Oil droplet algae and culture application thereof

Abstract

The present application relates to a strain of oil-rich chlorella vulgaris (Oocystis sp.) Graesiella emersonii ) SZLSi-3, which was preserved in China General Microbiological Culture Collection Center on April 23, 2021, and the preservation number is CGMCC No. 22392. The chlorella vulgaris provided by the present application can grow using CO2, can obtain oil-rich biomass, and has low-temperature resistance and NOx tolerance.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and bioenergy, specifically relating to an oil-rich algae and its cultivation and application. Background Technology

[0002] Since the beginning of the 21st century, economic development has been closely linked to energy consumption. Energy security is a strategic issue for all countries, and finding a clean, economical, efficient, and renewable new energy source has become an urgent problem to solve. Among these, bioenergy has always been an important reserve technology direction, and various countries have maintained interest in it. Among various biomass energy sources, microalgae have advantages such as high photosynthetic efficiency, fast growth rate, high biomass yield, and strong environmental adaptability. They have comprehensive utilization value and do not compete with food crops for land or food. The fatty acids obtained from microalgae can be converted into fatty acid methyl esters, i.e., biodiesel. Therefore, oil-rich microalgae are often considered an ideal raw material for third-generation biofuels.

[0003] Microalgae cells accumulate components such as lipids, starch, proteins, and pigments during their growth. In some algae, starch and glycogen account for 30% of the cell dry weight, while in others, the lipid content can reach as high as 80% under specific culture conditions. Microalgae have a solar energy conversion efficiency of up to 3.5%, making them a potential resource for the production of pharmaceuticals and fine chemicals. How to effectively screen for new algal strains with high lipid content, strong environmental adaptability, scalability, and low cost has become a key research focus for researchers worldwide.

[0004] CN105543099A discloses a strain of *Olecopodium* (… Graesiella sp. The isolation and screening method and application of WBG-1. This algal strain grows rapidly, adapts strongly to pH changes, and also possesses characteristics suitable for large-scale culture, such as large cell size, easy sedimentation, and resistance to protozoan ingestion. The *Oleocybe* species described herein... Graesiella sp. WBG-1 algae accumulates a large amount of lipids in its cells under low-nitrogen culture conditions, which can be used as a feedstock for microalgal biodiesel. Under high-nitrogen culture conditions, it accumulates a large amount of protein in its cells, which can be used for the production of microalgal proteins. Under higher pH conditions, it can efficiently fix exogenous carbon dioxide, which can be applied to the biofixation of carbon dioxide in flue gas. This algal strain can accumulate lipids and proteins under different culture conditions, and can effectively fix carbon dioxide at higher pH. This algal strain was cultured under natural light and temperature conditions with an average daily temperature of 21.8–30.5℃. The effect is poor or even death occurs in cold weather; moreover, when the flue gas contains toxic and harmful substances such as NOx, the effect of flue gas culture decreases or even leads to algal cell death.

[0005] Temperature has a significant impact on the growth and reproduction of microalgae. The optimal survival temperature for microalgae is typically 15-35℃. Temperatures that are too low can lead to slow or even halted growth. Furthermore, microalgae growth requires a greater amount of energy in cold winter weather. Therefore, selecting and breeding multifunctional microalgae that are tolerant to low temperatures, high pH levels, NOx, and SOx, making them more suitable for industrial applications, is of great importance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an oil-rich *Olecosella oleifera* strain and its cultivation applications. The *Olecosella oleifera* strain provided by this invention utilizes CO2 for growth, yielding oil-rich biomass, and also exhibits low-temperature tolerance and NOx tolerance.

[0007] This invention provides an oil-rich *Olecopodium* strain (… Graesiella emersonii SZLSi-3 was deposited on April 23, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 22392.

[0008] The *Oleocybe sZLSi-3* provided by this invention has green algal cells under a microscope. The cells are spherical or ellipsoidal, with a diameter of 6-8 μm. The cells contain pigments and have no flagella on the cell surface.

[0009] The SZLSi-3 oil-bearing algae provided by this invention can tolerate CO2 concentrations up to 40 v, NOx concentrations up to 0.09 v, and low temperatures as low as 5 °C.

[0010] The 18S rDNA gene sequencing analysis results of *S. oleifera* SZLSi-3 provided by this invention are shown in the sequence listing. According to sequence alignment, there are certain differences between the 18S rDNA of *S. oleifera* SZLSi-3 of this invention and previously published *S. oleifera* species.

