CN107841464B

CN107841464B - Algae culture method

Info

Publication number: CN107841464B
Application number: CN201711346620.0A
Authority: CN
Inventors: 何红娣; 马琴华; 陈浪军; 陈鹏; 方德和; 应仲元
Original assignee: Hangzhou Yusen Agricultural Technology Development Co ltd
Current assignee: HANGZHOU YUSEN AGRICULTURAL TECHNOLOGY DEVELOPMENT Co.,Ltd.
Priority date: 2017-12-15
Filing date: 2017-12-15
Publication date: 2020-04-24
Anticipated expiration: 2037-12-15
Also published as: CN107841464A

Abstract

The invention provides a method for culturing algae, which comprises the following steps: (1) selecting chlorella algae to be planted in a triangular flask containing a growth culture medium, culturing at the illumination of 5000lux and the temperature of 26 ℃, shaking the triangular flask 2-3 times every day, and obtaining a seed solution when the chlorella algae grow to a logarithmic phase, wherein each liter of the growth culture medium contains: 4g of glucose, 0.5g of yeast powder, 1.5g of ferrous sulfate, 1.6g of sodium acetate, 0.6g of sodium chloride, 1g of monopotassium phosphate, 2g of magnesium sulfate and 2g of ammonium chloride. (2) Inoculating the algae liquid obtained in the step (1) into an expanded culture medium according to the volume ratio of 10%; (3) the expanded culture is carried out under the conditions of 20 +/-10 ℃, 2000 +/-500 LUX and 20-100 rpm of shaking table vibration speed, and the culture method is simple, convenient and easy to operate, green and environment-friendly and is beneficial to large-scale production.

Description

Algae culture method

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a culture method of algae.

Background

Microalgae is a general term for microorganisms containing chlorophyll A and capable of photosynthesis, the individual is tiny, the form can be generally distinguished under a microscope, the microalgae is widely distributed, and are distributed in land lakes and ocean water areas, and floating microalgae plays a significant role in material circulation and energy flow of pond culture, and is indispensable for maintaining the normal function of a pond ecosystem and stabilizing the pond environment. The phytoplankton of chlorophyta and cyanophyta can become dominant species in natural water, for the culture pond, people hope that the phytoplankton of chlorophyta beneficial to culture objects in the culture pond can occupy dominant species in the water, and the excellent phytoplankton of the planktonic microalgae can promote the decomposition and conversion of nutritive salt in the water, reduce and eliminate various toxic substances such as ammonia nitrogen, nitrite nitrogen, organic pollutants and the like in the processes of stable population and continuous increase of biomass; but also can generate oxygen through photosynthesis to increase dissolved oxygen of the culture water body and promote the oxidative decomposition of oxygen-consuming organic matters in the water body.

Chlorella (Chlorella) is a kind of unicellular green algae of genus Chlorella of phylum Chlorophytum, is a kind of spherical unicellular freshwater algae, the diameter is 3-8 microns, one of the earliest life on the earth, appear more than 20 hundred million years ago, it is a kind of high-efficient photosynthetic plant, grow and reproduce with photoautotrophy, the distribution is extremely wide. The chlorella is born in fresh water, and the chlorella can divide into 4 cells at intervals of 20 hours by virtue of sunlight, water and carbon dioxide to generate vigorous reproductive capacity, continuously convert solar energy into chlorella bodies containing various nutritional ingredients and release a large amount of oxygen in proliferation; and its photosynthetic capacity is more than 10 times higher than that of other plants. The chlorella can be used as excellent natural bait for aquatic economic animals, and can absorb elements such as nitrogen and phosphorus in water, reduce eutrophication level of water body, and purify water quality. Moreover, the chlorella is rich in grease and can be used for producing biodiesel; also contains some hydrocarbon substances, and can be processed into gasoline and diesel oil after extraction. If the chlorella is cultured in a large amount by using the sewage and has high oil content and hydrocarbon content, not only can the deterioration of water quality be relieved, but also a large amount of high-quality raw materials can be provided for producing biomass.

