A kind of gymnodinium algae phycocyanin and its preparation method and method of using the same to generate hydrocarbons and produce oil

Abstract

The present invention discloses a kind of gymnodinium algae phycocyanin and its preparation method and a method of using the same to generate hydrocarbons and produce oil. The basic molecular structural unit of gymnodinium algae phycocyanin is C 94 H 165 N3O 10 . The aliphatic compound composition content of the gymnodinium algae is 87.4%. The gymnodinium algae in the present invention has a high hydrogen index (HI) and quality index (QI), which are 642 and 877 mg HC / g TOC, respectively, and its HI and QI indexes are significantly correlated with its oil yield and hydrocarbon generation potential. The gymnodinium algae in the present invention has a high oil yield (61.1%) and hydrocarbon generation potential (67%).

Description

Technical Field

[0001] The invention belongs to the technical field of ecological restoration of water bodies and development of biomass energy, and particularly relates to a gymnodinium algae phycocyanin and a preparation method thereof and a method for generating hydrocarbons and oil by utilizing the same. Background Art

[0002] Since the beginning of the 20th century, the acceleration of global industrialization has led to a rapid increase in the consumption of fossil fuels. The use of fossil fuels has led to the depletion of oil reserves and increasingly serious environmental problems. In particular, global warming caused by the increase in carbon dioxide content in the atmosphere has been identified as one of the major challenges of this century. Global warming will lead to increasingly serious eutrophication of water bodies, resulting in the massive reproduction of algae (cyanobacteria and dinoflagellates), frequent water blooms and red tides. On the other hand, water blooms or red tides may provide abundant renewable biomass energy. It is reported that my country can obtain up to 13,600 tons (dry weight) of algae biomass from Dianchi Lake alone each year. If the algae oil yield is 50%, the potential bio-oil production can reach about 6,800 tons. In addition, microalgae can also absorb and fix carbon dioxide through the process of photosynthesis, and convert inorganic carbon into organic carbon, which is used to build proteins, nucleic acids and lipids, and can be converted into other useful products such as chemicals, biofuels and biopolymers after processing. Therefore, microalgae as a third-generation biomass raw material is a promising source of biodiesel oil. So, is it possible to develop algae biomass such as dinoflagellates to achieve CO2 capture and water pollution repair and convert them into biomass resources to reduce dependence on fossil energy?

[0003] Dinoflagellates are widely distributed in marine and freshwater environments, showing great diversity and complexity in structure, morphology, nutritional status and living habits. Dinoflagellates distributed in the ocean play a key role in marine primary productivity and may be the main contributors to Ordovician marine kerogen and oil shale. In the ancient geological era of the Ordovician, many algae (green algae, yellow-green algae, diatoms) and vascular plants had not yet evolved, but fossil records dating back to the Ordovician show the composition of the dormant cyst walls of dinoflagellates. Therefore, it is speculated that some of the organic matter derived from dinoflagellates may be an important precursor of marine kerogen since the Ordovician. In addition, kerogen is the most abundant ancient organic matter on Earth to date and is also the main source of oil and natural gas. Therefore, dinoflagellates may also have certain hydrocarbon and gas production capabilities. However, if dinoflagellates are used as raw materials for oil refining, since algae generally contain a large amount of lipids, proteins and polysaccharides, they are easily decomposed by heat during pyrolysis, which may lead to high nitrogen and oxygen content in biodiesel, and high acidity index, which in turn affects the quality of biodiesel. Therefore, it is particularly important to remove proteins and polysaccharides from dinoflagellates. It is reported that after removing proteins and polysaccharides, some cyanobacteria and green algae will obtain a highly fatty and difficult-to-degrade organic matter, which is defined as "algaenan". There are few research reports on the existence of this difficult-to-degrade organic matter in dinoflagellates. Most studies focus on the dormant cyst walls of marine dinoflagellates, but its structure is highly aromatic and completely different from algaenan. However, there is a report that a non-hydrolyzable residue was isolated from a marine chain-like naked dinoflagellate. Its rapid pyrolysis product is mainly composed of alkane / olefin double bonds, with carbon chains ranging from C7 to C35, which is very similar to the cracking characteristics of algaenan, indicating that some dinoflagellate species may contain algaenan. Therefore, it is necessary to further study the existence, composition and chemical structure of resistant biopolymers in other marine dinoflagellates, as well as their ability to produce oil and hydrocarbons during thermal evolution. Summary of the invention

