obtains 2Flux values sends member (3) to;
The signal output of member (3) connects CO with the signal input of topworks (4) 2The delivery port and the CO of basin (7) 2The input aperture of flow metering mechanism (2) is communicated with, CO 2The delivery port of flow metering mechanism (2) is communicated with the input aperture of little algae incubator (5) through pipeline, and nutritive salt basin (6) is communicated with through pipeline with the input aperture of topworks (4), and the delivery port of topworks (4) is communicated with the input aperture of little algae incubator (5);
Member (3) is according to the pH value of the culture system of pH transmitter (1) transmission, to CO 2Flow metering mechanism (2) sends instruction, control CO 2Break-make or flow size, CO 2The actual CO that flow metering mechanism (2) will measure 2Flow signal sends back member (3), the CO of member (3) accumulative total culture system 2Additional amount; And calculate the addition of nutrition salt flowage liquid feeding according to formula (I); Member (3) sends instruction according to the addition of the nutrition salt flowage liquid feeding that calculates gained to topworks (4), and command adapted thereto is received by topworks (4) after nutrition salt flowage liquid feeding basin (6) carries out the interpolation of nutrition salt flowage liquid feeding.
9. describedly add the device of nutritive salt culturing microalgae based on the pH feedback control according to claim 7 or 8, it is characterized in that, said topworks (4) regulates and the fluid delivery system of the addition of metering nutrition salt flowage liquid feeding according to the instruction of member (3).
10. according to claim 9ly add the device of nutritive salt culturing microalgae based on the pH feedback control, it is characterized in that, said fluid delivery system is two, multidigit or regulates the volume pump or the peristaltic pump of aperture continuously.
11. the device of adding the nutritive salt culturing microalgae based on the pH feedback control according to claim 7; It is characterized in that; Said little algae incubator (5) is open cultivation pool or closed bioreactor, and wherein, open cultivation pool is runway pond or circular shallow pond; Said closed bioreactor is duct type bioreactor, flat bioreactor or cylinder shape bioreactor.
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CN114196546A (en) * 2022-02-16 2022-03-18 广东省科学院生态环境与土壤研究所 Application of DCMU (dendritic cell activator-responsive unit) in stabilizing microalgae polyculture growth pH (potential of hydrogen) or improving microalgae polyculture growth speed
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