CN110144296B

CN110144296B - Culture medium for culturing astaxanthin-producing microalgae, and economic culture method and application of astaxanthin-producing microalgae

Info

Publication number: CN110144296B
Application number: CN201910532454.6A
Authority: CN
Inventors: 洪喻; 于明然; 马冠秋
Original assignee: Beijing Forestry University
Current assignee: Zhongyu Jiaxuan (Beijing) Environmental Protection Technology Co.,Ltd.
Priority date: 2019-06-19
Filing date: 2019-06-19
Publication date: 2021-05-18
Anticipated expiration: 2039-06-19
Also published as: CN110144296A

Abstract

The invention provides a culture medium for culturing astaxanthin-producing microalgae, an economical culture method for the astaxanthin-producing microalgae and application of the culture method, and relates to the technical field of environmental science. The urine sewage is yellow water in the domestic sewage, can provide required nutrient contents such as nitrogen, phosphorus and potassium for culturing astaxanthin-producing microalgae, does not need to be additionally added, and effectively reduces the production cost. The invention also provides an economical culture method of the astaxanthin-producing microalgae, after the astaxanthin-producing microalgae are inoculated in the culture medium of the technical scheme, the astaxanthin-producing microalgae are stressed by changing the illumination time, astaxanthin can be extracted after the culture is finished, simultaneously the water quality can be synchronously purified, and the method has positive significance for solving the market gap problem of astaxanthin production by haematococcus pluvialis and the difficulty faced by yellow water treatment.

Description

Culture medium for culturing astaxanthin-producing microalgae, and economic culture method and application of astaxanthin-producing microalgae

Technical Field

The invention relates to the technical field of environmental science, in particular to a culture medium for culturing astaxanthin-producing microalgae, an economical culture method for the astaxanthin-producing microalgae and application of the culture method.

Background

In recent years, the problem of water pollution is particularly serious, wherein the urban domestic sewage is one of important water pollution sources. With the continuous development of Chinese economy and the continuous promotion of urbanization process, the urban domestic sewage becomes a main source of the continuous increase of the discharge amount of the Chinese wastewater. The domestic sewage is classified into black water (toilet sewage) and gray water (sewage from home kitchens, laundry, baths, and toilets), according to the source of the domestic sewage, wherein the black water is classified into yellow water (urine sewage) and brown water (feces sewage). The treatment of yellow water faces many difficulties, and even if the discharge is concentrated at night, the yellow water has great influence on people. The problems that the pollution of point sources is difficult to be treated and recovered in a centralized way are faced on mobile transport means such as airplanes, ships, trains and the like.

The yellow water has the characteristics of low carbon and high nitrogen and phosphorus, contributes about 80 percent of nitrogen, 50 percent of phosphorus and 90 percent of potassium in urban domestic sewage, and is suitable for resource recovery and reutilization of nutrient substances. The yellow water has the characteristics of low carbon and high nitrogen and phosphorus, and is beneficial to the growth of algae. Microalgae is proposed for purifying sewage from 20 th century 50 s by Oswald and the like, can overcome the defects of secondary pollution, potential nutrient loss, incomplete utilization of resources and the like easily caused by the traditional sewage treatment method, can effectively remove nitrogen, phosphorus and other nutrients causing water eutrophication, can be used as a new energy source for producing grease, and can be used for preparing fertilizers, breeding feeds and the like. A variety of microalgae are currently used as a source for producing feed and other health-promoting compounds.

Astaxanthin is an orange carotenoid, a fat-soluble pigment, is present in many microorganisms and underwater animals, is considered to be a "super antioxidant", and is currently widely used in the food, medical and health care industries. Some microalgae can produce astaxanthin, which is one of the synthetic sources of astaxanthin, but the culture medium for culturing microalgae to produce astaxanthin at present has high cost, and various nutrient substances such as carbon, nitrogen, phosphorus, trace elements and the like need to be added, so that the production cost of astaxanthin is high.