[0004] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a new type of algae with high hydrocarbon generation potential. The present invention successfully separated a new type of algae from Gymnodinium aerucyinosum Stein and used advanced solid 13 C NMR technology, Rock-Eval pyrolysis technology and gold tube-autoclave closed cracking system were used to characterize the structure and evaluate the oil and hydrocarbon generation potential of the original sample of naked dinoflagellates and the new algaenan isolated from it, and compared with the composition, structure and hydrocarbon generation capacity of algaenan isolated from other algae species.

[0005] Another object of the present invention is to disclose a method for preparing the above-mentioned gymnodinium phycocyanin.

[0006] Another object of the present invention is to provide a method for producing hydrocarbons and oil from the above-mentioned gymnodinium algae.

[0007] The purpose of the present invention is achieved through the following solutions:

[0008] A kind of gymnodinium phycocyanin, the basic unit of its molecular structure is C 94 H 165 N3O 10 .

[0009] The gymnodinium phycocyanin is mainly composed of aliphatic compounds, with a content of aliphatic compounds of 87.4%, but its structure is different from the phycocyanin separated from other algae species.

[0010] The hydrogen index and mass index of the gymnodinium phycocyanin are 642 and 877 mg HC / g TOC, respectively.

[0011] A method for preparing gymnodinium phycocyanin comprises the following steps:

[0012] (1) The original sample of naked dinoflagellates was extracted with a mixed solution of dichloromethane and methanol, and the obtained solid part (free fat, LF) was dried;

[0013] (2) adding 1-2 mol / L trifluoroacetic acid (TFA) to the obtained solid, heating at 90-120° C. for 1-3 h in a N2 protective atmosphere, and centrifuging to remove the supernatant;

[0014] (3) then heating the mixture with 2-4 mol / L and 4-6 mol / L trifluoroacetic acid in a nitrogen atmosphere at 100-120°C for 15-20 h respectively, and centrifuging to remove the supernatant;

[0015] (4) Finally, 4-6 mol / L HCl is used to heat at 110-130° C. for 18-24 h in a N 2 protective atmosphere, and the supernatant is discarded by centrifugation. The solid precipitate is the gymnodinium phycocyanin.

[0016] Preferably, the original sample of Gymnodinium in step (1) is obtained by culturing Gymnodinium aerucyinosum Stein algae species, collecting algae cell concentrate by centrifugation, and freeze-drying.

[0017] Preferably, the volume ratio of dichloromethane to methanol in the mixed solution of dichloromethane and methanol in step (1) is 80-100:5-10.

[0018] Preferably, the Soxhlet extraction in step (1) is performed for 24 to 72 hours; and the obtained solid portion is dried at 40 to 50°C.

[0019] Preferably, step (2) is repeated more than once.

[0020] Preferably, in step (4), the solid precipitate is washed with water until it becomes neutral.

[0021] A method for generating hydrocarbons and oil by utilizing gymnodinium algae phycocyanin, specifically comprising pyrolyzing the obtained gymnodinium algae phycocyanin at 40-60 MPa to generate hydrocarbons and oil.

[0022] The pyrolysis temperature is 300-500°C, preferably 350-480°C, specifically 350, 375, 400, 420, 440, 460, 480°C, etc.

[0023] Preferably, the oil yield of the gymnodinium phycocyanin in the gold tube-autoclave system is 61.1%, and the hydrocarbon generation potential is 67.0%.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] (1) The gymnodinone in the present invention is a new type of biopolymer containing a large amount of aliphatic carbon and aromatic carbon, and its basic structural unit molecular formula is C 94 H 165 N3O 10 , whose structure is different from any previously reported algaenan.

