Oil-producing foot-shaped algae and culture application thereof

Abstract

The present application relates to a strain of oil-producing legenidium (Legenidium sp. Kirchneriella lunaris ) TMJ-B4, which was preserved in the China General Microbiological Culture Collection Center on April 23, 2021, and the preservation number is CGMCC No. 22394. The oil-producing legenidium provided by the present application can grow by utilizing CO2, obtain biomass rich in oil, and has strong tolerance to copper ions and can adsorb a certain amount of copper ions.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and bioenergy, and specifically relates to an oil-producing hoof alga 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. Moreover, they 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] The literature "Study on Factors Affecting the Growth and Lipid Synthesis of *Hylocereus undatus* and *Synechocera*" (Xu Yang, Master's Thesis, 2013) explores the effects of nutrient elements on the growth of these two microalgae, *Hylocereus undatus*. Kirchneriella sp. zp-1) and mononephrite ( Monoraphidium sp. The effects of AGP experiments on Mg growth and oil production were investigated. 2+ Ca 2+ Fe 3+ Zn 2+ Mn 2+ NO3 - HPO4 2- The effects of plasma and three plant hormones—indoleacetic acid (IAA), naphthaleneacetic acid (NAA), and 6-benzylaminopurine (6-BA)—on the growth of *Houttuynia cordata* and *Syngonium moniliforme* were investigated, and the optimal concentrations of these factors for the growth of the two algae were determined.

[0005] However, the microalgae isolated so far often have relatively limited functions, and their resistance to biofouling (such as chemical defense capabilities and tolerance to extreme environments) needs to be improved. Therefore, breeding microalgae with strong tolerance and diverse functions will help expand the application fields of microalgae and make them more suitable for industrial applications. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an oil-producing hoof-shaped algae and its cultivation and application. The oil-producing hoof-shaped algae provided by this invention can utilize CO2 for growth, obtaining oil-rich biomass, and exhibits strong tolerance to copper ions, capable of adsorbing a certain amount of copper ions.

[0007] This invention provides an oil-producing hoof-shaped algae ( Kirchneriella lunaris TMJ-B4 was deposited on April 23, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 22394.

[0008] The hoof-shaped algae provided by this invention ( Kirchneriella lunaris Under a microscope, TMJ-B4 algal cells are green. Algal cells are often clustered in groups of 4-8. Individual cells are curved into a hoof or sickle shape, 4-6 μm long and 1-3 μm wide in the center.

[0009] The hoof-shaped algae provided by this invention ( Kirchneriella lunaris TMJ-B4 can tolerate CO2 concentrations up to 30% and copper ion concentrations up to 160 ppm.

[0010] The hoof-shaped algae provided by this invention ( Kirchneriella lunaris The 18S rDNA gene sequencing analysis results of TMJ-B4 are shown in the sequence listing. Based on sequence alignment, *Hylocereus undatus* (… Kirchneriella lunaris TMJ-B4 differs somewhat from the 18S rDNA of the previously published Algae horn.

[0011] This invention also provides a hoof-shaped algae ( Kirchneriella lunaris The TMJ-B4 algae were cultured in a photobioreactor using a freshwater medium, with a CO2 concentration of 1.0 v%-30 v%, preferably 5 v%-20 v%. The culture conditions were: light intensity of 1500-20000 Lux, pH of 6-10, temperature of 15-35℃, preferably 20-30℃, a photoperiod of 24 h, and a light-dark ratio of 14:10-10:14. The culture was continued until the stationary phase, at which point the microalgal cells were harvested. Analysis showed that the algal cell dry weight reached over 5 g / L, and the total lipid content accounted for over 45% of the cell dry weight.

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

[0013] The hoof-shaped algae described in this invention ( Kirchneriella lunarisApplication of TMJ-B4 in CO2 fixation. This algal strain can utilize CO2 for photoautotrophic growth, exhibits high CO2 fixation efficiency, and can tolerate CO2 concentrations up to 30%.

[0014] The hoof-shaped algae described in this invention ( Kirchneriella lunaris Application of TMJ-B4 in the production of microalgal lipids. This algal strain, under suitable growth conditions, undergoes photoautotrophic growth to obtain lipid-rich algal cells with a dry weight exceeding 5 g / L and a total lipid content exceeding 45% of the cell dry weight.

[0015] The hoof-shaped algae described in this invention ( Kirchneriella lunaris Application of TMJ-B4 in copper ion adsorption. This algal strain can adsorb a certain concentration of copper ions without affecting the normal growth of microalgae. The copper ion concentration in the system is no higher than 160 ppm, and the copper ion removal rate is higher than 80%.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The hoof-shaped algae obtained by screening in this invention ( Kirchneriella lunaris TMJ-B4 can efficiently grow under autotrophic conditions using CO2, with high carbon sequestration efficiency, thus alleviating the CO2 greenhouse effect problem brought about by the current industrial society.

[0017] (2) Hoof-shaped algae ( Kirchneriella lunaris TMJ-B4 can adsorb a certain concentration of copper ions, with a copper ion concentration not exceeding 160 ppm and a copper ion removal rate exceeding 80%.

