CN105969664B - Method for promoting microalgae grease accumulation by adding high-concentration organic wastewater into natural seawater - Google Patents
Method for promoting microalgae grease accumulation by adding high-concentration organic wastewater into natural seawater Download PDFInfo
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Abstract
The invention discloses a method for promoting microalgae grease accumulation by adding high-concentration organic wastewater into natural seawater, belonging to the technical field of microalgae biology. According to the invention, high-concentration organic wastewater with the proportion of 1% -10% is added into natural seawater to serve as a culture medium of an experimental group, the culture is carried out under the condition of continuous illumination, and when microalgae stops growing, the microalgae is harvested through centrifugal separation. The result shows that the oil yield of the microalgae cultured in the natural seawater added with the high-concentration organic wastewater is obviously improved, the culture cost of the microalgae is reduced, and the method is worthy of popularization and application.
Description
Technical Field
The invention relates to the technical field of microalgae biology, in particular to a method for promoting microalgae grease accumulation by adding high-concentration organic wastewater into natural seawater.
Background
With the rapid development of economy and society, the demand of human beings for fossil fuels such as coal, petroleum and natural gas is increasing. However, the large-scale use of fossil fuels seriously aggravates the energy crisis and global climate change, and thus, the search for a new renewable energy source is urgently needed. Microalgae are widely used as raw materials for producing biodiesel because of the characteristics of rapid cell propagation, short growth cycle, strong carbon fixation capacity, high photosynthetic efficiency, strong environmental adaptability, no occupation of agricultural arable land and the like. However, the large-scale commercial production of microalgae oil production is restricted due to the problem of high culture cost of microalgae. Therefore, the search for a low-cost microalgae culture medium becomes a hot spot and a focus of research.
Many researchers have studied the use of wastewater to culture microalgae. Ting Cai et al cultured Synechocystis with different proportions of industrial wastewater anaerobic high concentration organic wastewater added to artificial seawater, wherein Synechocystis achieved the maximum biomass yield (150.9 + -8.6 mgL-1d-1) and oil yield (19.9 + -1.2 mgL-1d-1) when the proportion of high concentration organic wastewater was 3% (Cai et al, 2013).
Joo-Young Jung et al cultivated Scenedesmus obliquus with varying proportions of natural seawater and BG11, where the maximum biomass (1.1g/L) and fatty acid methyl ester production (9%) was obtained when the proportion of natural seawater was 10% (Jung et al, 2015). However, the preparation of artificial seawater and BG11 consumes a large amount of fresh water resources. In contrast, seawater, abundant in reserves, covers 71% of the earth's surface, and contains abundant minerals beneficial to the growth of marine organisms, mainly Na +, Cl-, Mg2+, SO42-, Ca2 +. Wherein the seawater is known to have high salinity, which is beneficial to stimulating the accumulation of microalgae grease, so the seawater has great development prospect for culturing microalgae. However, the direct use of seawater for culturing microalgae is not desirable because microalgae growth requires minerals and abundant nutrients such as nitrogen and phosphorus. The content of nutrient substances such as nitrogen, phosphorus and the like in the seawater is quite low, which is not enough to meet the requirement of microalgae growth. The high-concentration organic wastewater contains rich nutrient substances such as nitrogen, phosphorus and the like. If the waste water is directly discharged, serious environmental pollution can be caused. If the high-concentration organic wastewater is added into seawater to be used as a culture medium for culturing microalgae, the seawater can be used as an inducer for microalgae grease accumulation, and the high-concentration organic wastewater can be used as a nutrient source for microalgae growth, so that the win-win purpose of microalgae growth and grease accumulation is achieved. And the seawater reserves are abundant, the cost is lower, and the high-concentration organic wastewater is also treated. Therefore, the method for culturing the microalgae by using the natural seawater added with the high-concentration organic wastewater is an economic, environment-friendly and effective method.
Disclosure of Invention
The invention provides a method for culturing microalgae by adding high-concentration organic wastewater into natural seawater, which greatly promotes the accumulation of microalgae grease and can well solve the problem of high microalgae culture cost.