[0011] This invention also provides an oil-bearing algae ( Graesiella emersonii The cultivation method for SZLSi-3 involves using a freshwater culture medium in a photobioreactor, with a CO2 concentration of 1.0 v%-40 v%, preferably 5 v%-30 v%. The cultivation conditions are: light intensity of 1500-20000 Lux, pH of 6.0-8.5, temperature of 5-35℃, preferably 5-15℃, a photoperiod of 24 hours, and a light-dark ratio of 14:10-10:14. After reaching the stationary phase, the microalgal cells are harvested. Analysis shows that the algal cell dry weight reaches over 6.5 g / L, and the total lipid content accounts for over 40% of the cell dry weight.

[0012] In the cultivation method of the present invention, the freshwater culture medium can be any one of BG11 medium, SE medium or D1 medium.

[0013] In the cultivation method of the present invention, the introduced gas also contains NOx, and the concentration of NOx is not higher than 0.09v.

[0014] The present invention relates to the application of *Oleocybe szolium* SZLSi-3 in CO2 fixation. This algal strain can utilize CO2 for photoautotrophic growth, tolerate CO2 concentrations up to 40% v%, and exhibits high CO2 fixation efficiency, exceeding 60%.

[0015] The present invention relates to the application of *Olecopodium sibiricum* SZLSi-3 in the production of microalgal oils. This algal strain undergoes photoautotrophic growth under suitable conditions to obtain oil-rich algal cells with a dry weight exceeding 6.5 g / L and a total lipid content exceeding 40% of the cell dry weight. This makes it suitable as a raw material for the production of biofuels and other oil products.

[0016] The present invention relates to the application of *Oleocybe siliflora* SZLSi-3 in purifying waste gas or flue gas containing CO2 and NOx. The CO2 concentration in the waste gas or flue gas is ≤40 vol%, and the NOx concentration is ≤0.09 vol%.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The oil-bearing algae selected in this invention ( Graesiella emersonii SZLSi-3 can grow efficiently under autotrophic conditions using CO2, with high carbon sequestration efficiency, thus alleviating the CO2 greenhouse effect problem brought about by the current industrial society.

[0018] (2) Oil-bearing algae ( Graesiella emersonii SZLSi-3 also possesses low-temperature resistance. This algal strain has strong low-temperature resistance and can still maintain a normal growth rate at 5-15℃, reducing cultivation costs during winter cultivation and solving the problem of seasonal limitations in microalgae production.

[0019] (3) Oil-bearing algae ( Graesiella emersonii SZLSi-3 possesses both NOx tolerance and removal capabilities. When CO2 and NOx are present in the exhaust gas, it can prevent the inhibition of microalgae photosynthesis by NOx during microalgae growth, maintain normal microalgae growth, and remove some NO.

[0020] (4) The product obtained from this algae strain is highly economical. After 6 days of cultivation, the dry weight of the algal cells reaches more than 6.5 g / L, and the total lipid content of the cells accounts for more than 40% of the dry weight of the cells, making it suitable for the production of biodiesel.

[0021] Instructions for the Preservation of Biological Materials The oil-bearing algae provided by this invention ( Graesiella emersoniiSZLSi-3 is deposited at the China General Microbiological Culture Collection Center (CGMCC); accession number: CGMCC No. 22392; deposit date: April 23, 2021; address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0022] The present invention and its effects will be further described in detail below with reference to specific embodiments. The embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures; however, the scope of protection of the present invention is not limited to the following embodiments.

[0023] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores. In this invention, v% refers to volume fraction.

[0024] Example 1: Isolation, domestication, and screening to obtain *Olecopodium spp.* SZLSi-3 (1) Obtaining the starting algal strain: In October 2015, 150 mL of water sample was collected from Donghu Lake of Liaoning Petroleum University in Fushun City. The water sample was filtered with gauze to remove large impurities. 50 mL of the filtered water sample was inoculated into 200 mL of BG11 medium for enrichment culture. The light intensity was 5000 Lux, the temperature was 15℃, the photoperiod was 24 h, and the light-dark ratio was 14:10. After about half a month of culture, the medium turned green. The enriched water sample was diluted to 10 -5 The algae were spread onto BG11 solid plates under aseptic conditions and cultured at a light intensity of 5000 Lux and a temperature of 15°C. After approximately 10 days of culture, single green algal colonies appeared on the plates. These colonies were then picked and cultured in shake flasks at 15°C and a light intensity of 5000 Lux. After 8 days of culture, microscopic examination was performed to determine if the algae were a pure culture. If not, the above steps were repeated until a pure culture was confirmed. After repeated culture, a pure algal strain was obtained and designated SZL-3.

