Pictocystis pictoricola as well as culture and application thereof

Abstract

The invention relates to a chlorocystis pichoricola strain as well as culture and application thereof, the chlorocystis pichoricola strain is oil-producing chlorocystis pichoricola FNH-5 which is preserved in the China General Microbiological Culture Collection Center on February 21, 2023, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.45485. The invention further discloses a preparation method of the chlorocystis pichoricola strain. The oil-producing picetocystis provided by the invention can immobilize CO2, especially has good petroleum hydrocarbon degradability, can grow in a petroleum hydrocarbon-containing system and enrich oil biomass, and realizes CO2 immobilization, pollution treatment and comprehensive utilization of oil prepared from microalgae.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology and petroleum pollution control technology, specifically involving a strain of Piccardia green algae and its cultivation and application. Background Technology

[0002] With the increasing expansion of offshore oil exploration and transportation, oil pollution has arisen due to well blowouts, pipeline leaks, and oil spills caused by collisions or sinkings of large oil tankers. Oil pollution poses long-term threats to the environment, natural resources, and aquaculture resources, and has become one of the environmental problems that humanity must address. Currently, common methods for treating oil pollution include physical, chemical, and biological methods. Physical and chemical methods often leave residues and cause secondary pollution. Biological methods have advantages such as simplicity, low cost, and no secondary pollution. However, the ocean is a dynamic and open system, leading to problems with biological treatment, such as the easy dilution of the microorganisms used and the difficulty in reusing them.

[0003] Recent studies have shown that using planktonic microalgae to purify oil-polluted waters can not only reduce oil pollution but also, based on research into microalgae fuels, utilize the resulting algal cells for biodiesel production. Compared to traditional treatment methods, using microalgae to treat oil-polluted waters has advantages such as low cost, no secondary pollution, low energy consumption, and high efficiency, making it a highly promising environmental protection project. Furthermore, it can provide algal biomass for research into new renewable energy sources.

[0004] Liang Yizhi et al. (“Study on the Enrichment of Lipids by Chlorella and Dunaliella using Petroleum Hydrocarbons”, Liaoning Normal University) obtained dominant strains of marine Chlorella and Dunaliella salina using single-cell isolation and ultraviolet mutagenesis techniques. These two microalgae can degrade hydrocarbons with conjugated double bonds and aromatic petroleum hydrocarbons, and can be cultured in petroleum wastewater to enrich microalgal lipids. However, the degradation efficiency of petroleum hydrocarbons by these two microalgae is not very high, and the lipid content is only about 20%.

[0005] Therefore, breeding more microalgae that can efficiently degrade petroleum hydrocarbons can provide a greener and more economical approach to the treatment of petroleum pollution. At the same time, they can also be used to obtain biodiesel and other products through petroleum hydrocarbon-oil conversion, thus realizing the resource utilization of microalgae biomass. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an oil-producing *Pick's Green Algae* strain, its cultivation, and its applications. The *Pick's Green Algae* strain provided by this invention can fix CO2, and in particular, exhibits excellent petroleum hydrocarbon degradation capabilities. It can grow and enrich oil biomass in petroleum hydrocarbon-containing systems, achieving comprehensive utilization of CO2 fixation, pollution control, and microalgae-based oil extraction.

[0007] The first aspect of this invention provides a strain of Picochlorum sp. FNH-5, which was deposited on February 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45485.

[0008] The Picochlorum sp. FNH-5 provided by this invention has green algal cells under a microscope. The cells are single cells, and each single cell is spherical or ellipsoidal with a smooth cell wall and a single chloroplast inside. The chloroplast is plate-like, and each cell contains a protein nucleus, which is sometimes not obvious. The cell diameter is 2-6 μm.

[0009] The Picochlorum sp. FNH-5 provided by this invention can fix CO2, tolerate CO2 concentrations up to 30v, and is suitable for growth at CO2 concentrations of 1.0v%-10v.

[0010] The Picochlorum sp. FNH-5 provided by this invention can tolerate petroleum hydrocarbon concentrations up to 50 mg / L, and when the petroleum hydrocarbon concentration in the system is below 30 mg / L, the petroleum hydrocarbon removal rate is above 90%.

[0011] The 18S rDNA gene sequencing analysis results of Picochlorum sp. FNH-5 provided by this invention are shown in the sequence listing. According to sequence alignment, Picochlorum sp. FNH-5 differs from previously published Picochlorum strains.