In order to solve the technical problems, the invention provides the following technical scheme:
a method for promoting microalgae oil accumulation by adding high-concentration organic wastewater into natural seawater is characterized by comprising the following steps:
(1) placing the small bicolor SDEC-6 in BG11 culture medium for enrichment culture;
(2) preparing a mixed culture medium, wherein the mixed culture medium is composed of natural seawater and high-concentration organic wastewater with the proportion of 2% -4%;
(3) inoculating the algae cell seed liquid prepared in the step (1) into the mixed culture medium prepared in the step (2), wherein the density of the inoculated biomass is 0.06-0.07 g/L;
(4) placing the reaction device prepared in the step (3) in an artificial climate chamber for culturing under the following culture conditions: the temperature is 25 +/-1 ℃, and continuous illumination is carried out on 81 mu mol of photons/m 2/s;
the natural seawater in the step (2) is subjected to suction filtration to remove algae therein;
the initial water quality of the high-concentration organic wastewater is as follows: 2326.72 + -20 mg/L, NH 3-N2153.07 + -25 mg/L, TP 20.24 + -0.1 mg/L, COD: 6096.1 + -65 mg/L, pH: 8.31 +/-0.1.
Preferably, the filtration membrane used for filtering the natural seawater is a 0.45 μm cellulose acetate filtration membrane.
The concentrations of various nutrient substances and mineral substances in the natural seawater are respectively as follows: 26.38 + -0.04 Mg/L of TN, 3.83 + -0.76 Mg/L of NH3-N, 0.016 + -0.00 Mg/L of TP, 9.37 + -0.02 of pH, 13920 + -142 Mg/L of Na, 1295 + -3 Mg/L of Mg, 988.8 + -13.6 Mg/L of Ca, 346.6 + -7.2 Mg/L of K and 78.65 + -0.34 Mg/L of Si.
Preferably, the high concentration organic wastewater in step (2) is filtered through six layers of gauze and centrifuged.
Preferably, the speed of centrifuging the high-concentration organic wastewater is 4500-.
Wherein the culture period of the step (4) is 12 days.
The invention has the following beneficial effects:
(1) the method for promoting the accumulation of the microalgae grease by adding the high-concentration organic wastewater into the natural seawater obviously improves the grease yield of the microalgae, and can be used as a better culture mode.
(2) The method for promoting the accumulation of the microalgae grease by adding the high-concentration organic wastewater into the natural seawater has the advantages of rich natural seawater reserves and low cost, and simultaneously, the microalgae obtains a relatively ideal growth rate, so the cost of microalgae culture is reduced.
(3) The method for promoting the accumulation of the microalgae grease by adding the high-concentration organic wastewater into the natural seawater enables the high-concentration organic wastewater to be treated at the same time, and is environment-friendly and economical.
Drawings
FIG. 1 is a graph of the growth of SDEC-6, a small bicolor, in different media.
FIG. 2 shows the lipid accumulation properties of SDEC-6 from Chromophyta microcystis in different media.
Detailed Description
The invention is further illustrated by the following examples.
1. The algae strain is obtained by selecting cultured microalgae as small bicolor SDEC-6, screening from Jinnanquan park, and storing in freshwater algae seed bank of China.
2. The natural seawater is selected from yellow sea (Shandong sunshine sea area), and the concentrations of various nutrient substances and mineral substances in the natural seawater are respectively as follows: 10-50Mg/L of TN, 1-10Mg/L of NH3-N, 0.01-1Mg/L of TP, 8-10 of pH, 15000Mg/L of Na 10000-.
3. The high-concentration organic wastewater is selected from environmental protection companies of ten prescriptions in Jinan province, and the initial water quality of the high-concentration organic wastewater is as follows: 3000mg/L of TN 2000-N, 3000mg/L of NH 3-N2000-N, 10-50mg/L of TP, COD: 5000-8000mg/L, pH: 8-10.
Example 1: growth characteristics of Dioscorea Minacissima SDEC-6
1. The algae body culture process:
(1) placing the small bicolor SDEC-6 in BG11 culture medium for enrichment culture, wherein the BG11 culture medium comprises the following components: NaNO31.5g/L, K2HPO 440 mg/L, MgSO4 & 7H2O 75mg/L, CaCl2 & 2H2O 36mg/L, Citricc acid6mg/L, Ferric ammonium citrate 6mg/L, EDTANa 21 mg/L, Na2CO 320 mg/L, A5 solution1mL/L. A5 solution: H3BO32.86g/L, MnCl2 & 4H2O 1.86.86 g/L, ZnSO4 & 7H2O 0.22g/L, Na2MoO4 & 2H2O 0.39.39 g/L, CuSO4 & 5H2O 0.08.08 g/L, Co (NO3)2 & 6H2O 0.05.05 g/L.
(2) Diluting the algae seed solution obtained in the step (1) ten times, and then, the absorbance at 680nm is 0.172, and the dry weight is 0.06-0.07 g/L.
