CN105018349A - Microalgae cycling breeding technology - Google Patents
Microalgae cycling breeding technology Download PDFInfo
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- CN105018349A CN105018349A CN201510502753.7A CN201510502753A CN105018349A CN 105018349 A CN105018349 A CN 105018349A CN 201510502753 A CN201510502753 A CN 201510502753A CN 105018349 A CN105018349 A CN 105018349A
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Abstract
The invention discloses a microalgae cycling breeding technology. The technology comprises the steps of 1, adding ammonia to a culture solution where microalgae are cultured, so that flocculation of the microalgae is achieved by means of the ammonia; 2, separating microalgae floccules from the culture solution to obtain culture liquor, and then adding PO43- and Mg2+ to the culture liquor to enable NH4+ in the culture liquor to react with PO43- and Mg2+ to generate NH4MgPO4.6H2O sediment; 3, separating the NH4MgPO4.6H2O sediment from the culture liquor to obtain treating liquor, adding certain constituents to the treating liquor to form a new culture solution, and conducting cycling culture of the microalgae by means of the new culture solution; 4, repeating the steps 1, 2 and 3 in sequence to achieve cycling breeding of the microalgae. By the adoption of the method, cost is low, efficiency is high, and environment friendliness is realized. The technology is suitable for large-scale breeding of microalgae and harvesting of microalgae biomass.
Description
Technical field
The present invention relates to a kind of processing method of producing microalgae biomass, particularly relate to the cyclic culture technique of a kind of micro-algae.
Background technology
Along with the day by day exhausted and increasingly serious environmental problem of non-renewable fossil energy, the whole world faces global problems such as affecting energy dilemma, climate warming, food shortage, soil anxiety, environmental pollution widely.Thus, increasing physicism concentrates on the field such as exploitation, the biological restoration of environment, the exploitation of recovered material of new forms of energy.In the exploitation of new forms of energy and the biological restoration of environment, micro-algae becomes study hotspot material because possessing the feature of some uniquenesses.These features comprise: (1) micro-algae is extensively distributed in the waters such as ocean, lake, beach, of a great variety.Micro-algae directly utilizes the simple nutritive substance of the elements such as sunlight, carbonic acid gas and nitrogenous, phosphorus to grow fast, and in cell, synthesize a large amount of grease; (2) compare with oilseed plants such as soybean, rape and maniocas, the growth cycle of micro-algae is short, and oleaginousness is high, and grease productive rate is high, and unit surface oil offtake is hundreds of times of soybean, and per hectare can be produced several ten thousand per year and rise biofuel.Micro-algae can utilize the non-cheap water source such as underground water tilling the land and utilize seawater, Desert Area such as beach, saltings, desert, does not strive ground with farm crop, strives water; (3) micro-algae also can fix great amount of carbon dioxide in culturing process.According to calculating, often cultivate 1 ton of micro-algae, about ton carbonic acid gas can be consumed.Therefore, utilize micro-algae to manufacture biofuel and can reduce Carbon emission in a large number.In addition, micro-algae also can utilize the nutritive ingredients such as the nitrogen in waste water, phosphorus and purify various trade effluent and city domestic sewage in light autotrophy culturing process.Therefore, the purposes of micro-algae is very extensive, throughout industry and fields such as aquaculture, medicine manufacture, food-processing, environment protection, energy refinings.But a large amount of acquisitions of microalgae biomass are the maximum bottlenecks of both culturing microalgae, embody a concentrated reflection of the high-density culture of micro-algae and efficient, low cost and to gather link.Wherein, account for 20% ~ 30% of total cost based on centrifugal recovery process, and flocculation technique can significantly concentrate algae liquid, increase micro-algae particle diameter, thus reduce cost of gathering.Therefore, present invention applicant proposes the ammonia flocculation technique (patent No.: 201110390674.3) in invention early stage.Ammonia flocculation technique lures flocculation mechanism based on alkali, have flocculation efficiency high, do not introduce external source metal ion, do not affect the advantages such as subsequent product refining.In addition, Knuckey etc. also indicate and lure the algae powder of flocculation to be more suitable for aquaculture from alkali.However, this technology still Shortcomings: is the treatment process that ammonia nitrogen reclaims with collaborative ammonia still process method in the CO2 adopted, and efficiency is too low, energy consumption is excessive; Two is in follow-up cultivation, and the growth conditions of micro-algae is not good; Three is that the algae liquid secondary ammonia flocculation feasibility of gathering of follow-up cultivation was not verified.
The struvite precipitator method, namely add PO in ammonia nitrogen waste water
4 3-and Mg
2+, make it and NH
4 +form the double salt magnesium ammonium phosphate sediment (being commonly called as struvite, a kind of high-quality slow release fertilizer) being insoluble in water.
Summary of the invention
Therefore, the present invention intends with struvite precipitator method high efficiente callback ammonia nitrogen and improves the growth conditions of micro-algae in follow-up cultivation, thus series connection of the flocculation of the ammonia of ammonia flocculation-recovery ammonia-follow-up cultivation of micro-algae-again being gathered is integrated.