[0012] The second aspect of this invention provides a method for culturing *Picochlorum sp.* FNH-5. The method involves culturing the algae in a photobioreactor using a seawater microbial culture medium, with a CO2 concentration of 1.0 v%-30 v%, preferably 5 v%-20 v%, and culturing under the following conditions: light intensity of 1500-20000 Lux, pH of 5-8, temperature of 15-35℃, preferably 20-30℃, a photoperiod of 24 h, and a light-dark ratio of 14:10-10:14. The algae are cultured until the stationary phase, at which point the microalgal cells are harvested. Testing shows that the dry weight of the algal cells reaches over 5 g / L, and the total lipid content of the cells accounts for over 45% of the dry weight.

[0013] In the cultivation method of the present invention, the seawater microbial culture medium is a conventional culture medium for culturing marine microalgae, preferably an enriched f / 2 medium.

[0014] A third aspect of this invention provides the application of *Picochlorum* sp. in CO2 fixation. This algal strain is capable of photoautotrophic growth using CO2, exhibits high CO2 fixation efficiency, and can tolerate CO2 concentrations up to 30% v%.

[0015] The fourth aspect of this invention provides the application of Picochlorum sp. FNH-5 in a petroleum hydrocarbon degradation system. This algal strain can tolerate petroleum hydrocarbon concentrations up to 50 mg / L without affecting the normal growth of the microalgae. When the petroleum hydrocarbon concentration in the system is not higher than 30 mg / L, the petroleum hydrocarbon removal rate is higher than 90%.

[0016] The fifth aspect of this invention provides an application of Picochlorum sp. FNH-5 in the production of microalgal lipids. This algal strain undergoes photoautotrophic growth under suitable growth conditions to obtain lipid-rich algal cells, with a dry cell weight exceeding 5 g / L and a total lipid content exceeding 45% of the cell dry weight.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The Pick Chlorella FNH-5 strain selected in this invention can grow efficiently under autotrophic conditions using CO2, and has high carbon fixation efficiency, which helps to achieve the dual carbon target.

[0019] (2) Picq Green Cell Algae FNH-5 can tolerate a concentration of petroleum hydrocarbons up to 50 mg / L. When the concentration of petroleum hydrocarbons in the system is not higher than 30 mg / L, the degradation rate of petroleum hydrocarbons is higher than 90%.

[0020] (3) Pick's green algae FNH-5 has high economic value. Under normal culture conditions, the dry weight of algal cells can reach 5g / L, and the total lipid content of cells accounts for more than 45% of the dry weight of cells. It can be used as a raw material for the production of biodiesel and other oil products.

[0021] (4) Pick's green algae FNH-5 can cultivate microalgae in oil-polluted seawater. While purifying and repairing oil-polluted seawater, it also enriches the microalgae with oil, thus achieving comprehensive utilization of pollution control, CO2 fixation and biodiesel production.

[0022] Instructions for the Preservation of Biological Materials

[0023] The *Picochlorum* sp. FNH-5 provided by this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC); accession number: CGMCCNo.45485; deposit date: February 21, 2023; deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0024] 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.

[0025] 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.

[0026] Example 1: Screening, isolation and identification of Pick's green algae FNH-5

[0027] (1) Obtaining the starting algal strain: In October 2018, 150 mL of water sample was collected from a seaside location in Dalian. The water sample was filtered through gauze to remove large impurities. 50 mL of the filtered water sample was inoculated into 200 mL of enriched f / 2 medium for enrichment culture. The formulation of the enriched f / 2 medium is shown in Tables 1, 2, and 3. The seawater in the medium was taken from the World Peace Park in Lushunkou District. The light intensity for culture was 5000 Lux, the temperature was 25℃, the photoperiod was 24 h, and the light-dark ratio was 14:10. After 12 days of culture, the medium turned green. The enriched water sample was diluted to 10... -5 The culture medium was spread onto enriched f / 2 solid plates under aseptic conditions and cultured at a light intensity of 5000 Lux and a temperature of 25°C. After approximately 9 days of culture, single green algal colonies appeared on the plates. These colonies were then picked and cultured in shake flasks at 25°C under a light intensity of 5000 Lux. After 8 days of culture, microscopic examination was performed to determine if the algal strain was pure. If not, the above steps were repeated until a pure culture was confirmed. After repeated culture, a pure algal strain was obtained and designated FNH-0.

[0028] (2) Domestication and cultivation of petroleum hydrocarbons: 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, the CO2 content in the introduced gas was 5v%, the culture temperature was 25℃, the concentration of petroleum hydrocarbons added to the culture system was 10 mg / L, and the concentration was increased by 5 mg / L every day. The culture was ended after 8 days, and the domestication and cultivation were repeated 3 times.

[0029] (3) The algal culture obtained in step (2) 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 FNH-5.