[0018] (3) Hoof-shaped algae ( Kirchneriella lunaris The algal cells obtained from TMJ-B4 are highly economical. Under normal culture conditions, the dry weight of algal cells can reach 5 g / L, and the total lipid content of cells accounts for more than 45% of the dry weight of cells. They can be used as raw materials for the production of biodiesel and other oil products.

[0019] Instructions for the Preservation of Biological Materials The hoof-shaped algae provided by this invention ( Kirchneriella lunaris TMJ-B4 is deposited at the China General Microbiological Culture Collection Center (CGMCC); accession number: CGMCC No. 22394; deposit date: April 23, 2021; deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0020] The method and its effects of the present invention will be further described in detail below with reference to specific embodiments. These 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.

[0021] 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.

[0022] Example 1: Isolation, domestication, and screening to obtain the hoof algae TMJ-B4 (1) Obtaining the starting algal strain: In October 2013, 150 mL of water sample was collected from the Tumen River in Tumen City, Jilin Province. The water sample was filtered through 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 25℃, 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 25°C. After about 10 days of culture, single green algal colonies appeared on the plates. These colonies were then picked and cultured in shake flasks at 25°C and a light intensity of 5000 Lux. After 8 days of culture, microscopic observation was performed to determine if it was a pure algal strain. If not, the above steps were repeated until a pure culture was confirmed. After repeated culture, a pure algal strain was obtained and designated TMJ-4.

[0023] (2) Copper ion acclimatization culture: The pure algae cultured in the shake flask in step (1) is introduced into the microalgae aeration culture device for acclimatization culture. The light intensity is 5000 Lux, the CO2 content in the introduced gas is 5v%, the culture temperature is 25℃, the copper ion concentration in the culture system is 50ppm, which is increased by 10ppm every day. The culture is ended after 8 days, and the acclimatization culture is repeated 3 times.

[0024] (3) The algal culture solution acclimatized in step (2) was cultured in a streak plate 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 TMJ-B4.

[0025] Example 2 Identification of Algal Strains DNA was extracted from *Tectus hoofiae* TMJ-B4 algal 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... Kirchneriella lunaris It exhibits the highest similarity, with a BLASTn value of 2158 and a Max index value of 99.41%, confirming that TMJ-B4 is an algae known as Hoofia. Kirchneriella lunaris ).

[0026] Example 3: Cultivation and application of *Houttuynia cordata* TMJ-B4 The logarithmic growth phase of *Hylocereus undatus* TMJ-B4 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 0.2. A CO2 gas with a concentration of 10v% was introduced from the bottom of the reactor. During cultivation, the light intensity was 8000 Lux, the cultivation temperature was 25℃, the pH was controlled at 7-8, the photoperiod was 24h, the light-dark ratio was 14:10, and the cultivation time was 8 days, reaching a stable period. 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 TMJ-B4 was 5.59 g / L, and the total lipid content of the cells accounted for 45.59% of the cell dry weight.

[0027] Table 1 BG11 culture medium *The composition of A5+Co solution in Table 1 is shown in Table 2. Example 4: Comparison of the cultivation effects of TMJ-4 and TMJ-B4 Algal cultures of TMJ-4 and TMJ-B4 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 0.2. Different concentrations of CO2 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 pH was controlled at 7-8, the photoperiod was 24 h, the light-dark ratio was 14:10, and the cultivation time was 8 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.

[0028] Table 3 The results show that the TMJ-B4 algal strain selected in this invention has better biomass and lipid content than the initial algal strain TMJ-4, and also has a certain ability to remove copper ions.

Claims

1. An oil-producing hoof-shaped algae, which is a hoof-shaped algae ( Kirchneriellalunaris TMJ-B4 was deposited on April 23, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 22394.

2. The hoof-shaped algae as described in claim 1 ( Kirchneriellalunaris The cultivation method of TMJ-B4 is characterized by: In the photobioreactor, freshwater culture medium is used for cultivation, and gas with a CO2 content of 1.0v%-30v% is introduced.

3. The cultivation method according to claim 2, characterized in that: A gas with a CO2 content of 5v%-20v% is introduced.

4. The cultivation method according to claim 2, characterized in that: The cultivation conditions are as follows: light intensity 1500-20000 Lux, pH value 6-10, temperature 15-35℃, photoperiod 24h, light-dark time ratio 14:10-10:14, and microalgal cells are harvested after the culture reaches the stationary phase.

5. The cultivation method according to claim 4, characterized in that: The temperature is 20-30℃.

6. The cultivation method according to claim 2, characterized in that: The freshwater culture medium is any one of BG11 medium, SE medium or D1 medium.

7. The hoof-shaped algae as described in claim 1 ( Kirchneriellalunaris Application of TMJ-B4 in CO2 fixation.

8. The hoof-shaped algae as described in claim 1 ( Kirchneriellalunaris Application of TMJ-B4 in the production of microalgae oil.

9. The hoof-shaped algae as described in claim 1 ( Kirchneriellalunaris Application of TMJ-B4 in the adsorption of copper ions.

10. The application according to claim 9, characterized in that: This algal strain can adsorb a certain concentration of copper ions, with the copper ion concentration in the system not exceeding 160 ppm and the copper ion removal rate exceeding 80%.

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