At present, the growth of microalgae belongs to the hot research discipline, and the optimization of microalgae culture conditions and the accumulation of oil and fat become the key points of research. At present, the large-scale culture of microalgae mainly has two ways, namely autotrophic culture and heterotrophic fermentation, wherein the autotrophic culture can fix greenhouse gas carbon dioxide and release oxygen, and is environment-friendly, but the utilization of light energy is often greatly limited due to the mutual shielding effect among microalgae cells. The higher the cell concentration is, the more obvious the shielding effect is, and the growth and fat synthesis of the cells are seriously influenced, which makes it very difficult to realize high-density culture of oil-containing microalgae in a photo-bioreactor. For heterotrophic culture, the growth of cells depends mainly on the absorption of organic carbon sources by the cells, and since it is not limited by light, high-density fermentation and efficient synthesis of fat can be achieved by feeding organic carbon. Although heterotrophic culture has the advantages of high growth rate, short culture period, easy control of the culture process and the like, the method for producing biodiesel by using heterotrophic microalgae has the main problem that the method depends on organic carbon sources (such as glucose, starch and the like) as raw materials, and the production cost is increased. In the prior art, most of microalgae are produced by utilizing a photobioreactor, the research focus of the research is shifted from the research of general production technology to the research of deep processing and special substance extraction of the microalgae, the technology for producing chlorella in Taiwan in China is quite mature, products of some chlorella are accepted by the masses, although the research on the chlorella is carried out in the 20 th century and the 60 th years in China, the cost is reduced because the large-scale production cannot be carried out, so the method only stays in the stage of producing primary products, the input-output ratio is extremely unbalanced, the development and research process of the chlorella is restricted, the biomass of a large-scale open culture mode in China reaches about 1000 ten thousand/ml, the method is limited by high and low input yield, the difference from the foreign culture level is large, and in addition, the prior art is also beneficial to a method for preparing biodiesel by quickly pyrolyzing heterotrophic algae cultured by using starch enzymolysis, the method comprises the steps of preparing culture solution by using low-quality grain starch as a raw material and using enzymolysis starch to prepare glucose aqueous solution, and obtaining heterotrophic chlorella by a heterotrophic conversion technology; then the heterotrophic algae cells with high fat content are used for fast pyrolysis to obtain the biodiesel with high yield and high quality. The method selects low-quality grain starch to hydrolyze and then provides glucose to be used as an organic carbon source, so that the cost is high and the large-scale production is difficult to realize.

In recent years, the breeding industry in China is rapidly developed, and breeding wastewater contains a large amount of nitrogen and phosphorus and organic matters with extremely high concentration and is regarded as high-concentration sewage, and direct discharge can cause water eutrophication and water source pollution. In addition, the aquaculture wastewater carries a large amount of pathogenic bacteria, emits extremely strong odor and increasingly causes serious pollution to the environment. The protection of ecological environment is not slow enough.

The microalgae has high oil content, can be cultivated by various water bodies, has the advantages of not occupying cultivated land and the like, and the microalgae is cultivated by using the cultivation wastewater, so that the problem of environmental pollution caused by direct discharge of the wastewater is solved, the resource utilization of the cultivation wastewater is realized, and positive influences are generated on both energy and environment.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a culture method of microalgae. The invention is realized by adopting the following technical scheme:

a culture method of chlorella is characterized by comprising the following steps:

(1) and (3) selecting chlorella algae seeds to be cultured in a triangular flask containing 100mL of growth medium under the illumination of 5000lux at 26 ℃, and shaking the triangular flask for 2-3 times every day. The color of the culture solution can be observed to gradually turn green after 3-4 days. And (3) obtaining a seed solution (algae solution) when the algae grows to the logarithmic growth phase, wherein each liter of the growth culture medium contains: 4g of glucose, 0.5g of yeast powder, 1.5g of ferrous sulfate, 1.6g of sodium acetate, 0.6g of sodium chloride, 1g of monopotassium phosphate, 2g of magnesium sulfate and 2g of ammonium chloride.

