TWI648400B - Micractinium sp. and uses thereof - Google Patents

Abstract

The present invention relates to a isolated Micractinium sp. which can grow on different materials to form a biofilm and produce a high amount of triglyceride. The invention also relates to the use of the isolated Micractinium sp. for producing biomass fuel and edible oil.

Description

Micromania (MICRACTINIUM SP.) and its use

The invention relates to a novel Micractinium sp. isolate, which can grow on different materials to form a biofilm, and can be cultured by using a biofilm culture mode to reduce water resources and land. Demand and streamline the harvesting process and reduce harvesting costs. The isolate can produce high amount of triacylglycerol, can absorb carbon dioxide during culture, can be used as carbon fixation, and has high calorific value, low ash and low sulfur, and can be used as raw diesel. And the source of edible oil.

Since the industrial revolution of the 18th century, human demand for petrochemical energy has increased. However, the natural reserves of petrochemical energy are limited, so it is gradually necessary to seek new alternative energy sources. The first generation of biomass energy is based on sugarcane, sugar beet, corn, soybean and other food crops. It uses biomass, transesterification and other technologies to produce biomass energy, but it will increase the demand for agricultural land and food. The problem of crop prices is high; the second generation of biomass energy is based on non-food crops such as rice straw, corn stalks, wood chips and bagasse, using cellulite hydrolysis technology to produce biomass energy, but such biomass energy has The source of raw materials is limited and the cost of treatment is too high. The third generation of germplasm originated from the 1970 energy crisis. The algae biodiesel development plan promoted by the United States uses algae as the main raw material and extracts with oil. Hydrogenation and transesterification technologies are used to produce biomass energy.

Microalgae belongs to a kind of unicellular algae. Its cell size ranges from a few micrometers (μm) to several hundred micrometers. It is generally not directly visible to the naked eye. It needs microscopic assistance. Observed. The distribution of microalgae is very wide. It can be found in fresh water, ocean or wet soil. It is estimated that there are about 200,000 to 800,000 species of microalgae on the earth, of which only about 3 have been found and recorded. With 5,000 species, its biodiversity is very complex, from a basic research or development perspective to an area that is barely actively developed.

Microalgae has the advantages of fast growth rate, high carbon dioxide utilization rate, high-density culture, low probability of contamination by pathogens and small required land area, and can accumulate a large amount of biomass (biomass) in a short period of time. Produces raw materials for biodiesel, bioethanol, and biofuel. In addition, microalgae can be cultivated using non-agricultural land, seawater, domestic wastewater, agricultural and animal husbandry wastewater, and flue gas, which greatly reduces the demand for land and fresh water, thereby reducing competition for resources with food and cash crops. In addition, its cell structure is simple and lacks cell differentiation. Compared with oil-producing crops such as palm, rapeseed, soybean and sugar cane, its operation is easier than that of plant cells, and it has a similar post-glycation translation modification mechanism to plants. gene expression (Yen, HW et al, Microalgae -based biorefinery-From biofuels to natural products, Bioresour.Technol, 2013,135:... 166-174).

Currently, algae polysaccharides, carotenoids, phycobilins, Eicosapentaenoic Acid (EPA) and docosahexaenoic acid are contained in microalgal biomass. acid (DHA)), etc., have been widely used in health care, beauty, food processing, aquaculture and bio-energy industries (Spolaore, P. et al. , Commercial applications of microalgae., J.Biosci.Bioeng., 2006, 101: 87-96).

The algae oil produced by microalgae is mainly composed of triglyceride, various fatty acids and sterols, and can be converted into liquid biofuel by hydrogenation or transesterification. Different algae species have different proportions of algae oil composition. Therefore, studies have indicated that the fatty acid profile of microalgae algae oil can be used as one of the indicators for screening algae species (Ramos, MJ et al ., Influence of fatty acid composition of Raw materials on biodiesel properties., Bioresour . Technol ., 2009, 100: 261-268). However, not all algae oil produced by microalgae is suitable for the preparation of biofuels. The degree of unsaturation of fatty acids and the ratio of triglycerides may also affect the suitability of the algae. For the preparation of biofuels. Studies have indicated that Monoraphidium contortum (SAG 47.8) has a biomass yield of 300 mg/L/day, and the algae oil content accounts for 22.2% (w/w) of the dry weight of algae. consisting of C16: 0 to C18:. 1 fatty acid, as a potential for producing biofuel algae strains (Bogen, C et al, Identification of Monoraphidium contortum as a promising species for liquid biofuel production Bioresour.Technol, 2013,... 133:622-626).