Disclosure of Invention

The invention provides a culture medium for culturing astaxanthin-producing microalgae, aiming at overcoming the defect that the existing microalgae culture medium for producing astaxanthin has higher cost, the components of the culture medium are derived from yellow water in domestic sewage, the cost is low, the preparation cost of astaxanthin can be obviously reduced after the culture medium is used for culturing the astaxanthin-producing microalgae, and the urine sewage can be purified by using the astaxanthin-producing microalgae.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a culture medium for culturing astaxanthin-producing microalgae, which consists of urine sewage and water in a volume ratio of 1: 99-199.

Preferably, the water is aerated tap water.

Preferably, the volume ratio of the urine sewage to the water is 1: 140-160.

Preferably, the urine sewage and water are respectively filtered by microporous filter membranes and then mixed according to the volume ratio to obtain the culture medium.

The invention provides an economical culture method of astaxanthin-producing microalgae, which comprises the following steps:

(1) inoculating astaxanthin-producing microalgae into the culture medium in the technical scheme, so that the density of the astaxanthin-producing microalgae in the culture medium is not less than 4 multiplied by 10⁴cells/mL to obtain an inoculation culture medium;

(2) carrying out first culture on the inoculation culture medium under the condition that the illumination time is 10-14 h/d to obtain a first culture;

(3) the first culture was subjected to a second culture under illumination for 24 h/d.

Preferably, the astaxanthin-producing microalgae is haematococcus pluvialis.

Preferably, the culture temperature of the first culture and the culture temperature of the second culture are 18-22 ℃ independently, and the illumination intensity of the first culture and the second culture are 4000-4800 lux independently.

Preferably, the culture time of the first culture is 5-7 d.

Preferably, the time of the second culture is 8-12 days.

The invention also provides application of the culture method in the technical scheme in purifying urine sewage.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a culture medium for culturing astaxanthin-producing microalgae, which consists of urine sewage and water in a volume ratio of 1: 99-199. The urine sewage is yellow water in the domestic sewage, can provide required nutrient contents such as nitrogen, phosphorus and potassium for culturing astaxanthin-producing microalgae, does not need to be additionally added, and effectively reduces the production cost.

The invention also provides an economical culture method of the astaxanthin-producing microalgae, the astaxanthin-producing microalgae is inoculated in the culture medium of the technical scheme, the culture medium containing urine sewage is taken as a stress source, the mode of changing illumination time is combined to stress the astaxanthin-producing microalgae, astaxanthin can be extracted after the culture is finished, simultaneously, the water quality can be synchronously purified, and the method has positive significance for solving the market gap problem of astaxanthin production by haematococcus pluvialis and the difficulty in yellow water treatment.

The invention also provides the application of the culture method in the technical scheme in purifying urine sewage, and the culture method is convenient to operate, low in cost, economical, environment-friendly and good in application prospect.

Drawings

FIG. 1 is a graph showing the growth of Haematococcus pluvialis in the culture medium according to the different urine wastewater addition ratios in examples 1 to 4 and comparative examples 1 to 4.

FIG. 2 is a photograph of a conical flask when microalgae were cultured;

FIG. 3 is microscopic examination of astaxanthin-producing microalgae after 15 days of culture;

FIG. 4 shows astaxanthin extracted from microalgae cultured in examples and comparative examples; wherein FIG. 4-1 shows astaxanthin obtained after the cultivation in the method shown in example 7, FIG. 4-2 shows astaxanthin obtained after the cultivation in the method shown in comparative example 5, FIG. 4-3 shows astaxanthin obtained after the cultivation in the method shown in comparative example 6, FIG. 4-4 shows astaxanthin obtained after the cultivation in the method shown in example 8, and FIG. 4-5 shows astaxanthin obtained after the cultivation in the method shown in comparative example 7.

Detailed Description

The invention provides a culture medium for culturing astaxanthin-producing microalgae, which consists of urine sewage and water in a volume ratio of 1: 99-199. The culture medium provided by the invention can realize the culture of astaxanthin-producing microalgae without additionally adding carbon, nitrogen, phosphorus and other trace elements, so as to obtain astaxanthin. The culture medium provided by the invention only consists of water and urine sewage, the cost of the water is low, and the urine sewage is yellow water in domestic sewage, the source is wide, and the cost is low, so that the culture medium can obviously reduce the production cost of astaxanthin, and can realize the treatment of the urine sewage and the recycling of wastes.