The object of this invention is to provide the cyclic culture technique of a kind of micro-algae, the present invention is by reclaiming the ammonia in the nutrient solution after the flocculation of micro-algae based on the struvite precipitator method, and utilize the substratum after recovery ammonia again to carry out the cultivation of micro-algae, and ammonia flucculation process can be reused carry out microalgae.Can solve after the flocculation of micro-algae ammonia the problem of recovery ammonia difficulty, and by follow-up cultivation and again ammonia flocculation gather and be connected into integral system, the significantly commercial viability of raising ammonia flocculation process.
For this reason, technical scheme provided by the invention is:
A cyclic culture technique for micro-algae, comprises the steps:
Step one, to have in the nutrient solution of micro-algae to cultivation and add ammonia, utilize ammonia by described micro-algae flocculation; Enter step 2 afterwards,
Step 2, the flocculation of micro-algae to be separated with nutrient solution, to obtain cultivation clear liquid, backward cultivation clear liquid in add PO
4 3-and Mg
2+, make the NH in cultivation clear liquid
4 +with PO
4 3-and Mg
2+reaction is to generate NH
4mgPO
46H
2o precipitates; Enter step 3 afterwards,
Step 3, by NH
4mgPO
46H
2o precipitation is separated with cultivation clear liquid, obtains processing clear liquid, backward process clear liquid in add certain component and form new nutrient solution, and recycle this new micro-algae of nutrient solution Cyclic culture; And,
The method of step 4, successively repeating step one, step 2 and step 3 is to realize the cyclic culture of micro-algae.
Preferably, in the cyclic culture technique of described micro-algae, in described step 2, in cultivation clear liquid, add Na
2hPO
412H
2o is to provide PO
4 3-, in cultivation clear liquid, add MgCl
26H
2o is to provide Mg
2+, and the molar ratio of N:P:Mg is 1:1.05:1.10.
It is further preferred that in the cyclic culture technique of described micro-algae, in described step 2, first in cultivation clear liquid, add Na
2hPO
412H
2o and MgCl
26H
2o, 100r/min stir 10min, afterwards natural subsidence 5h, then enter described step 3.
Preferably, in the cyclic culture technique of described micro-algae, in described step one, first there is the pH of the nutrient solution of micro-algae to be adjusted to 9.00 cultivation, then have in the nutrient solution of micro-algae to described cultivation and add ammonia, utilize ammonia to be flocculated by described micro-algae,
Described ammonia to be concentration be 12 ~ 28% ammoniacal liquor or ammonia.
It is further preferred that in the cyclic culture technique of described micro-algae, described ammoniacal liquor is added cultivate have in the nutrient solution of micro-algae to ammonia nitrogen final concentration be 59.85 ~ 159.60mmolL
– 1.
Preferably, in the cyclic culture technique of described micro-algae, in described step one, described nutrient solution is f/2 nutrient solution.
It is further preferred that in the cyclic culture technique of described micro-algae, in described step 3, described certain component comprises trace element and VITAMIN, and described trace element is identical with the usage ratio of VITAMIN with described f/2 nutrient solution medium trace element with the consumption of VITAMIN.
It is further preferred that in the cyclic culture technique of described micro-algae, in described step 3, described certain component also comprises PO
4 3-ion, described PO
4 3-pO in the consumption of ion and described f/2 nutrient solution
4 3-usage ratio identical.
It is further preferred that in the cyclic culture technique of described micro-algae, in described step 3, described certain component also comprises Ca
2+and Mg
2+ion, described Ca
2+and Mg
2+ca in the consumption of ion and described f/2 nutrient solution
2+and Mg
2+the usage ratio of ion is identical.
It is further preferred that in the cyclic culture technique of described micro-algae, in described step 3, described certain component also comprises NO
3 -, Ca
2+and Mg
2+ion, wherein, described NO
3 -, Ca
2+and Mg
2+ca in the consumption of ion and described f/2 nutrient solution
2+and Mg
2+the usage ratio of ion is identical.
The present invention at least comprises following beneficial effect:
The cyclic culture technique of the micro-algae of the present invention comprises: the flocculation of micro-algae ammonia gather after ammonia reclaiming process; The follow-up culture process of micro-algae after recovery ammonia; Ammonia flocculation process again.The flocculation of micro-algae ammonia gather after ammonia reclaiming process, be the mode of the high efficiente callback ammonia based on the struvite precipitator method.Substratum after recovery ammonia is conducive to the follow-up cultivation of micro-algae, and can reuse ammonia flucculation process and carry out microalgae.The present invention can solve the problem of recovery ammonia difficulty of ammonia flocculation, and by follow-up cultivation and again ammonia flocculate to gather and be connected into integral system.