[0030] Table 1. Formulation of enriched f / 2 culture medium

[0031]

[0032] Table 2. F / 2 Trace Metal Solution

[0033] reagents Mother liquor Added amount <![CDATA[FeCl3·6H2O]]> — 6g <![CDATA[Na2EDTA·2H2O]]> — 4.36g <![CDATA[CuSO4·5H2O]]> 9.8g / L 1.0mL <![CDATA[Na2MoO4·2H2O]]> 6.3g / L 1.0mL <![CDATA[ZnSO4·7H2O]]> 22.0g / L 1.0mL <![CDATA[CoCl2·6H2O]]> 10.0g / L 1.0mL <![CDATA[MnCl2·4H2O]]> 180.0g / L 1.0mL Distilled water (to the final volume) — 1.0L

[0034] Table 3. Jiafu f / 2 Vitamin Solution

[0035] reagents Mother liquor Added amount Concentration after addition final concentration of culture medium <![CDATA[Vitamin B 12 ]]> 1.0g / L 1.0mL 1mg / L 0.5 μg / L Biotin 0.1g / L 10.0mL 1mg / L 0.5 μg / L <![CDATA[Thiamine HCl B1]]> — 200.0mg 300mg / L 150 μg / L Distilled water (to the final volume) — 1.0L — —

[0036] DNA was extracted from the obtained *Picochlorum sp.* FNH-5 algal cells using the CTAB method, and the 18S rDNA gene was cloned and sequenced. 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. The results showed the greatest similarity to *Picochlorum sp.*, with a BLASTn value of 3425 and a Max index value of 99.97%, confirming that FNH-5 is *Picochlorum sp.*.

[0037] Example 3: Cultivation and application of Pict's green algae FNH-5

[0038] The logarithmic growth phase of *Picochlorum* sp. FNH-5 was inoculated into enriched f / 2 medium and cultured in a photobioreactor. The OD of the culture medium after inoculation was measured. 690 The concentration was 0.21. 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 6-7, 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 FNH-5 was 5.79 g / L, and the total lipid content of the cells accounted for 46.12% of the cell dry weight.

[0039] Example 4: Comparison of the application effects of FNH-5 and FNH-0

[0040] Algal cultures of FNH-0 and FNH-5 in the logarithmic growth phase were inoculated into f / 2 enriched medium containing different concentrations of petroleum hydrocarbons and cultured in a photobioreactor. The OD values ​​of the inoculated culture media were measured. 690The value was 0.23. 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 6000 Lux, the pH was controlled at 6-7, 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.

[0041] Table 3. Effects of FNH-0 and FNH-5 under different conditions

[0042]

[0043] The results show that the FNH-5 algal strain selected in this invention has a better ability to degrade petroleum hydrocarbons than the initial algal strain FNH-0, and also has better biomass and oil content.

Claims

1. A strain of Pick's green algae, characterized in that... For oil-producing green cells (Pick's green cells) Picochlorum sp. FNH-5 was deposited on February 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45485.

2. The *Pickella greenia* as described in claim 1 (… Picochlorum sp. FNH-5, characterized in that: Under a microscope, the algal cells of this strain are green, single-celled, and spherical or ellipsoidal in shape with smooth cell walls. Each cell contains a single chloroplast, which is plate-like. Each cell contains a protein nucleus, which is sometimes not obvious. The cell diameter is 2-6 μm.

3. A type of Pick's green algae ( Picochlorum sp. The cultivation method of FNH-5 is characterized by: In the photobioreactor, seawater microbial culture medium is used for cultivation, and a gas with a CO2 content of 1.0v%-30v%, preferably 5v%-20v%, is introduced for cultivation.

4. The cultivation method according to claim 3, characterized in that: The cultivation conditions are as follows: light intensity 1500-20000 Lux, pH value 5-8, temperature 15-35℃, preferably 20-30℃, light cycle 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 3, characterized in that: The seawater microbial culture medium is a culture medium for culturing marine microalgae, preferably an enriched f / 2 medium.

6. A type of Pictella ( Picochlorum sp. Application of CO2 fixation.

7. The application according to claim 6, characterized in that: This algal strain can fix CO2, tolerates CO2 concentrations up to 30v, and is suitable for growth at CO2 concentrations of 1.0v%-10v.

8. A type of Pick's green algae ( Picochlorum sp. Application of FNH-5 in the degradation of petroleum hydrocarbon systems.

9. The application according to claim 8, characterized in that: This algal strain can tolerate a petroleum hydrocarbon concentration of up to 50 mg / L in the system; when the petroleum hydrocarbon concentration in the system is not higher than 30 mg / L, the petroleum hydrocarbon degradation rate is higher than 90%.

10. A type of Pick's green algae ( Picochlorum sp. Application of FNH-5 in the production of microalgae oil.

11. The application according to claim 10, characterized in that: This algal strain undergoes photoautotrophic growth under suitable conditions to obtain oil-rich algal cells with a dry weight of over 5 g / L and a total lipid content of over 45% of the cell dry weight.

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