(3) Inoculating the seed solution obtained in the step (2) into a mixed culture medium with the proportion of high-concentration organic wastewater of 1% -10%, taking BG11, natural seawater and the high-concentration organic wastewater as culture media as control experiments respectively, carrying out constant-temperature culture in an artificial climate chamber, sampling every day during the culture process, measuring biomass, and carrying out water quality analysis.
(4) And (4) after the algae in the step (3) stop growing, carrying out centrifugal harvesting on the algae liquid, and pouring out the supernatant to obtain the algae mud.
(5) And (4) freeze-drying the algae mud obtained in the step (4) to obtain dry algae powder.
2. Measuring algal biomass:
centrifuging 20mL of algae solution at the same time every day for 10min under the condition of 5000r/min for layering, taking out algae mud, drying at constant temperature in a constant temperature drying oven at 60 ℃, weighing, and determining biomass concentration (g/L). The supernatant was ultrafiltered through a 0.45 μm cellulose acetate membrane for analysis of nitrogen and phosphorus concentrations. The total nitrogen is measured by an alkaline potassium persulfate digestion ultraviolet spectrophotometry (HJ636-2012), the ammonia nitrogen is measured by a Nashiner reagent spectrophotometry (HJ535-2009), and the total phosphorus is measured by an ammonium molybdate spectrophotometry (GB 11893-89).
3. Biomass measurement results:
(1) as shown in FIG. 1, the small bicolor SDEC-6 did not grow in the pure high concentration organic wastewater; in pure seawater, the growth of the small bicolor SDEC-6 is slow due to too low nutrient content. In natural seawater with the proportion of 1 to 10 percent of high-concentration organic wastewater, the small bicolor SDEC-6 grows.
(2) As shown in Table 1, in the natural seawater with the high concentration organic wastewater adding ratio of 1% -10%, the biomass of the small bicolor SDEC-6 is obviously higher than that in BG11, the natural seawaterBiomass; wherein the biomass of the SDEC-6 of the microchromis is up to 0.38g/L in natural seawater with the addition proportion of 4 percent of high-concentration organic wastewater, the average biomass yield is 26.08mg/L/d, and the specific growth rate is 0.18d-1。
4. And (4) analyzing results:
the natural seawater with the proportion of the high-concentration organic wastewater of 1 to 10 percent can be used as an ideal substitute culture medium to culture the dichotomous algae SDEC-6 to obtain the biomass, because the nitrogen and phosphorus concentration in the range of the addition proportion is more suitable for the growth of the dichotomous algae SDEC-6, and the NaCl in the seawater can increase the salt concentration in the mixed culture medium so as to stimulate the metabolic activity of the algae to accelerate the growth of the algae, and meanwhile, the high salinity in the seawater can avoid the invasion of other microorganisms.
Table 1 final dry weight, average biomass yield, specific growth rate of SDEC-6, small diplodia in different media.
Example 2: oil accumulation characteristics of Dioscorea opposita SDEC-6
1. And (3) measuring the oil content:
about 0.1g of the dried algal powder obtained in example 1 was weighed into a 50mL centrifuge tube, 10mL of chloroform/methanol (v/v ═ 2:1) solution was added, treated with a sonicator for 10min (frequency 20%), centrifuged at 4000r/min for 10min, the solution was separated into two phases after centrifugation, the supernatant was transferred to a 60mL separatory funnel, and the whole extraction process was repeated twice. Adding 0.9% sodium chloride solution (about 4-5mL sodium chloride solution with volume of 1/5 of the oil extract) according to the volume of the oil extract, shaking thoroughly for 1min, and standing for 15 min. The volume of the low-phase solution was measured and 5mL of the low-phase solution was placed in a 10mL glass tube, blown dry with nitrogen, and the tube was placed in an oven at 60 ℃ and dried to constant weight (about 30 min).
2. Calculating the oil content:
in the formula: LW-oil content on a dry weight basis, g/g
m 1-algal powder Dry weight, g
m 2-Dry weight of greasy 10mL glass tube, g
m 0-10 mL glass tube dry weight, g
v-volume of Low phase oil, mL
3. Analyzing the oil content result:
as shown in FIG. 2, the oil content of the SDEC-6 of the small bicolor algae in the natural seawater added with the organic wastewater with different proportions and high concentrations is obviously higher than that in BG11 and natural seawater, because the salinity in the seawater is favorable for stimulating the synthesis of oil in algae cells. When the addition proportion of the high-concentration organic wastewater is 2%, the oil content of the Chromolaena minutissima SDEC-6 reaches the highest (50.13%) and is 1.72 times of the oil content in BG 11. When the addition proportion of the high-concentration organic wastewater is 4%, the grease yield of the Chromococcus laurentii SDEC-6 reaches the highest (13.36mg/L/d), which is 2.20 times of that in BG 11. Therefore, the oil accumulation data show that the natural seawater added with high-concentration organic wastewater can be used as a low-cost alternative culture medium to culture the small bicolor SDEC-6, and the obtained oil is far higher than the oil content in BG11 and natural seawater.