The ammonia nitrogen that the struvite precipitator method can solve the flocculation of micro-algae ammonia reclaims problem, and the rate of recovery reaches 98.8%, comparatively invention in the early stage (patent No.: CO 201110390674.3)
2the collaborative ammonia still process method of neutralization is more effective, convenient.The struvite precipitator method that its treating processes more once celebrated the report such as the tinkling of pieces of jade etc., Jiang Jingdong etc., Li Jin page are advanced.First, the method for the report such as Zeng Qingling etc., Jiang Jingdong etc., Li Jin page is under external source sodium hydroxide maintains the condition of pH, reclaims chlorination ammonium type ammonia nitrogen in fresh water.And the present invention directly realizes high-efficient deamination in the high nitrogenous sea water medium of alkalescence, save the interpolation of external source sodium hydroxide.Secondly, the pH of solution is down to best cultivation pH, after the completion of reaction without the need to the CO of previous work
2neutralizing treatment just can directly cultivate micro-algae.Moreover struvite particle just can be gathered in the crops by simple filtration, still needs the problem of evaporation, crystalline sulfuric acid ammonium dilute solution after avoiding film exchange process deamination.Finally, the substratum after process, its phosphorus source and magnesium source are supplemented.On the one hand, substratum just can directly cultivate micro-algae without the need to adding nitrogen, phosphorus, magnesium source specially; On the other hand, the magnesium ion needed for ammonia flocculation is provided again.And be better than the frond growth perfonnance of control group and more balanced metabolome in follow-up cultivation and become micro-algae high-density culture, sewage disposal, carotenoid production etc. and provide new method and inspiration.Therefore, the struvite precipitator method effectively improve the follow-up cultivation of micro-algae ammonia flocculation, and have certain superiority.
The method low cost, efficient, environmental protection, saving artificial seawater resource, and significantly improve the commercial viability of ammonia flocculation process, be applicable to the micro-algae of large-scale cultivation and microalgae biomass.
The present invention is perfect further micro-algae ammonia flocculation technique, thus facilitate the application of ammonia flocculation on industrially scalable, there is great directive significance.
Part is embodied by explanation below by other advantage of the present invention, target and feature, part also will by invention of the present invention and practice by those skilled in the art is understood.
Accompanying drawing explanation
Fig. 1 is the schema of the cyclic culture technique of micro-algae of the present invention.
Fig. 2 (a) is the Mg of algae liquid under different ammonia concn in one of them embodiment of the present invention
2+, Ca
2+concentration.
The flocculating rate that Fig. 2 (b) is algae liquid under different ammonia concn in one of them embodiment of the present invention and pH.
Fig. 3 is the growth curve chart of N.oculata under Different Nutrition condition in one of them embodiment of the present invention.
Fig. 4 is the total lipid content figure of N.oculata under Different Nutrition composition condition in one of them embodiment of the present invention.
Fig. 5 is the protein content figure of N.oculata alkali extract under Different Nutrition composition condition in one of them embodiment of the present invention.
Fig. 6 is the pigment content figure of N.oculata acetone extract under Different Nutrition composition condition in one of them embodiment of the present invention.
Fig. 7 is that the ammonia flocculation of N.oculata in follow-up cultivation in one of them embodiment of the present invention is gathered figure.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, can implement according to this with reference to specification sheets word to make those skilled in the art.
Should be appreciated that used hereinly such as " to have ", other element one or more do not allotted in " comprising " and " comprising " term or the existence of its combination or interpolation.
The invention provides the cyclic culture technique of a kind of micro-algae, comprise the steps:
Step one, to have in the nutrient solution of micro-algae to cultivation and add ammonia, utilize ammonia by described micro-algae flocculation; Enter step 2 afterwards,
Step 2, the flocculation of micro-algae to be separated with nutrient solution, to obtain cultivation clear liquid, backward cultivation clear liquid in add PO
4 3-and Mg
2+, make the NH in cultivation clear liquid
4 +with PO
4 3-and Mg
2+reaction is to generate NH
4mgPO
46H
2o precipitates; Enter step 3 afterwards,
Step 3, by NH
4mgPO
46H
2o precipitation is separated with cultivation clear liquid, obtains processing clear liquid, backward process clear liquid in add certain component and form new nutrient solution, and recycle this new micro-algae of nutrient solution Cyclic culture; And,
The method of step 4, successively repeating step one, step 2 and step 3 is to realize the cyclic culture of micro-algae.
Route of the present invention as shown in Figure 1, namely after micro-algae ammonia flocculation is gathered, reclaims upper strata with the struvite precipitator method and cultivates the ammonia nitrogen of clear liquid and obtain struvite particle and process clear liquid simultaneously; Add proper nutrition component subsequently in process clear liquid, and access fresh seeds and carry out follow-up cultivation; By measuring growth indexes and analyzing frond metabolite composition, evaluate the quality of each trophic component mode; Finally, in collection period, the flocculation of secondary ammonia is carried out to algae liquid and gather, realize ammonia flocculation and gather and the repeatedly circulation of microdisk electrode.Method low cost of the present invention, efficient, environmental protection, saving artificial seawater resource, and significantly improve the commercial viability of ammonia flocculation process, be applicable to the micro-algae of large-scale cultivation and microalgae biomass.
In one of them embodiment of the present invention, as preferably, in described step 2, in cultivation clear liquid, add Na
2hPO
412H
2o is to provide PO
4 3-, in cultivation clear liquid, add MgCl
26H
2o is to provide Mg
2+, and the molar ratio of N:P:Mg is 1:1.05:1.10.