In the present invention, the water is preferably aerated tap water. The tap water is fully aerated in the sun to remove residual chlorine and increase dissolved oxygen, which is beneficial to the growth of microalgae. In the present invention, the aerated tap water production method preferably comprises the steps of: a certain amount of tap water is taken and placed under the sunlight, and a small aerator is used for aeration. In the invention, the aeration time of 1L of tap water is preferably 1-3 h, and more preferably 2 h.

The urine sewage is directly collected, and the content of nutrient elements in the urine sewage has certain difference due to individual difference of collected crowds. The urine sewage is urine sewage generated by a healthy human body (without renal dysfunction, diabetes and the like), and the average content of main elements in the urine sewage in multiple experiments is as follows: 6730-9326 mg/L TN, 417-845 mg/L TP, 3016-4268 mg/L Na and 1853-2539 mg/L K.

However, as shown in the specific embodiment of the invention, the rapid proliferation of the astaxanthin-producing microalgae can be realized within the volume ratio of 1: 99-199 of urine wastewater to water. In the invention, the ratio of the urine wastewater to the water is preferably 1: 140-160, and the culture medium at the ratio can ensure that the astaxanthin-producing microalgae has the maximum specific growth rate.

In the present invention, the preparation method of the medium preferably comprises the steps of: filtering urine sewage and water by using microporous filter membranes respectively, and mixing according to a volume ratio to obtain the culture medium for culturing the astaxanthin-producing microalgae. In the present invention, the pore size of the microfiltration membrane is preferably 0.22. mu.m.

The invention also provides a culture method of the economical astaxanthin-producing microalgae, which comprises the following steps:

The astaxanthin-producing microalgae is inoculated to the culture medium in the technical scheme, so that the density of the astaxanthin-producing microalgae in the culture medium is not lower than 4 multiplied by 10⁴cells/mL to obtain the inoculation medium.

In the present invention, the species of the astaxanthin-producing microalgae include, but are not limited to, Haematococcus pluvialis. In the invention, the astaxanthin-producing microalgae can be preferably subjected to early culture by adopting a BG11 liquid culture medium, and then inoculated after the density of the microalgae is increased. The culture process of the astaxanthin-producing microalgae before inoculation is not specially limited, and the conventional method in the field is adopted.

The density of the astaxanthin-producing microalgae in the inoculation culture medium is not low and is not suitable to be 4 multiplied by 10⁴cells/mL or more, more preferably 6X 10⁴～10×10⁴cells/mL and too low density easily cause the over-strong light radiation of single algae cells, which is not beneficial to the proliferation of haematococcus pluvialis.

After the inoculation culture medium is obtained, the first culture is carried out on the inoculation culture medium under the condition that the illumination time is 10-14 h/d, and a first culture is obtained. The first cultivation is carried out in the invention, the aim is to obtain algae with high density and high biomass, and a material basis is provided for the astaxanthin accumulation of the algae cells in the second stage cultivation.

In the invention, the culture temperature of the first culture is preferably 18-22 ℃, and more preferably 19 ℃; the illumination intensity of the first culture is preferably 4000-4800 lux, and more preferably 4500 lux; the illumination time of the first culture is preferably 12 h/d; the culture time of the first culture is preferably 5-7 d, and more preferably 6 d.

After the first culture is obtained, the first culture is subjected to second culture under the condition of illumination time of 24 h/d. The astaxanthin-producing microalgae are stressed by changing the illumination time, and haematococcus pluvialis can be promoted to enter a cyst formation stage from a vegetative cell growth stage as soon as possible by uninterrupted illumination, so that a plurality of pigment particles including astaxanthin are accumulated.

In the invention, the culture temperature of the second culture is preferably 18-22 ℃, and more preferably 19 ℃; the illumination intensity of the second culture is preferably 4000-4800 lux, and more preferably 4500 lux; the culture time of the second culture is preferably 8-12 d, and more preferably 9 d.