Said Chlorella is Chlorella sp ATCC30412

(2) Inoculating the algae liquid obtained in the step (1) into an expanding culture medium according to the volume ratio of 10%, wherein the expanding culture medium comprises the following components: and (3) fungus residue hydrolysate: growth medium: mixing the bacillus culture solutions according to the volume ratio =5-6:2-3: 1-2;

the fungus residue hydrolysate is as follows: and (2) centrifuging the amino acid mother liquor obtained after the amino acid fermentation is finished, collecting mycoprotein, adjusting the solid content of the mycoprotein to 10%, adding 5-10wt% of 5mol/L NaOH, and hydrolyzing at normal temperature for 5-8 days to obtain a bacterial residue hydrolysate.

The bacillus culture solution is as follows: taking bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ATCC23843, which is cultured by activated culture, then inoculated into a flask and shake-cultured, and then cultured to a concentration of 1X 10⁸Bacterial liquid of each ml;

(3) performing scale-up culture at 20 + -10 deg.C, 2000 + -500 LUX, and shaking at 20-100 rpm;

the traditional chlorella culture medium contains more than 20 chemicals such as macroelements, trace elements, vitamins and partial growth regulators, and has the disadvantages of troublesome operation, large workload, especially few trace elements and elements, small dosage, large error and inconvenience in transportation, commercialization and marketization. The growth culture medium disclosed by the invention only contains 8 nutrient elements, is convenient to use, small in workload, convenient to store and transport, convenient for factory, standard, marketized, commercialized and specialized production of the culture medium, accurate in quantification of trace elements and small in error. And can also realize better chlorella culture effect;

after the microalgae is cultured by adopting a culture medium, a cheap carbon source is added in the process of expansion culture as a growth carbon source in the process of heterotrophic culture, the cheap carbon source is processed mushroom dregs, the carbon source has better growth advantages than the method of simply adding the heterotrophic carbon source such as glucose or starch hydrolysate, meanwhile, the method realizes the recycling of the mushroom dregs in the conventional biological fermentation production, solves the pollution problem of the mushroom dregs, can also change waste into valuable, and is more favorable for the absorption of nutrient substances by adopting an alkaline hydrolysis mode in order to be better absorbed and utilized by the chlorella because the mushroom dregs are basically solid substances;

the strain can be purchased from China general microbiological culture Collection center (CGMCC) and American mode culture collection and storage (ATCC).

The bacteria of the invention can obtain the bacteria liquid with required concentration by a conventional culture method, is limited by space and is not described in detail.

Detailed Description

Example 1

(2) Inoculating the algae liquid obtained in the step (1) into an expanding culture medium according to the volume ratio of 10%, wherein the expanding culture medium comprises the following components: and (3) fungus residue hydrolysate: growth medium: mixing the bacillus culture solutions according to the volume ratio =5:2: 1;

the fungus residue hydrolysate is as follows: and (2) centrifugally collecting mycoprotein from the amino acid mother liquor obtained after the amino acid fermentation is finished, adjusting the solid content of the mycoprotein to 10%, adding 5wt% of 5mol/L NaOH, and hydrolyzing for 5d at normal temperature to obtain a bacterial residue hydrolysate.

(3) performing scale-up culture at 25 deg.C, 2000 + -500 LUX, and shaking table vibration speed of 20-100 rpm for 3-5 d;

example 2

(2) Inoculating the algae liquid obtained in the step (1) into an expanding culture medium according to the volume ratio of 10%, wherein the expanding culture medium comprises the following components: and (3) fungus residue hydrolysate: growth medium: mixing the bacillus culture solutions according to the volume ratio =6:3: 2;

the fungus residue hydrolysate is as follows: and (2) centrifuging the amino acid mother liquor obtained after the amino acid fermentation is finished, collecting mycoprotein, adjusting the solid content of the mycoprotein to 10%, adding 10wt% of 5mol/L NaOH, and hydrolyzing at normal temperature for 5-8 days to obtain a bacterial residue hydrolysate.

(3) the expanded culture is carried out at 30 deg.C, 2000 + -500 LUX, and shaking table vibration speed of 20-100 rpm for 3-5 d.