In such scheme, as preferably, in described step 2, first in cultivation clear liquid, add Na
2hPO
412H
2o and MgCl
26H
2o, 100r/min stir 10min, afterwards natural subsidence 5h, then enter described step 3.
In one of them embodiment of the present invention, as preferably, in described step one, first there is the pH of the nutrient solution of micro-algae to be adjusted to 9.00 cultivation, then have in the nutrient solution of micro-algae to described cultivation and add ammonia, utilize ammonia to be flocculated by described micro-algae,
Described ammonia to be concentration be 12 ~ 28% ammoniacal liquor or ammonia.
In such scheme, as preferably, described ammoniacal liquor is added cultivate have in the nutrient solution of micro-algae to ammonia nitrogen final concentration be 59.85 ~ 159.60mmolL
– 1.
In one of them embodiment of the present invention, as preferably, in described step one, described nutrient solution is f/2 nutrient solution.
In such scheme, as preferably, in described step 3, described certain component comprises trace element and VITAMIN, and described trace element is identical with the usage ratio of VITAMIN with described f/2 nutrient solution medium trace element with the consumption of VITAMIN.
In such scheme, as preferably, in described step 3, described certain component also comprises PO
4 3-ion, described PO
4 3 -pO in the consumption of ion and described f/2 nutrient solution
4 3-usage ratio identical.
In some of them embodiment of the present invention, as preferably, in described step 3, described certain component, except comprising trace element and VITAMIN, also comprises Ca
2+and Mg
2+ion, described Ca
2+and Mg
2+ca in the consumption of ion and described f/2 nutrient solution
2+and Mg
2+the usage ratio of ion is identical.
In such scheme, in some of them embodiment of the present invention, in described step 3, described certain component, except comprising trace element and VITAMIN, also comprises Ca2+ and Mg2+ ion and PO43-ion.
In some of them embodiment of the present invention, as preferably, in described step 3, described certain component is except comprising trace element and VITAMIN and PO43-ion, in described step 3, described certain component also comprises NO3-, Ca2+ and Mg2+ ion, and wherein, described NO3-, Ca2+ are identical with the usage ratio of Ca2+ with Mg2+ ion in described f/2 nutrient solution with the consumption of Mg2+ ion.
In one of them embodiment of the present invention, concrete grammar is as follows:
1.1 test materials
Examination material is supplied to be a kind of rich oil marine microalgae Nannochloropsis oculata (Austin, TX, USA).
1.2 test method
1.2.1 being incubated in 250mL triangular flask of marine microalgae loads 180mL f/2 substratum (Guillard andRyther 1962, Guillard 1975), and accesses 40mL seed liquor (OD
680=0.8 ~ 0.9, logarithmic phase latter stage), pass into CO
2/ air gas mixture (3%, V/V), ventilation flow rate is 60 ~ 70mLmin
– 1, be 60 ~ 70 μm of olm in intensity of illumination
– 2s
– 1, temperature cultivates when being and carrying out full light under the condition of 25 DEG C ~ 26 DEG C.During to mid-term stationary phase, stop cultivating.Filter after being dissolved in distilled water for the artificial seawater of substratum by sea crystal and obtain (adjustment salinity to 3%).Obtained artificial seawater, its Ca
2+, Mg
2+concentration is about 2mmolL
-1and 13mmolL
-1, measuring method adopts EDTA volumetry (GB/T15452-2009).
1.2.2 the pH of algae liquid that 1.2.1 obtains by the determination of best ammonia concn is adjusted to 9.00, respectively by ammoniacal liquor with 0.00,7.98,15.96,23.94,31.92,39.90,47.88,59.85,79.80,99.75,159.60mmolL
– 1ammonia nitrogen final concentration add algae liquid, mixing.Reference
deng method measure the flocculating rate after 2h, the supernatant simultaneously getting each group of algae liquid carries out centrifugal, gets supernatant liquor and measure Ca after centrifugal
2+, Mg
2+concentration and pH.Reagent colorimetric method (HJ535-2009) is adopted to measure the concentration of ammonia nitrogen.
1.2.3 ammonia nitrogen final concentration is got in the process of the struvite precipitator method is 159.60mmolL
– 1flocculation after supernatant liquor carry out centrifugal, get supernatant liquor after centrifugal, to cultivate in clear liquid to upper strata with the molar ratio of N:P:Mg=1:1.05:1.10 and add Na
2hPO
412H
2o and MgCl
26H
2o, 100r/min stir 10min, natural subsidence 5h, get upper strata cultivation clear liquid and carry out centrifugal.Measure the ammonia nitrogen concentration of centrifugal clear liquid before and after process.
1.2.4 follow-up cultivation and growth measurement cultivate micro-algae according to the method for 1.2.1, and are set to control group N0.Get the deamination centrifugal clear liquid that 1.2.3 obtains, divide into groups according to the array mode of table 1, and correspondingly add nutritive element to each group, the amount of addition needed for equal volume control group N0, in table 1, trace metals represents trace element, and Vitamins represents VITAMIN.Inoculate according to the method for 1.2.1, then measure nitre nitrogen, total phosphorus, ammonia nitrogen, the Ca of substratum
2+, Mg
2+concentration.Total phosphorus yield adopts ammonium molybdate spectrophotometric method (GB11893-89).