The culture method of the astaxanthin-producing microalgae can successfully obtain astaxanthin, the yield can reach 59.55mg/L, the used culture medium is low in cost and simple and convenient to operate, and therefore the culture cost of the astaxanthin-producing microalgae is effectively reduced.

The invention also provides application of the culture method in the technical scheme in purifying urine sewage. The culture method provided by the invention can effectively utilize the nutrient components such as nitrogen and phosphorus in the urine sewage.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

The haematococcus pluvialis strain used in the following experiments was purchased from Qingdao and was expanded to 10 ℃ by BG11 medium at 20 ℃ and 1500lux⁴cells/mL。

The urine sewage used in the following tests is from three different sources, and the average content of the main elements of the urine sewage from the three sources is as follows:

source A: collected in summer in Beijing urban cultural and educational district (TN:7500mg/L, TP:742mg/L, Na:4123mg/L, K:2345 mg/L);

source B: collected in the Beijing urban cultural and educational district toilet in winter (TN:8823mg/L, TP:645mg/L, Na:3567mg/L, K:2430 mg/L);

source C: collected in the toilet of cultural relics area of Beijing city in spring (TN:7200mg/L, TP:703mg/L, Na:3234mg/L, K:2120 mg/L).

Example 1

Taking 200mL of tap water, aerating for 1 hour, and filtering through a 0.22 mu m filter membrane for later use; taking 10mL of urine sewage (source A) generated by a healthy human body and filtering the urine sewage through a 0.22 mu m filter membrane for later use; and mixing the filtered urine sewage with aerated tap water according to the volume ratio of 1:99 to obtain the culture medium for the astaxanthin-producing microalgae.

Example 2

Taking 200mL of tap water, aerating for 1 hour, and filtering through a 0.22 mu m filter membrane for later use; taking 10mL of urine sewage (source A) generated by a healthy human body and filtering the urine sewage through a 0.22 mu m filter membrane for later use; and mixing the filtered urine sewage with aerated tap water according to the volume ratio of 1:124 to obtain a culture medium for the astaxanthin-producing microalgae.

Example 3

Taking 200mL of tap water, aerating for 1 hour, and filtering through a 0.22 mu m filter membrane for later use; taking 10mL of urine sewage (source A) generated by a healthy human body and filtering the urine sewage through a 0.22 mu m filter membrane for later use; and mixing the filtered urine sewage with aerated tap water according to the volume ratio of 1:149 to obtain a culture medium for the astaxanthin-producing microalgae.

Example 4

Taking 200mL of tap water, aerating for 1 hour, and filtering through a 0.22 mu m filter membrane for later use; taking 10mL of urine sewage (source A) generated by a healthy human body and filtering the urine sewage through a 0.22 mu m filter membrane for later use; and mixing the filtered urine sewage with aerated tap water according to the volume ratio of 1:199 to obtain a culture medium for the astaxanthin-producing microalgae.

Comparative example 1

Taking 200mL of tap water, aerating for 1 hour, and filtering through a 0.22 mu m filter membrane for later use; taking 10mL of urine sewage (source A) generated by a healthy human body and filtering the urine sewage through a 0.22 mu m filter membrane for later use; and mixing the filtered urine sewage with aerated tap water according to the volume ratio of 1:24 to obtain a culture medium for the astaxanthin-producing microalgae.

Comparative example 2

Taking 200mL of tap water, aerating for 1 hour, and filtering through a 0.22 mu m filter membrane for later use; taking 10mL of urine sewage (source A) generated by a healthy human body and filtering the urine sewage through a 0.22 mu m filter membrane for later use; and mixing the filtered urine sewage and aerated tap water according to the volume ratio of 1:49 to obtain a culture medium for the astaxanthin-producing microalgae.

Comparative example 3

Taking 200mL of tap water, aerating for 1 hour, and filtering through a 0.22 mu m filter membrane for later use; taking 10mL of urine sewage (source A) generated by a healthy human body and filtering the urine sewage through a 0.22 mu m filter membrane for later use; and mixing the filtered urine sewage and aerated tap water according to the volume ratio of 1:74 to obtain a culture medium for the astaxanthin-producing microalgae.