Example 3

Symbiotic experiment of chlorella and bacillus and large-scale treatment of bacterial residues: the chlorella is symbiotically cultured by adopting the bacillus amyloliquefaciens and the chlorella, the growth of the chlorella is promoted, and the system of the chlorella and the bacillus amyloliquefaciens is added, so that the growth of the bacillus amyloliquefaciens can synergistically promote the growth of the chlorella.

And (3) measuring the oil content: centrifuging the algae liquid in a stabilizer at 3000r/min, cleaning the algae mud with deionized water, drying at 65 ℃ in an electric heating blower drying box to obtain algae powder, crushing cell walls of the algae powder, placing the algae powder in a test tube with a plug mill, adding absolute ethyl alcohol, uniformly mixing, extracting at room temperature for 3h, properly and uniformly mixing during the period, transferring supernatant into a centrifuge tube, adding activated clay for decolorization, centrifuging to collect supernatant, placing the supernatant in the electric heating blower drying box to evaporate the absolute ethyl alcohol, weighing, and calculating the oil content, wherein the oil content = oil content/algae powder mass%;

experimental groups: following the procedure of example 1;

comparative group 1: a bacillus symbiotic system is not used, namely the amylolytic bacillus culture solution is deleted from the expanded culture medium, and the rest is the same;

comparative 2 group: the expanded culture medium does not contain mushroom residue hydrolysate, and the rest is the same;

comparative 3 group: the expanded culture medium only contains the mushroom dreg hydrolysate and the amylolytic bacillus culture solution, and the rest is the same;

in comparison group 4, the same procedure was followed except that paenibacillus strain disclosed in CN2015100638727 was added to the medium.

All the above were cultured in culture flasks, and after 2 days, color recording was started, and microalgae cells in each flask were counted every 24h using a microscope and a red blood cell counting plate. Counting the counting result and drawing a growth curve for comparison. The results are shown in Table 1:

TABLE 1 symbiotic experiments

	Color of culture flask	Chlorella cell concentration	The oil content wt%
				Experimental group	The color of the culture bottle turns green after 2 days, and the color gradually deepens	5.5×10¹⁰Per mL	44.3
Comparative example 1	Day 4 the culture flask was green in color	1.2×10⁹Per mL	31.7
				Comparative example 2	The color of the culture bottle turns green after 2 days, and the color gradually deepens	4.9×10⁹Per mL	42.7
Comparison 3	Day 3 color green	1.7×10⁹Per mL	40.8
				Comparative example 4	Day 3 color change to green	1.8×10¹⁰Per mL	37.8

According to the experiment, the bacillus and chlorella symbiotic system can produce larger chlorella concentration, the color of the culture bottle turns green 2 days after inoculation, and compared with a comparative experiment 1-3 group, the group shows deeper green, which shows that the number of chlorella cells is obviously improved compared with the latter group, due to the symbiotic promotion relationship of bacillus amyloliquefaciens and chlorella, the growth microenvironment of chlorella is improved by the existence of bacillus amyloliquefaciens, so that the biomass of chlorella is more, and the oil content is more obvious than that of the comparative group. In the control group 1, due to the removal of symbiotic conditions of bacillus amyloliquefaciens, the growth of chlorella is relatively retarded, the logarithmic phase biomass is obviously reduced compared with that of an experimental group, and the oil content of the produced chlorella is relatively low. Compared with the experiment group of the comparison group 2, the strain dreg culture solution is used in the enlarged culture, so that the waste utilization is realized, the result which is basically similar to that obtained by the conventional culture is obtained, the effect on the oil content, the growth speed and the biomass is equivalent to that of a growth culture medium, the cheap carbon source is utilized, and the energy is greatly saved. Comparing 3 groups with the experimental group, directly feeding the obtained chlorella liquid into the dreg hydrolysate and the amylolytic bacillus culture solution for culture without any previous culture solution components, and because a certain domestication environment is lacked, the growth of the chlorella is slower than that of the experimental group, and finally the biomass is also influenced; compared with the experimental group, the symbiotic bacillus is replaced by the bacillus amyloliquefaciens in the application to be the paenibacillus, the biomass obtaining and the oil content are improved to a certain extent compared with the symbiotic system without using bacteria and algae, however, the application obtains more excellent effects in terms of the biomass and the oil content of the produced chlorella, the biomass of chlorella cells is improved by 2.05 times, the oil content is improved by 14.6%, and the more excellent synergistic effect of the chlorella in the application and the bacillus amyloliquefaciens is shown.