Carry out follow-up cultivation according to the condition of 1.2.1 to each group, within every 2 days, measure the OD of algae liquid
680value, and compare as time OD
680value and last time OD
680value is to judge growth period.During to logarithmic phase latter stage, mensuration frequency is changed into and measures, with the OD of continuous 2 days every day
680value, without during significant difference being plateau (p>0.05), stops cultivating.
Drawing 5mL plateau algae liquid to drying on 0.45 μm of filter membrane of constant weight through 105 DEG C in advance, after vacuum filtration, in 105 DEG C, filter membrane being dried to constant weight.Heavy divided by sample volume by filter membrane difference, obtain biomass.Represent biomass with unit volume dry weight, unit is gL
– 1; Represent Biomass yield with unit time biomass, unit is gL
– 1d
– 1.
The micro-algae N of table 1
.oculatathe interpolation combination of follow-up cultivation Middle nutrition element
Note: "+" represents this nutritive element of interpolation; This nutritive element is not added in "-" expression.
1.2.5 the compositional analysis of frond meta-bolites
(1) total lipid content analysis: in plateau centrifugal algae liquid of gathering, obtains algae mud, through vacuum lyophilization, obtains dry algae powder.Adopt chloroform methanol method to extract total fat, by total fat weight divided by algae grain weight amount, obtain TL amount mark.
(2) pigment content analysis: take 10mg algae powder in centrifuge tube, adds 5mL 80% acetone/water solution (volume fraction, v/v), and under condition of ice bath, ultrasonication is extracted into algae powder and turns greyish white, centrifugal, gets supernatant.With reference to the pigment content of this acetone extract of spectrophotometry that Mac í as-S á nchez etc. proposes.Frond pigment content (μ gmg
-1)=acetone extract pigment concentration (μ gmL
-1) × 5mL/10mg.
(3) protein content analysis: take 10mg algae powder in centrifuge tube, add 0.5molL
– 1naOH solution 2mL, extract 10min under boiling water bath, cooling, centrifugal, get supernatant.The protein concn of this alkali extract is measured by Bradford method.Frond protein content (mgmg
-1)=alkali extract protein concentration (mgmL
-1) × 2mL/10mg.
1.2.6 micro-algae secondary ammonia flocculation is gathered with each group of micro-algae algae liquid of 1.2.4 cultivation for material, with 159.60mmolL
– 1ammonia nitrogen final concentration to algae liquid carry out ammonia flocculation gather.Reference
deng method, in 0,10,20,30min place measures flocculating rate.
1.3 data analysing method
Software Excel 2007 and Origin 8.5 is adopted to carry out data analysis and mapping.
2 results and analysis
2.1 ammonia concns are to Mg in micro-algae flocculating rate, pH and supernatant liquor
2+, Ca
2+the impact of concentration
Under different ammonia concn, Mg in micro-algae flocculating rate, pH and supernatant liquor
2+, Ca
2+the change of concentration is as shown in Fig. 2 (a) He Fig. 2 (b).Fig. 2 (a) and Fig. 2 (b) illustrate flocculating rate, pH and Mg of N.oculata under different ammonia concn
2+, Ca
2+concentration.As can be seen from Fig. 2 (a) and Fig. 2 (b), the Mg in supernatant liquor
2+concentration along with the increase of ammonia concn be minimizing trend, when not adding ammoniacal liquor, Mg
2+concentration is about 13mmolL
-1; Ammoniacal liquor final concentration is 0.159molL
-1time, Mg
2+concentration is down to 1.5mmolL
-1left and right, its concentration should be supplemented in subsequent disposal.Ca in supernatant liquor
2+concentration remains stable along with the increase of ammonia concn, is 2mmolL
-1left and right.PH value tends towards stability along with after the first sharply rising of increase of ammonia concn, remains between 10.00 ~ 10.70.Obviously, this pH scope is conducive to struvite precipitator method recovery ammonia nitrogen.Micro-algae flocculating rate rises along with the increase of ammonia concn, when ammonia concn is 59.85mmolL
-1time, flocculating rate tends towards stability.Get 59.85,79.80,99.75,159.60mmolL
-1the flocculating rate at ammonia nitrogen final concentration place carries out variance analysis, and result shows, the flocculating rate between these 4 process is all without significant difference (p>0.05).Use 159.60mmolL
-1ammonia nitrogen final concentration flocculation microalgae N.oculata can obtain the highest, stable flocculating rate.Therefore, 159.60mmolL is used
-1ammoniacal liquor be rational as the former ammonia nitrogen concentration of struvite precipitator method process.