Comparative example 4

Taking 200mL of tap water, aerating for 1 hour, and filtering through a 0.22 mu m filter membrane for later use; taking 10mL of urine sewage (from A) generated by a healthy human body and filtering the urine sewage with a 0.22 mu m filter membrane for later use; and mixing the filtered urine sewage with aerated tap water according to the volume ratio of 1:224 to obtain a culture medium for the astaxanthin-producing microalgae.

Example 5 Effect of the volume ratio of urine wastewater to Water in the Medium according to the invention on astaxanthin-producing microalgae

The culture media prepared in examples 1 to 4 and comparative examples 1 to 4 were placed in six well plates at 5 mL/well, and the initial algal density was 5X 10⁴cells/mL haematococcus pluvialis, culturing for 10d under the conditions of illumination intensity of 4500lux, temperature of 20 ℃ and illumination time of 12h/d, and shaking the six-well plate for 2 times every day during the culturing period.

Samples were taken every other day during the culture and cells were counted using a 0.1mL phytoplankton counting frame under a binocular microscope. Each sample was counted in parallel 3 times.

As a result, as shown in FIG. 1, the algal cells were hard to grow and the algal density rapidly decreased in comparative examples 1, 2 and 3. In example 1, example 2, example 3, example 4 and comparative example 4, algal cells proliferated rapidly and reached substantially maximum biomass within 6 days. Wherein, the algae of the example 3 has the maximum specific growth rate in the logarithmic growth phase, which can reach 0.31d^-1. Example 1 other experimental groups with initial hysteresis of algal cells after inoculation are more evident; comparative example 4 the maximum biomass of algal cells was relatively low due to the low concentration of salt ions in the medium.

Example 6

Taking 1500mL of tap water, aerating for 6 hours, and then filtering with a 0.22-micron filter membrane for later use; 50mL of healthy human urine sewage (source B) is taken to pass through a 0.22 mu m filter membrane for standby; and mixing the filtered urine sewage with aerated tap water according to the volume ratio of 1:149 to obtain a culture medium for the astaxanthin-producing microalgae.

Example 7

100mL of the medium prepared in example 6 was placed in a 250mL conical flask, and the initial algal density was inoculated at 8.05X 10⁴cells/mL haematococcus pluvialis is cultured for 6 days under the conditions of the temperature of 19 ℃, the illumination intensity of 3500lux and the illumination time of 12h/d, and the illumination time of 24h/d is cultured for 9 days. Six well plates were shaken 2 times daily during the incubation period. The microalgae cultured in Erlenmeyer flask are shown in FIG. 2. The cultured haematococcus pluvialis is stressed in a mode of changing the photoperiod for 7-10 days, and the stress period is increased rapidly to 12L:12D (Day0-6) and 24L: 0D. The duration of the experiment was 15 days.

Samples were taken every other day during the culture and cells were counted using a 0.1mL phytoplankton counting frame under a binocular microscope. Each sample was counted in parallel 3 times. Astaxanthin production was measured on day 10 and day 15, respectively. Transferring 30mL of algae solution into 50mL centrifuge tube in conical flask, centrifuging for 15min at 10000r/min with ultracentrifuge, discarding supernatant, adding 5mL of 4mol/L H into the lower precipitate₂SO₄The solution is placed in water bath at 80 deg.C for 5min to break the cell wall of algae.

Centrifuging for 15min at 10000r/min with ultracentrifuge after breaking wall, discarding supernatant, adding 3mL DMSO solution into lower precipitate, and extracting in 45 deg.C water bath for 1 h. Centrifuging for 15min at 10000r/min with ultracentrifuge, collecting supernatant, heating 3mL DMSO solution in 45 deg.C water bath for 1h to extract if precipitate remains at lower part, and repeating the operation until precipitate can not be centrifuged.

Comparative example 5

The initial density of inoculation of Haematococcus pluvialis is 1 × 10⁴The procedure was the same as in example 7 except for the steps of cells/mL.

Comparative example 6

The procedure of example 7 was repeated except that the culture temperature of H.pluvialis was 31 ℃.