[0026] (2) The algae quality index (QI) of the gymnodinium phycocyanin of the present invention is very high, and its HI and QI indexes are significantly correlated with its oil production rate and hydrocarbon generation potential.

[0027] (3) The gymnodinium phycocyanin in the present invention has a high oil yield (61.1%) and hydrocarbon generation potential (67%).

[0028] (4) Rock-Eval pyrolysis technology, solid 13 C NMR and closed pyrolysis are effective techniques for evaluating the oil and hydrocarbon generation potential of algae. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the NMR comparison spectrum of phycocyanin from Gymnodinium and phycocyanin isolated from other algae species.

[0030] Figure 2 This is a comparison chart of the oil and gas production rates of the original sample of naked dinoflagellates and its algaenan as a function of temperature during the cracking process. The original sample in the figure refers to the original sample of naked dinoflagellates, and the algaenan refers to the algaenan of naked dinoflagellates.

[0031] Figure 3 This is a comparison chart of the oil components of the original sample of naked dinoflagellates and its algaenan during the cracking process. The original sample in the figure refers to the original sample of naked dinoflagellates, and the algaenan refers to the algaenan of naked dinoflagellates.

[0032] Figure 4 The HI and QI indexes of different fractions of Gymnodinium phycocyanin and other algae are compared with solid 13 Linear fitting diagram of oil production rate and hydrocarbon generation potential evaluated by CNMR technology and closed pyrolysis technology.

[0033] Figure 5 This is a graph showing the changes in the n-alkane content of the original sample of naked dinoflagellates and its algaenan produced during the pyrolysis process as a function of temperature.

[0034] Figure 6 It is the basic molecular structural unit model of gymnodinium phycocyanin.

[0035] Figure 7 This is the gas chromatogram of liquid hydrocarbons produced by the pyrolysis of gymnodinium phycocyanin at different pyrolysis temperatures. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0037] Unless otherwise specified, the reagents used in the examples can be purchased from the market.

[0038] Example 1

[0039] 1. Prepare raw materials: original sample of Gymnodinium aerucyinosum Stein, dichloromethane, methanol, trifluoroacetic acid, HCl, ultrapure water. Gymnodinium aerucyinosum Stein was purchased from the Institute of Oceanology, Chinese Academy of Sciences.

[0040] Prepare algae culture solution according to the f / 2 medium formula and transfer it to several small conical bottles. Then, place the conical bottles containing the algae culture solution in a high-pressure steam sterilizer at 110-120°C for 20-30 minutes. After the algae culture solution cools to room temperature, transfer the naked dinoflagellate (Gymnodinium aerucyinosum Stein) into it and place it in a light incubator (temperature 25°C, light intensity 200umol m -2 s -1 , the light cycle is 16 hours of light period and 8 hours of dark period) for cultivation. After about 15-30 days of cultivation, the algae cells enter the stable growth period, and the algae liquid and algae culture fluid are expanded into a 100L photobioreactor at a volume ratio of 1:50 for further cultivation. Finally, the algae cell concentrate is collected by centrifugation and frozen, and then the algae cell concentrate is freeze-dried using a freeze dryer to obtain a solid algae sample, which is ground into powder to obtain the original sample of Gymnodinium aerucyinosum Stein.

[0041] (1) Separation of phycocyanin: Take a certain amount of original sample (OS) of naked dinoflagellates (Gymnodinium aerucyinosum Stein) and use a mixed solution of dichloromethane and methanol (93:7, volume ratio) for Soxhlet extraction for 72 hours. The solid part (free lipids, LF) is placed in an oven at 50°C for drying overnight, and a portion is preserved. Take the LF fraction, add 2M trifluoroacetic acid (TFA), heat at 100°C for 3h under nitrogen protection, centrifuge to remove the supernatant, and repeat the above steps again; then use 4M and 6M TFA to heat at 100°C for 18h under nitrogen protection, respectively, and centrifuge to remove the supernatant. Finally, use 6M HCl to heat at 110°C for 24h under nitrogen protection, centrifuge and discard the supernatant. The solid precipitate (phycocyanin, NHOM) is washed with ultrapure water until neutral, freeze-dried, and stored.