[0025] (2) Domestication and cultivation of CO2: The pure algae cultured in the shake flask in step (1) was introduced into the microalgae aeration culture device for domestication and cultivation. The light intensity was 5000 Lux and the temperature was 10℃. The CO2 content in the introduced gas was gradually increased from 5v% to 40v%, each time by 5v%. The culture was completed after 8 days and repeated 3 times.

[0026] (3) The algal culture obtained in step (2) is cultured by streak plating to obtain pure algal strains. The culture steps are the same as in (1).

[0027] (4) NOx acclimatization culture: The algal solution in the logarithmic growth phase of step (3) was introduced into a mixed gas with a CO2 content of 20v%, a light intensity of 5000 Lux, and a temperature of 10℃. NO gas was injected into the mixed gas to cultivate the algal species to tolerate NOx. During the cultivation process, the NO content in the mixed gas was gradually increased from 0.01% to 0.09%, and increased by 0.01% every day during the cultivation process. After the cultivation was completed, the acclimatization culture was repeated 3 times to harvest the NOx-tolerant algal solution.

[0028] (5) The algal culture obtained in step (4) was cultured by streak plating to obtain a pure algal strain. The culture steps were the same as in (1). After the culture was completed, larger algal colonies were selected for shake flask culture to obtain the target algal strain, which was named SZLSi-3.

[0029] Example 2 Identification of Algal Strains DNA was extracted from the obtained SZLSi-3 microalgae cells using the CTAB method, and the 18S rDNA gene was cloned. Three positive clones were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The 18S rDNA gene sequencing analysis results are shown in the sequence listing. The 18S rDNA sequence was entered into the GenBank database for BLAST alignment, and the results showed that it was consistent with... Graesiella emersonii With the highest similarity (BLASTn value of 2375 and Max index value of 99.92%), SZLSi-3 can be identified as *Olecopodium oleiferum*. Graesiella emersonii ).

[0030] Example 3: Application of *Oleocybean* SZLSi-3 The logarithmic growth phase of *Oleocybe sZLSi-3* algal culture was inoculated into BG11 medium and cultured. The formulation of BG11 medium is shown in Tables 1 and 2. The culture was carried out in a photobioreactor. The OD of the culture medium after inoculation was... 690 The concentration was 0.27. Flue gas with a CO2 content of 40% v% and a NO content of 0.09% was introduced from the bottom of the reactor. During cultivation, the light intensity was 8000 Lux, the cultivation temperature was 15℃, the pH was controlled at 7-8, the photoperiod was 24h, the light-dark ratio was 14:10, and the cultivation time was 6 days. After cultivation, the algal solution was collected by centrifugation, and then freeze-dried under vacuum at -60℃ to constant weight. The dry weight of the algal powder was measured, the biomass yield was calculated, and the total lipid content was determined using the n-hexane:ethyl acetate method. The results showed that the biomass yield of the SZLSi-3 algal strain was 6.89 g / L, and the total lipid content of the cells accounted for 42.39% of the cell dry weight.

[0031] Table 1 BG11 culture medium *Table 2 Composition of A5+Co solution in Table 1 Example 4: Comparison of the cultivation effects of SZL-3 and SZLSi-3 Algal cultures of SZL-3 and SZLSi-3 in the logarithmic growth phase were inoculated into BG11 medium and cultured in a photobioreactor. The OD values ​​of the inoculated culture media were measured. 690 The concentration was 0.31. Different concentrations of CO2 and NO mixed gas were prepared according to experimental requirements and then introduced from the bottom of the reactor. During cultivation, the light intensity was 8000 Lux, the temperature was 15℃, the pH was controlled at 7-8, the photoperiod was 24 h, the light-dark ratio was 14:10, and the cultivation time was 7 days. After cultivation, algal cells were collected. The algal powder was then freeze-dried under vacuum at -60℃ to constant weight, and the dry weight was measured to calculate the biomass. The total lipid content of the cells was determined using the n-hexane:ethyl acetate method. The results are shown in Table 3.

[0032] Table 3 As shown in Table 3, with the increase of CO2 content in the gas, the SZLSi-3 strain selected in this invention exhibits more stable tolerance to CO2 than the initial SZL-3 strain, with higher biomass and total lipid content. Under conditions where the NO concentration is no higher than 0.09 v%, it has almost no impact on the normal growth and lipid accumulation of the microalgae, and also demonstrates a certain NO removal rate.