The comparison of substratum composition difference and micro algae growth state in 2.2 follow-up culturing process
Supernatant liquor after ammonia flocculation after the process of the struvite precipitator method, the change of its leading ion composition and the follow-up cultivation situation of each group as shown in table 2.As shown in Table 2, after the process of the struvite precipitator method, the ammonia nitrogen concentration of ammonia flocculation supernatant liquor is from 159.60mmolL
-1be down to 1.843mmolL
-1, ammonia nitrogen reaches efficient recovery, and the rate of recovery reaches 98.8%.(patent No.: 201110390674.3) more effective, quick, has very much progress in comparatively invention in early stage.The ion composition of each test group differs greatly with control group N0 (i.e. f/2 substratum).Wherein, the nitrogen concentration of test group is about 2.1 ~ 3.1 times of control group; Phosphorus concentration is about 18.6 ~ 19.0 times of control group, belongs to excessive even stress level.Ca
2+the change of concentration is little.Mg
2+concentration is from the 1.5mmolL of (see Fig. 2 (a)) before process
-1rise to 10.0mmolL
-1.Mg is described
2+supplemented in the process of struvite precipitator method process, but still than control group N
0little 3mmolL
-1left and right.PH is down to 7.50 after process from before treatment 10.70, cultivates pH 8.0, can directly cultivate micro-algae without the need to neutralization close to best.The salinity of supernatant liquor increases about 2% after treatment, and this should be because use Na
2hPO
412H
2o and MgCl
26H
2o causes.
The growth perfonnance of N.oculata under table 2 Different Nutrition condition
As shown in Table 2, the growth conditions of the micro-algae of N5 group is optimum, its biomass and Biomass yield are all maximum, and biomass is 2.5,1.8,2.1,2.1,2.1 times of N0 ~ N4 group respectively, and Biomass yield is 1.4,1.4,1.6,1.6,1.6 times of N0 ~ N4 group respectively; But the growth cycle of control group is the shortest, N1 ~ N4 group is taken second place, and the growth cycle of N5 group is the longest.As shown in Figure 3, the growth curve of the micro-algae of each test group and control group have very big difference.In the cultivation of front 8d, comparatively N1 ~ N5 group is large for the speed of growth of the micro-algae of control group, and enters plateau at first; But after 8d, the speed of growth of the micro-algae of test group exceedes control group, and growth cycle is than contrast group leader.Wherein, the speed of growth of the micro-algae of N2 group is the fastest.In general, the upgrowth situation of the micro-algae of each test group in follow-up cultivation is better than control group.
In 2.3 follow-up cultivations, Different Nutrition composition is on the impact of N.oculata meta-bolites composition
2.3.1 Different Nutrition composition is in the follow-up cultivation of impact of N.oculata total fat accumulation, and Different Nutrition composition on the impact of N.oculata total lipid content as shown in Figure 4.As can be seen from Figure 4, the total lipid content of N1 ~ N5 group is respectively 49.3%, 49.3%, 54.0%, 47.0%, 36.6%, suitable with control group, and group difference is not significantly (p>0.05).Illustrate that the nutritional condition of N1 ~ N5 group all can ensure that microalgae cell accumulates certain grease.According to existing invention, find that the total lipid content of N0 ~ N5 group has reached the requirement of microalgae biodiesel refining, belong to medium level.
2.3.2 Different Nutrition composition on the impact of N.oculata protein accumulation as shown in Figure 5, and the alkali extract protein content of control group and test group exists notable difference.The alkali extract protein content of N1 ~ N5 group is 2.12,1.96,1.95,2.06,1.65 times of control group respectively, and the trophic component of obvious test group is more conducive to accumulating albumen.According to the data of Fig. 5 and the conversion relation of 1.2.5 (3), the frond protein content that can obtain N0 ~ N5 group is respectively 0.040,0.085,0.078,0.078,0.083,0.066mgmg
-1.
2.3.3 Different Nutrition composition on the impact of N.oculata pigment accumulation as shown in Figure 6, and the pigment content of the acetone extract of test group is all high than control group.Wherein, the chlorophyll content of N1 ~ N5 group is 10.3,3.7,3.2,2.9,6.5 times of control group respectively, and the carotenoid content of N1 ~ N5 group is 6.0,2.6,2.2,1.8,4.7 times of control group respectively.Obviously, the trophic component of N1 and N5 group is more conducive to the accumulation of the high value added products such as carotenoid.According to the data of Fig. 6 and the conversion relation of 1.2.5 (2), the chlorophyll content that can obtain N0 ~ N5 group frond is respectively 0.61,6.31,2.30,2.00,1.77,3.48 μ gmg
-1, and the carotenoid content of N0 ~ N5 group frond is respectively 0.31,1.89,0.95,0.68,0.58,1.47 μ gmg
-1.
2.4 micro-algae secondary ammonia flocculations are gathered
As shown in Figure 7, strong aqua (0.159molL is being added
-1ammonia nitrogen final concentration) after 10min, the flocculating rate of each group all reaches more than 90%, and tends towards stability in the 20th subsequently, 30min.Show the follow-up cultivation of micro-algae still by ammonia flocculation harvesting biomass, thus likely realize the flocculation of microdisk electrode-ammonia and gather-repeatedly the circulation of follow-up cultivation.