[0042] 2. Establishment of the molecular structure model and molecular formula of phycocyanin

[0043] The isolated phycocyanin is a complex biomacromolecule that cannot dissolve in acid or alkali and cannot be degraded by microorganisms. At present, there is no report on the accurate molecular weight of phycocyanin at home and abroad. The few reports use C90 as the unit to determine the molecular formula of phycocyanin. Therefore, in the present invention, we use Chemsketch 12.0 (ACD / Labs 2016) software and g NMR software to fit the molecular structure model of phycocyanin from Gymnodinium sphaerocephalum, and determine the molecular formula of phycocyanin in C94 as the unit.

[0044] 3. Gold tube-autoclave pyrolysis, the specific experimental process is as follows:

[0045] (1) Pyrolysis process:

[0046] 80-120 mg of algae quality sample was sealed in gold tubes under argon protection, and the gold tubes were placed in 7 autoclaves respectively, and then placed in the same pyrolysis furnace for pyrolysis reaction. During the pyrolysis process, a constant pressure of 50 MPa was maintained. The autoclave was placed in an electric furnace controlled by a programmed temperature rise, and 7 temperature points (350, 375, 400, 420, 440, 460, and 480°C) were set. Each sample used 1 gold tube at each temperature point. The pyrolysis heating program was as follows: the autoclave was quickly heated from room temperature to 250°C within 8 hours, and then heated to the target temperature at a heating rate of 20°C / h. The pressure control valve corresponding to the autoclave was closed, the autoclave was taken out of the electric furnace, and the temperature was immediately cooled in a water bath. The pyrolysis gold tube was taken out of the autoclave for the next experimental analysis. After the temperature was raised from room temperature to the target temperature, it was taken out for sampling analysis.

[0047] (2) Analysis of gaseous hydrocarbon components:

[0048] The collection of gaseous hydrocarbon components is completed in a vacuum glass system. The gold tube with a clean surface is placed in a fixed volume vacuum system and punctured with a needle under vacuum conditions. After the pyrolysis gas products are completely released from the gold tube, the connection channel between the vacuum system and the Agilent 8890N gas chromatography (GC) instrument is opened to analyze the gas composition. The quantitative method uses the external standard method for quantification.

[0049] (3) Analysis of light hydrocarbon components:

[0050] The volatile hydrocarbon components of the gold tube punctured under vacuum conditions were collected in a 4mL glass bottle. After complete evaporation, it was cooled with liquid nitrogen and 3mL of n-pentane was added immediately. The gold tube was taken out, cut open with scissors, placed in the above-mentioned 4mL glass bottle, and the standard sample was added for injection and analysis.

[0051] (4)C 14+ Component Analysis:

[0052] After the analysis of the light hydrocarbon components, the n-pentane in the 4 mL glass bottle was evaporated to dryness, and the residue in the bottle was taken out and Soxhlet extracted with dichloromethane: methanol (93:7, v / v) for 72 h. After extraction, the solvent was rotary evaporated and dried and weighed. The extract was separated into saturates, aromatics and resins using a silica gel column. These three parts were dried and weighed. The insoluble residue was recovered from the filter paper, dried and weighed.

[0053] Parameter comparison

[0054] The gymnodinium phycocyanin prepared in Example 1 of the present invention is solid 13 The comparison of the oil-producing carbon and gas-producing carbon quantified by C NMR technology with the algae of other algae species is listed in Table 1, and the relative contents of the original samples and various carbon-containing functional groups of the algae are listed in Tables 2 and 3. In addition, the composition (protein, fatty compounds and sugars) and other parameters (hydrogen index, quality index, oil yield, hydrocarbon generation potential) of the algae of Gymnodinium phytoplankton were compared with some cyanobacteria, cyanobacterial phytoplankton and green algae studied by previous researchers, and the comparison results are listed in Table 4. Among them, the composition and other parameters of Schizothrix fasciatus, duckweed and Cladophora were analyzed according to the method described in Example 1.