In a comparative example, in a method for culturing chromococcus by diluting high-concentration organic wastewater with domestic wastewater (application No. 201610034390.3), the high-concentration organic wastewater is used as a culture medium main body, and the chromococcus is cultured by adding domestic wastewater in different proportions to obtain a certain biomass yield and oil accumulation, and the result shows that when the high-concentration organic wastewater is diluted by 10 times, the oil content (38.65%) and the oil yield (11.79mg/L/d) reach the maximum value. According to the invention, natural seawater is used as a culture medium main body, and the microalgae is cultured by adding high-concentration organic wastewater with different proportions, wherein the oil content (50.13%) reaches the maximum value when the proportion of the added high-concentration organic wastewater is 2%, and the oil yield (13.36mg/L/d) reaches the maximum value when the proportion of the added high-concentration organic wastewater is 2%. The comparison shows that the method for promoting the oil accumulation of the microalgae by adding the high-concentration organic wastewater into the natural seawater can obviously improve the oil content and the oil yield of the algae. And the natural seawater has extremely large storage capacity and is easy to obtain, the culture cost is reduced, and the large-scale commercial production of microalgae oil production is facilitated.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (6)
1. A method for promoting microalgae oil accumulation by adding high-concentration organic wastewater into natural seawater is characterized by comprising the following steps:
(1) placing the small bicolor SDEC-6 in BG11 culture medium for enrichment culture;
(2) preparing a mixed culture medium, wherein the mixed culture medium is composed of natural seawater and high-concentration organic wastewater with the proportion of 2% -4%;
(3) inoculating the algae cell seed liquid prepared in the step (1) into the mixed culture medium prepared in the step (2), wherein the density of the inoculated biomass is 0.06-0.07 g/L;
(4) placing the reaction device prepared in the step (3) in an artificial climate chamber for culturing under the following culture conditions: the temperature is 25 +/-1 ℃, and the temperature is 81 mu molphosons/m2Continuous illumination for the second time;
the natural seawater in the step (2) is subjected to suction filtration to remove algae therein;
the initial water quality of the high-concentration organic wastewater is as follows: 2326.72 + -20 mg/L, NH 3-N2153.07 + -25 mg/L, TP 20.24 + -0.1 mg/L, COD: 6096.1 + -65 mg/L, pH: 8.31 +/-0.1.
2. The method for promoting microalgae oil accumulation by adding high-concentration organic wastewater into natural seawater as claimed in claim 1, wherein the filtration membrane used for filtering natural seawater is 0.45 μm cellulose acetate filtration membrane.
3. The method for promoting microalgae oil accumulation by adding high-concentration organic wastewater into natural seawater as claimed in claim 2, wherein the concentrations of each nutrient and mineral in the natural seawater are respectively as follows: 26.38 + -0.04 Mg/L of TN, 3.83 + -0.76 Mg/L of NH3-N, 0.016 + -0.00 Mg/L of TP, 9.37 + -0.02 of pH, 13920 + -142 Mg/L of Na, 1295 + -3 Mg/L of Mg, 988.8 + -13.6 Mg/L of Ca, 346.6 + -7.2 Mg/L of K and 78.65 + -0.34 Mg/L of Si.
4. The method for promoting microalgae lipid accumulation by adding high-concentration organic wastewater into natural seawater as claimed in claim 1, wherein the high-concentration organic wastewater in the step (2) is filtered by six layers of gauze and centrifuged.
5. The method for promoting microalgae oil accumulation by adding high-concentration organic wastewater into natural seawater as claimed in claim 4, wherein the speed of centrifuging the high-concentration organic wastewater is 4500-6500r/min, and the duration is 8-10 min.
6. The method for promoting microalgae lipid accumulation by adding high-concentration organic wastewater into natural seawater as claimed in claim 1, wherein the culture period of the step (4) is 12 days.
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| CN105483015A (en) * | 2016-01-19 | 2016-04-13 | 山东大学 | Method for cultivating chroococcus by domestic-wastewater-diluted kitchen waste anaerobic digestion solution |
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| CN105483015A (en) * | 2016-01-19 | 2016-04-13 | 山东大学 | Method for cultivating chroococcus by domestic-wastewater-diluted kitchen waste anaerobic digestion solution |
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