3 conclusions and discussion
Combination and potential advantages thereof are added in nutrition best in 3.1 follow-up cultivations
From the productive rate that table 2 is listed, N1 and N5 group is better than control group N0 and other experimental group.And in grease productive rate, protein yields, chlorophyll productive rate, carotenoids yield, the advantage of N1 and N5 still obviously (Fig. 4 ~ 6).Through calculating: the Biomass yield of N1 group, grease productive rate, protein yields, chlorophyll productive rate, carotenoids yield, again ammonia flocculating rate (10min) are 0.97,1.02,2.06,10.08,5.78,0.97 times of control group respectively; And the Biomass yield of N5 group, grease productive rate, protein yields, chlorophyll productive rate, carotenoids yield, again ammonia flocculating rate (10min) are 1.39,1.08,2.28,9.08,6.44,1.03 times of control group respectively.Obviously, the nutrition interpolation combination of N1 and N5 group is more excellent.Compared with control group, the algae powder of N1 and N5 group is rich in grease, albumen and pigment simultaneously, can be used for the field such as animal-feed, aquaculture.And the substratum of N1 and N5 group also can be used as the substratum that sets out of following medium optimization.
It is worth mentioning that, the new growth perfonnance feature of the micro-algae of N1 with N5 group has attracted the inspiration of cultivating micro-algae and the accumulation of inducible metabolism thing about mixing nitrogenous source.Have invention to show, when micro-algae N.oculata is using nitroxyl nitrogen and oxynitroso nitrogen as nitrogenous source, its biomass yield is 108% and 104% when taking ammonia as nitrogenous source, and the respective adaptive phase is suitable with the logarithmic phase time.Obviously, except N5 group, this conclusion of the data fit of table 2.Therefore, the difference of nitrogenous source composition is not the reason causing test group and control group micro algae growth there are differences.On the contrary, 2.1 ~ 3.1 times of the N/P ratio of N1 ~ N5 group to be 2.6 ~ 3.9, N source concentration be control group, P source concentration is 18.6 ~ 19.0 times of control group.This is perhaps the reason that test group and control group micro algae growth there are differences.The invention such as Andersen shows, the N/P of marine microalgae substratum is than being generally greater than 16:1.Therefore, have reason to think that irrational N/P ratio, abundant nutrition and various abiotic stress (salt stress, P deficiency, calcium ions and magnesium ions are coerced) can cause the frond speed of growth relatively slowly (Fig. 3, during 0 ~ 8d), but frond can be made again to accumulate certain grease, protein and pigment simultaneously.This result is consistent with the result of Piorreck etc., and namely the accumulation of albumen and pigment obviously raises along with the increase of N source concentration.In addition, the carotenoid content of N1 ~ N5 group is also apparently higher than N0 group.This salt stress all existed N1 ~ N5 group just, a kind of self-protection reaction that P coerces, calcium ions and magnesium ions is coerced etc.Result of the present invention or can be applicable to the induction algae such as Haematocoocus Pluvialls, Dunaliella salina kind and produce carotenoid.
Result of the present invention fails to illustrate which kind of nutritive element and nutritive element combination can promote the follow-up cultivation of micro-algae, because this test relates to 3 kinds of elements (nitrogen, phosphorus, calcium ions and magnesium ions) and the growth of combination to micro-algae thereof.By the rule of permutation and combination, total
plant combination.Therefore, the optimization of substratum is the content beyond the present invention.But the ammonia nitrogen that the struvite precipitator method improve ammonia flocculation really reclaims problem, and achieves the follow-up cultivation of micro-algae, reaches invention target simultaneously.In addition, result of the present invention is better than the high fat content (induction of N hunger) of the reports such as Piorreck and higher protein content (N is more than needed to be induced) conflicting result on metabolite composition.
The 3.2 struvite precipitator method are improved micro-algae ammonia and to be flocculated the Analysis of Advantages of follow-up cultivation
The ammonia nitrogen that the struvite precipitator method can solve the flocculation of micro-algae ammonia reclaims problem, and the rate of recovery reaches 98.8%, comparatively invention in the early stage (patent No.: CO 201110390674.3)
2the collaborative ammonia still process method of neutralization is more effective, convenient.The struvite precipitator method that its treating processes more once celebrated the report such as the tinkling of pieces of jade etc., Jiang Jingdong etc., Li Jin page are advanced.First, the method for the report such as Zeng Qingling etc., Jiang Jingdong etc., Li Jin page is under external source sodium hydroxide maintains the condition of pH, reclaims chlorination ammonium type ammonia nitrogen in fresh water.And the present invention directly realizes high-efficient deamination in the high nitrogenous sea water medium of alkalescence, save the interpolation of external source sodium hydroxide.Secondly, the pH of solution is down to best cultivation pH, after the completion of reaction without the need to the CO of previous work
2neutralizing treatment just can directly cultivate micro-algae.Moreover struvite particle just can be gathered in the crops by simple filtration, still needs the problem of evaporation, crystalline sulfuric acid ammonium dilute solution after avoiding film exchange process deamination.Finally, the substratum after process, its phosphorus source and magnesium source are supplemented.On the one hand, substratum just directly can cultivate micro-algae (N1 group) without the need to adding nitrogen, phosphorus, magnesium source specially; On the other hand, the magnesium ion needed for ammonia flocculation is provided again.And be better than the frond growth perfonnance of control group and more balanced metabolome in follow-up cultivation and become micro-algae high-density culture, sewage disposal, carotenoid production etc. and provide new method and inspiration.Therefore, the struvite precipitator method effectively improve the follow-up cultivation of micro-algae ammonia flocculation, and have certain superiority.