[0055] Table 1. Comparison of oil and gas carbon production between Gymnodinium phycocyanin and other algae species phycocyanin

[0056]

[0057] Note: a Oil-producing carbon = methylidene (36-45ppm) + methylene (24-36ppm); b Gas-generating carbon = methyl (0-24ppm); c Oil and gas producing carbon = oil-producing carbon + gas-producing carbon.

[0058] [1]Kong,

[0059] [2] Zhang, D., Yang, Y., Hu, J., Ran, Y., & Mao, J. (2019). Occurrence of aliphatic biopolymer in chlorophyceae algae and cyanobacteria-richphytoplankton. Organic Geochemistry, 135, 1-10.

[0060] [3]Hu, S., Kong,

[0061] Table 2. Relative contents of carbon-containing functional groups in the original samples of Gymnodinium and its algaenan structure

[0062]

[0063] Table 3. Relative contents of carbon-containing functional groups in the original samples of Gymnodinium and its algaenan structure

[0064]

[0065] Table 4. Comparison of parameters of Gymnodinium phycocyanin with other cyanobacteria, cyanobacterial algae mixture and green algae

[0066]

[0067] Note: [1] Kong,

[0068] [2] Zhang, D., Yang, Y., Hu, J., Ran, Y., & Mao, J. (2019). Occurrence of aliphatic biopolymer in chlorophyceae algae

[0069] and cyanobacteria-rich phytoplankton.Organic Geochemistry,135,1-10.

[0070] [3]Hu, S., Kong,

[0071] [4] Behar, F., Derenne, S., & Largeau, C. (1995). Closed pyrolyses of the isoprenoid algaenan of Botryococcus braunii, L race: geochemical implications for derived kerogens. Geochimica et Cosmochimica Acta, 59(14), 2983-2997.

[0072] The hydrogen index and mass index of the gymnodinium algaenan are 642 and 877 mg HC / g TOC, respectively, which are significantly higher than the hydrogen index and mass index of the original sample and the algaenan isolated from other algae species.

[0073] The content of fat compounds in the gymnodinium algae is higher and the content of proteins and polysaccharides is lower than that of its original sample and other algae species.

[0074] Figure 1 This is a comparison of the NMR spectra of phycocyanin from Gymnodinium and phycocyanin isolated from other algae species. Figure 1 It can be seen that the NMR spectrum of the gymnodinium phycocyanin is different from the NMR spectrum of other phycocyanin isolated from cyanobacteria and green algae. In addition to containing a large number of aliphatic functional groups, the gymnodinium phycocyanin in the present invention also contains a portion of aromatic functional groups.

[0075] Figure 2 This is a comparison of the oil and gas production rates of the original sample of naked dinoflagellates and its algae quality as a function of temperature during the pyrolysis process. Figure 3 This is a comparison of the oil components of the original sample of Gymnodinium and its algaenan during the lysis process. Figure 2 and Figure 3 It can be seen from the above that the Gymnodinium algae is more inclined to produce oil, while its original sample is more inclined to produce gas (carbon dioxide and gaseous hydrocarbons), and from Figure 3 It can also be seen that the oil generated by the pyrolysis of the naked dinoflagellate algae is mainly composed of saturated hydrocarbons and aromatic hydrocarbons with low polarity, while the oil generated by its original sample is mainly composed of colloids (polar compounds).

[0076] Figure 4 The HI and QI indexes of different fractions of Gymnodinium phycocyanin and other algae are compared with solid 13 Linear fitting diagram of CNMR technology and closed pyrolysis technology for evaluating oil production rate and hydrocarbon generation potential. Figure 4 It can be seen that the HI and QI indices determined by the Rock-Eval pyrolysis technology are consistent with the oil yield and hydrocarbon generation potential obtained by the closed pyrolysis technology, as well as the solid 13 There is a significant correlation between the oil-producing carbon and the oil-producing and gas-producing carbon quantified by C NMR technology, indicating that Rock-Eval pyrolysis technology, solid-state 13 C NMR and closed pyrolysis are effective techniques for evaluating the oil and hydrocarbon generation potential of organic matter.