[0033] Example 5: Comparison of culture temperatures for SZLSi-3 and SZL-3 The logarithmic growth phase algal cultures of SZLSi-3 and SZL-3 were inoculated into BG11 medium and cultured in a photobioreactor. The OD of the inoculated culture medium was measured. 690 The concentration was 0.25. Flue gas with a CO2 content of 10v% and a NO content of 0.05v% was introduced from the bottom of the reactor. During cultivation, the light intensity was 8000 Lux, the pH was controlled at 7-8, the photoperiod was 24 h, the light-dark ratio was 14:10, and the cultivation time was 87 days. After cultivation, algal cells were collected by centrifugation, and then freeze-dried under vacuum at -60℃ to constant weight. The dry weight of the algal powder was measured, the biomass was calculated, and the total lipid content of the cells was determined using the n-hexane:ethyl acetate method. The results are shown in Table 4.

[0034] Table 4 As can be seen from Table 4, the SZLSi-3 strain of the present invention has better low-temperature tolerance than the initial algal strain SZL-3.

[0035] Comparative Example 1 Same as Example 3, except that it uses Scenedesmus HCS-02 disclosed in CN107177505A ( Scenedesmus sp. The culture, with accession number CGMCC No. 10766, was completed after 8 days. Analysis showed a biomass yield of 3.1 g / L and a total lipid content of 20.78% of the cell dry weight. However, the culture was unsuccessful due to the microalga's inability to tolerate NO.

[0036] Comparative Example 2 Similar to Example 3, but using Chlorella SF-B1 disclosed in CN109576158A, with accession number CGMCC No. 11005, and cultured for 8 days. The biomass yield was measured at 4.5 g / L, and the total lipid content of the cells accounted for 21.65% of the cell dry weight. Because this microalga is intolerant to low temperatures, the culture results were poor.

Claims

1. A species of oil-rich algae, known as *Olecoscoleum* (or *Olecoscoleum*). Graesiella emersonii SZLSi-3 was deposited on April 23, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 22392.

2. The *Olecopodium* according to claim 1, characterized in that: Under a microscope, algal cells appear green, spherical or ellipsoidal in shape, with a diameter of 6-8 μm. They contain pigments and have no flagella on their surface.

3. The *Olecopodium* according to claim 1, characterized in that: The oil-bearing algae SZLSi-3 can tolerate CO2 concentrations up to 40 v, NOx concentrations up to 0.09 v, and low temperatures down to 5 °C.

4. The *Olecopodium* as described in claim 1 ( Graesiella emersonii The cultivation method of SZLSi-3 is characterized by: In the photobioreactor, freshwater culture medium is used for cultivation, and gas with a CO2 content of 1.0v%-40v% is introduced.

5. The method according to claim 4, characterized in that: A gas with a CO2 content of 5v%-30v% is introduced.

6. The method according to claim 4, characterized in that: The cultivation conditions were as follows: light intensity 1500-20000 Lux, pH 6.0-8.5, temperature 5-35℃, photoperiod 24h, and light-dark time ratio 14:10-10:

14.

7. The method according to claim 6, characterized in that: The temperature is 5-15℃.

8. The method according to claim 4 or 5, characterized in that: After culturing until the stable period, the microalgal cells were harvested. Tests showed that the dry weight of the algal cells was over 6.5 g / L, and the total lipid content of the cells accounted for over 40% of the dry weight of the cells.

9. The method according to claim 4, characterized in that: The freshwater culture medium is any one of BG11 medium, SE medium or D1 medium.

10. The method according to claim 4, characterized in that: The introduced gas also contains NOx, and the concentration of NOx is not higher than 0.09v.

11. The application of the oil-bearing algae SZLSi-3 as described in claim 1 in CO2 fixation.

12. The application according to claim 11, characterized in that: The aforementioned *Oleocybe sZLSi-3* can utilize CO2 for photoautotrophic growth, tolerate CO2 concentrations up to 40% v%, and has a high CO2 fixation efficiency of over 60%.

13. The application of the oil-bearing algae SZLSi-3 as described in claim 1 in the production of microalgae oil.

14. The application according to claim 13, characterized in that: The oil-rich algae SZLSi-3 were obtained by photoautotrophic growth under suitable growth conditions, with a dry weight of over 6.5 g / L and a total lipid content of over 40% of the dry weight of the cells.

15. The application of the oil-bearing algae SZLSi-3 as described in claim 1 in purifying waste gas or flue gas containing CO2 and NOx.

16. The application according to claim 15, characterized in that: The CO2 concentration in the exhaust gas or flue gas is ≤40v, and the NOx concentration is ≤0.09v.

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