Example 8

The procedure of example 7 was repeated except that Haematococcus pluvialis was maintained at a temperature of 25 ℃ and a light intensity of 4500 lux.

Comparative example 7

The procedure of example 8 was repeated except that Haematococcus pluvialis was irradiated with 2500lux of light.

The test results are shown in table 1, from which it can be seen that: as can be seen from the comparison between example 7 and comparative examples 5 and 6, the lower temperature (19 ℃) is favorable for the growth and proliferation of microalgae, and the higher initial density is favorable for the production of astaxanthin; as can be seen from comparison of example 8 with comparative example 7, the higher light intensity (4500lux) is beneficial for astaxanthin accumulation and production, but the effect is not very significant.

TABLE 1 algal specific growth rates and astaxanthin production under different treatment conditions

Example 9

Taking 1500mL of tap water, aerating for 6 hours, and then filtering with a 0.22-micron filter membrane for later use; 50mL of healthy human urine sewage (source C) is taken to pass through a 0.22 mu m filter membrane for standby; and mixing the filtered urine sewage with aerated tap water according to the volume ratio of 1:149 to obtain a culture medium for the astaxanthin-producing microalgae.

Example 10

100mL of the medium prepared in example 9 was placed in a 250mL conical flask, and the initial algal density was inoculated in each flask separately at 8.05X 10⁴cells/mL haematococcus pluvialis is cultured for 6 days under the conditions of the temperature of 19 ℃, the illumination intensity of 4500lux and the illumination time of 12h/d, and the illumination time of 24h/d is cultured for 9 days. Six well plates were shaken 2 times daily during the incubation period.

Samples were taken every other day during the culture and cells were counted using a 0.1mL phytoplankton counting frame under a binocular microscope. Each sample was counted in parallel 3 times. Astaxanthin production was measured on day 10 and day 15, respectively.

In conclusion, the urine sewage adding ratio is 1:149 is most suitable for the growth of haematococcus pluvialis, the proliferation of algae cells is fast, the consumption of nutrient salts is fast, and the growth cycle is short; and (4) obtaining an optimal condition combination through response surface optimization, and analyzing to obtain an optimal temperature, light intensity and initial inoculation algae density combination condition. The urine sewage addition ratio is 1:149 is most suitable for the growth of haematococcus pluvialis, the proliferation of algae cells is fast, the consumption of nutrient salts is fast, and the growth cycle is short; the culture conditions of example 10 were optimized (initial density 8.05X 10)⁴cells/mL, the culture temperature is 19 ℃, the illumination intensity is 4500lux), the response value-specific growth rate can reach 0.102d^-1The yield of astaxanthin can reach 59.55 mg/L.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The economical culture method of astaxanthin-producing microalgae is characterized by comprising the following steps:

(1) inoculating astaxanthin-producing microalgae into culture medium to make the density of astaxanthin-producing microalgae in the culture medium not less than 4 × 10⁴cells/mL to obtain an inoculation culture medium;

the culture medium is used for culturing astaxanthin-producing microalgae and consists of urine sewage and water in a volume ratio of 1: 99-1: 199;

(3) carrying out second culture on the first culture under the condition of 24h/d of illumination time;

the astaxanthin-producing microalgae is haematococcus pluvialis;

the culture temperature of the first culture and the culture temperature of the second culture are 18-22 ℃ independently, and the illumination intensity of the first culture and the illumination intensity of the second culture are 4000-4500 lux independently.

2. The culture method according to claim 1, wherein the water is aerated tap water.

3. The culture method according to claim 1 or 2, wherein the volume ratio of the urine wastewater to water is 1:140 to 1: 160.

4. The culture method according to claim 1, wherein the culture medium is obtained by filtering urine wastewater and water separately with a microporous membrane and mixing the filtered urine wastewater and water at a volume ratio.

5. The culture method according to claim 1, wherein the culture time of the first culture is 5 to 7 days.

6. The culture method according to claim 1, wherein the second culture time is 8 to 12 days.

7. Use of the culture method of any one of claims 1 to 6 for purifying urine wastewater.