[0077] Figure 5 The following is a graph showing the changes in the content of n-alkanes produced by the original sample of naked dinoflagellates and its algaenan during the pyrolysis process. Figure 5 It can be seen that the molecular structure of the original sample of naked dinoflagellates and its algae quality contains saturated hydrocarbon chains with a carbon number of up to 31, and it also shows that its structure is formed by cross-linking carbon chains of different chain lengths.

[0078] Figure 7 This is a gas chromatogram of liquid hydrocarbons generated by pyrophyll in the different cracking temperatures. Figure 7It can be seen that although there are some isoalkanes in the liquid hydrocarbons at low temperature (below 400℃), with the increase of cracking temperature, the distribution of normal alkanes becomes more and more obvious, and its production gradually increases and finally reaches the maximum value at the optimal oil production temperature (440℃). Figure 3 The results reflected are consistent.

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A gymnodinium phycocyanin, characterized in that: The basic molecular structural unit is C 94 H 165 N3O 10 ; The gymnodinium phycocyanin is prepared by the following steps: (1) extracting the original sample of naked dinoflagellates with a mixed solution of dichloromethane and methanol, and drying the obtained solid part; (2) adding 1-2 mol / L trifluoroacetic acid to the obtained solid, heating at 90-120° C. for 1-3 h in a N2 protective atmosphere, and centrifuging to remove the supernatant; (3) then heating the mixture with 2-4 mol / L and 4-6 mol / L trifluoroacetic acid in a nitrogen atmosphere at 100-120°C for 15-20 h respectively, and centrifuging to remove the supernatant; (4) Finally, 4-6 mol / L HCl is used to heat at 110-130° C. for 18-24 h in a N 2 protective atmosphere, and the supernatant is discarded by centrifugation. The solid precipitate is the gymnodinium phycocyanin.

2. The gymnodinium phycocyanin according to claim 1, characterized in that: The aliphatic compound content of the gymnodinium phycocyanin is 87.4%.

3. The gymnodinium phycocyanin according to claim 1, characterized in that: The hydrogen index and mass index of the gymnodinium phycocyanin are 642 and 877 mg HC / g TOC, respectively.

4. A method for preparing gymnodinium phycocyanin, characterized in that: The following steps are involved: (1) extracting the original sample of naked dinoflagellates with a mixed solution of dichloromethane and methanol, and drying the obtained solid part; (2) adding 1-2 mol / L trifluoroacetic acid to the obtained solid, heating at 90-120° C. for 1-3 h in a N2 protective atmosphere, and centrifuging to remove the supernatant; (3) then heating the mixture with 2-4 mol / L and 4-6 mol / L trifluoroacetic acid in a nitrogen atmosphere at 100-120°C for 15-20 h respectively, and centrifuging to remove the supernatant; (4) Finally, 4-6 mol / L HCl is used to heat at 110-130° C. for 18-24 h in a N 2 protective atmosphere, and the supernatant is discarded by centrifugation. The solid precipitate is the gymnodinium phycocyanin.

5. The preparation method according to claim 4, characterized in that: The original sample of naked dinoflagellates in step (1) is obtained by culturing naked dinoflagellates species, collecting algae cell concentrate by centrifugation, and freeze-drying.

6. The preparation method according to claim 4, characterized in that: In step (1), the volume ratio of dichloromethane to methanol in the mixed solution of dichloromethane and methanol is 80-100:5-10.

7. The preparation method according to claim 4, characterized in that: In step (1), the Soxhlet extraction is performed for 24 to 72 hours; and the obtained solid portion is dried at 40 to 50°C.

8. The preparation method according to claim 4, characterized in that: Step (2) is repeated more than once, and in step (4), the solid precipitate is washed with water until it becomes neutral.

9. A method for producing hydrocarbons and oil using algae-derived phycocyanin, characterized in that: Specifically, the gymnodinium algae phycocyanin described in any one of claims 1 to 3 can be pyrolyzed at 40 to 60 MPa to generate hydrocarbons and oil.

10. The method according to claim 9, characterized in that The pyrolysis temperature is 300-500°C.

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