Here the quantity illustrated and treatment scale are used to simplify explanation of the present invention.The application of method of the present invention, modifications and variations be will be readily apparent to persons skilled in the art.
As mentioned above, according to the present invention, owing to adopting the ammonia in struvite precipitator method recovery nutrient solution, the cyclic culture of micro-algae therefore can be realized.
Although embodiment of the present invention are open as above, but it is not restricted to listed in specification sheets and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the legend described.
Claims (10)
1. a cyclic culture technique for micro-algae, is characterized in that, comprise the steps:
Step one, to have in the nutrient solution of micro-algae to cultivation and add ammonia, utilize ammonia by described micro-algae flocculation; Enter step 2 afterwards,
Step 2, the flocculation of micro-algae to be separated with nutrient solution, to obtain cultivation clear liquid, backward cultivation clear liquid in add PO
4 3-and Mg
2+, make the NH in cultivation clear liquid
4 +with PO
4 3-and Mg
2+reaction is to generate NH
4mgPO
46H
2o precipitates; Enter step 3 afterwards,
Step 3, by NH
4mgPO
46H
2o precipitation is separated with cultivation clear liquid, obtains processing clear liquid, backward process clear liquid in add certain component and form new nutrient solution, and recycle this new micro-algae of nutrient solution Cyclic culture; And,
The method of step 4, successively repeating step one, step 2 and step 3 is to realize the cyclic culture of micro-algae.
2. the cyclic culture technique of micro-algae as claimed in claim 1, is characterized in that, in described step 2, in cultivation clear liquid, adds Na
2hPO
412H
2o is to provide PO
4 3-, in cultivation clear liquid, add MgCl
26H
2o is to provide Mg
2+, and the molar ratio of N:P:Mg is 1:1.05:1.10.
3. the cyclic culture technique of micro-algae as claimed in claim 2, is characterized in that, in described step 2, first in cultivation clear liquid, adds Na
2hPO
412H
2o and MgCl
26H
2o, 100r/min stir 10min, afterwards natural subsidence 5h, then enter described step 3.
4. the cyclic culture technique of micro-algae as claimed in claim 1, is characterized in that, in described step one, first has the pH of the nutrient solution of micro-algae to be adjusted to 9.00 cultivation, then has in the nutrient solution of micro-algae to described cultivation and add ammonia, utilizes ammonia to be flocculated by described micro-algae,
Described ammonia to be concentration be 12 ~ 28% ammoniacal liquor or ammonia.
5. the cyclic culture technique of micro-algae as claimed in claim 4, is characterized in that, described ammoniacal liquor is added cultivate have in the nutrient solution of micro-algae to ammonia nitrogen final concentration be 59.85 ~ 159.60mmolL
– 1.
6. the cyclic culture technique of micro-algae as claimed in claim 1, is characterized in that, in described step one, described nutrient solution is f/2 nutrient solution.
7. the cyclic culture technique of micro-algae as claimed in claim 6, it is characterized in that, in described step 3, described certain component comprises trace element and VITAMIN, and described trace element is identical with the usage ratio of VITAMIN with described f/2 nutrient solution medium trace element with the consumption of VITAMIN.
8. the cyclic culture technique of micro-algae as claimed in claim 7, is characterized in that, in described step 3, described certain component also comprises PO
4 3-ion, described PO
4 3-pO in the consumption of ion and described f/2 nutrient solution
4 3-usage ratio identical.
9. the cyclic culture technique of the micro-algae as described in as arbitrary in claim 7 or 8, it is characterized in that, in described step 3, described certain component also comprises Ca
2+and Mg
2+ion, described Ca
2+and Mg
2+ca in the consumption of ion and described f/2 nutrient solution
2+and Mg
2+the usage ratio of ion is identical.
10. the cyclic culture technique of micro-algae as claimed in claim 8, is characterized in that, in described step 3, described certain component also comprises NO
3 -, Ca
2+and Mg
2+ion, wherein, described NO
3 -, Ca
2+and Mg
2+ca in the consumption of ion and described f/2 nutrient solution
2+and Mg
2+the usage ratio of ion is identical.
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| CN107345209A (en) * | 2017-08-08 | 2017-11-14 | 新奥科技发展有限公司 | A kind of both culturing microalgae method |
| CN107723242A (en) * | 2017-08-28 | 2018-02-23 | 北京联合大学 | A kind of method for comprehensively utilizing rubbish from cooking zymotic fluid culture microalgae |
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| CN107723242B (en) * | 2017-08-28 | 2021-05-25 | 北京联合大学 | Method for culturing microalgae by comprehensively utilizing kitchen garbage fermentation liquor |
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