Example 4

Growth medium screening assay

BG-11 is a common culture medium for culturing algae generally, however, it contains macroelements, microelements, vitamins and part of growth regulators, etc., the operation is complicated, the workload is large, 15-20 kinds of nutrient elements are needed in the growth process of chlorella, most of the elements can not become restrictive, among them C, N, P are main elements for growth, have great influence on growth and accumulation of chlorella, the applicant of this application deletes and selects simple chlorella culture medium components by orthogonal optimization experiment, glucose is 4g, yeast powder is 0.5g, ferrous sulfate is 1.5g, sodium acetate is 1.6g, sodium chloride is 0.6g, potassium dihydrogen phosphate is 1g, magnesium sulfate is 2g, ammonium chloride is 2g, compared with the prior art, the use is convenient, the workload of preparation is small, and a good chlorella culture effect can also be realized.

Experimental groups: and (3) selecting chlorella algae seeds to be cultured in a triangular flask containing 100mL of growth medium under the illumination of 5000lux at 26 ℃, and shaking the triangular flask for 2-3 times every day. Obtaining seed liquid (algae liquid) when the algae grows to logarithmic phase;

control 1 group: replacing the growth medium with BG-11 for the rest of the experimental groups;

control 2 group: the growth medium is prepared by taking ammonium bicarbonate and calcium superphosphate cultured in a conventional soil pond as nutrient sources for culture, and the rest of the experimental groups are the same. The results are shown in Table 2.

TABLE 2 growth Medium screening test

	Growth rate (after 5 d)	Growth state
			Experimental group	0.341d^-1	After 3 days, the green color of the leaves is changed to be green, and the green color content of the leaves is obviously increased
Control 1 group	0.347 d^-1	After 3 days, the green color of the leaves is changed to be green, and the green color content of the leaves is obviously increased
			Control 2 group	0.102 d^-1	The surface forms an algae membrane, the wall attachment is serious, microscopic cells are adhered, and the cell bodies are decomposed and die in a certain proportion

As can be seen, the culture medium of the application achieves basically similar results compared with BG-11, greatly simplifies the components of the culture medium and reduces the cost.

Example 5 feeding experiment

Experimental groups: adding the mixture of the chlorella and the bacillus amyloliquefaciens cultured in the example 2 into the crushed and sieved crucian feed raw material according to the mass ratio of 30%, uniformly mixing, preparing into granulated feed by using a granulator, drying, and feeding crucian with the daily feeding amount of 3-5% of the weight of the crucian.

The experimental crucian fries are respectively cultured in aquarium with the volume of 500L, and 20 fish are cultured in each aquarium.

Control group: in the process of culturing chlorella, bacillus amyloliquefaciens is not added, and the rest of the experimental groups are the same.

Each feed is repeated for three times, after 8 weeks of culture, the experimental fish is fasted for one day and then weighed, after dissection, muscle samples are taken for conventional nutrient component analysis, and liver samples are taken for nonspecific immunity index and antioxidant index determination, and the results are shown in Table 3.

TABLE 3 feeding experiment

	Rate of weight gain	Coefficient of bait	Survival rate	Moisture (muscle nutrient)
					Experimental group	146.82±6.33	2.01±0.13	95%	76.53±1.24
Control group	103.27±5.43	2.98±0.16	90%	78.32±1.33

The experiments show that the chlorella prepared by the method has obvious effect in crucian feeding experiments, and compared with the method for culturing the chlorella only, the method for culturing the chlorella improves the weight gain rate and the survival rate of crucians, reduces the feed coefficient, improves the feed utilization rate, reduces the moisture content of fed crucians and enhances the protein quality.

Example 6 algal bacteria System Sewage treatment experiment

Adding 10% v/v of the phycomycetes system subjected to the enlarged culture in the example 1 into artificial sewage, wherein the pH of the artificial sewage is 7.0-8.0, and the COD content is 500mg/L, TN content and 100 mg/L, TP content and 5.3 mg/L; and (2) aerating simulated flue gas into the reactor, wherein the simulated flue gas comprises 5-20% of CO2 and 50-100ppm of NO, and the other components are N2, wherein after the reactor is operated for one day, the average concentration of gas components at the outlet of the reactor is stabilized at 2.5% of CO2 and 33ppm of NO (nitrogen equilibrium), culturing for 3 days, collecting microalgae by adopting a centrifugal method, and discharging treated water. The removal rates of COD, TN and TP of the treated sewage are respectively 88.0%, 89% and 97%, and the effluent reaches the secondary discharge standard of pollutant discharge Standard of municipal wastewater treatment plant (GB 8978-2002).

In the control group, 10% v/v of the obtained chlorella system was added to the artificial sewage at pH 7.0-8.0 and COD content 500mg/L, TN content 100 mg/L, TP content 5.3mg/L in the same manner as in example 1 except that no Bacillus amyloliquefaciens was added; and (3) aerating simulated flue gas into the reactor, wherein the simulated flue gas comprises 5-20% of CO2 and 50-100ppm of NO, and the rest components are N2, culturing for 3 days, collecting microalgae by adopting a centrifugal method, and discharging treated water. The removal rates of COD, TN and TP of the treated sewage are respectively 55.0%, 62% and 73%, and compared with the experimental group, the difference is obvious.

The treatment effect of the symbiotic system of the chlorella and the bacillus amyloliquefaciens on sewage is obviously greater than that of a single chlorella system.

Although the present invention has been described in detail with respect to the general description and the specific embodiments, it will be apparent to those skilled in the art that modifications and improvements can be made based on the present invention. Accordingly, it is intended that all such modifications and variations as fall within the true spirit of this invention be included within the scope thereof.

Claims

1. A method for culturing algae, which is characterized by comprising the following steps:

step (1): selecting chlorella, inoculating the chlorella into a triangular flask containing a growth culture medium, culturing at 26 ℃ under the illumination of 5000lux, shaking the triangular flask 2-3 times every day, and obtaining a seed solution when the chlorella grows to a logarithmic phase, wherein each liter of the growth culture medium contains: 4g of glucose, 0.5g of yeast powder, 1.5g of ferrous sulfate, 1.6g of sodium acetate, 0.6g of sodium chloride, 1g of monopotassium phosphate, 2g of magnesium sulfate and 2g of ammonium chloride;

step (2): inoculating the algae liquid obtained in the step (1) into an expanded culture medium according to the volume ratio of 10%;

the expansion medium contains the growth medium components of step (1);

the expanding culture medium comprises the following components: and (3) fungus residue hydrolysate: growth medium: mixing the bacillus culture solutions according to the volume ratio =5-6:2-3: 1-2;

the fungus residue hydrolysate is as follows: collecting mycoprotein generated after amino acid fermentation, adjusting the solid content of the mycoprotein to 10%, adding 5-10wt% of 5mol/L NaOH, and hydrolyzing at normal temperature for 5-8d to obtain a mushroom residue hydrolysate;

the bacillus culture solution is as follows: activating and culturing Bacillus amyloliquefaciens, inoculating into triangular flask, and culturing to concentration of 1 × 10⁸Bacterial liquid of each ml;

the chlorella is ATCC 30412; the bacillus amyloliquefaciens is ATCC 23843;

and (3): the scale-up culture is carried out at 20 + -10 deg.C, 2000 + -500 lux, and shaking speed of 20-100 rpm.

2. Use of the product obtained by the process of claim 1 for the treatment of sewage.

3. Use of the product obtained by the process of claim 1 for feeding feed.