The growth of microalgae depends on the photosynthesis. Therefore, light, carbon dioxide, water, nitrogen, phosphorus and potassium are the essential elements for cultivating microalgae. The breeding methods include self-operated, multi-operated and mixed culture, etc. Culture refers to the energy required for microalgae to grow by photosynthesis using a light source and inorganic carbon (such as carbon dioxide). The isoculture means that the microalgae use organic carbon in the medium as a carbon source (such as acetic acid under no light). Sodium, glucose) to grow; mixed culture refers to microalgae can carry out self-operated and hetero-growth at the same time. In general, the amount of algae in the microalgae increases approximately every 6 to 72 hours, and the faster the microalgae grows, the higher the frequency of harvesting. However, the growth rate of algae species with higher oil content is slower than that of algae species with lower oil content. Therefore, it is necessary to consider the growth rate and oil content of microalgae to select algae species suitable for preparing biomass fuel. In addition, different culture methods and medium components have different effects on the growth rate of microalgae, oil accumulation, yield and composition (Dhup, S. & Dhawan, V., Effect of nitrogen concentration on lipid productivity and fatty . acid composition of Monoraphidium sp, Bioresour.Technol , 2014,152:. 572-575).

At present, microalgae is used as a raw material for biomass fuel, and its production cost and technology still have to be overcome and breakthrough. Taking the self-supporting culture model as an example, the self-cultivation of microalgae mainly involves the suspension culture of microalgae in the culture solution. However, the current culture technology still cannot transfer the algae in the culture solution. The concentration is increased to 10 g/L (1%) or more. In addition, because the concentration of algae in the culture solution is low and the cells of the algae are small, it is necessary to separate the algae from the culture solution through complicated processes and equipment when the algae is recovered and dried, which is time consuming and expensive, so that the algae The recovery cost of the body can account for 20~30% of the total production cost of the microalgae biodiesel.

The microalgae biofilm culture system utilizes microalgae to attach to the culture mode that grows on the surface of the carrier. It was first applied to the treatment of nitrogen and phosphorus in industrial wastewater in the 1980s (Przytocka-Jusiak M. et al ., Removal of nitrogen from industrial Waste waters with the use of algal rotating disks and denitrification packed bed reactor., Water Res ., 1984, 18: 1077-1082). The culture mode does not need to suspend the microalgae in the culture solution, and the algae body can directly obtain the high concentration algae mud when harvesting, which can simplify the traditional complicated algae harvesting process and greatly reduce the algae recovery. Energy consumption and cost are micro-algae farming models with great development potential. For example, in 2010, Johnson et al. developed a microalgae biofilm attachment culture model in which Chlorella sp. was cultured in wastewater with styrofoam as a carrier, and it was found that the chlorella was attached to the cultured wastewater. More than 60% of nitrogen and phosphorus, and algae can be harvested directly by scraping (Johnson, MB & Wen, Z., Development of an attached microalgal growth system for biofuel production, Appl . Microbiol . Biotechnol ., 2010, 82:525-534); In 2012, Ozkan et al. cultured a microalgae biofilm photoreactor with low energy consumption and low water demand to culture Botryococcus braunii , and found that its biofilm light was compared with the open pond culture system. The reactor culture process reduces water requirements by 45% and significantly reduces the energy consumption required to remove water from algae (99.7%) (Ozkan, A. et al ., Reduction of water and energy requirement of algae cultivation using an algae) biofilm photobioreactor, 2012, Bioresour.Technol, 114 : 542-548); Hungary MFKK engineering company since 2012, launched ALGADISK project aimed at developing an modular, easy-to-zoom, low operation and low installation costs. Automated microalgae biofilm reactor, which was published in 2014 by the cooperative Bay-Bio Institute in Hungary, and the average concentration of algae collected directly from the microalgae biofilm reactor can reach 100g/L ( Sebestyén, P. et al ., Biomass and lipid production by microalgae in a new biofilm based photobioreactor., presented at Young Biotechnologist National Conference, 2014, Szeged), this concentration is more than 10 times the concentration of general suspended microalgae; and 2013 In the United States, the University of Iowa Gross et al. used the Revolving Algal Biofilm (RAB) growth system to culture Chlorella vulgaris , and found that the algae harvested by the RAB system compared to the open pond culture system. The moisture content of the mud is about 90.3%, which is close to the moisture content of the algae collected after the traditional centrifugation (about 88.6%). Therefore, the microalgae biofilm culture mode can directly harvest high-concentration algae mud by means of direct scraping, eliminating the traditional sedimentation, flocculation and centrifugation harvesting processes, saving time, space, and reducing water resources and harvesting costs. Advantages such as energy consumption. However, the content of algae oil recovered by the above RAB system is about 40% (w/w) lower than that of the open pond culture system, and the algae oil content collected in the ALGADISK plan is also higher than that of the suspension culture algae oil. The content of the oil in both culture modes is about 10% (w/w) (Gross, M. & Wen, Z., Yearlong evaluation of performance and durability of a pilot-scale Revolving Algal Biofilm (RAB) cultivation system , Bioresour.Technol, 2014,171:... 50-58; and Sebestyén, P et al, Biomass and lipid production by microalgae in a new biofilm based photobioreactor, presented at Young Biotechnologist National Conference, 2014, Szeged). When microalgae is used as a source of biofuel, not all algae strains are suitable for cultivation in a biofilm culture mode.

Another advantage of using microalgae as a source of biofuels is the high calorific value of microalgae (greater than 25 MJ/Kg), which is used in power plants (including thermal power plants and cogeneration plants). Compared with coal, the calorific value of microalgae is about 1.044 times higher than that of bituminous coal (heat value 23.94 MJ/Kg), which is about 1.215 times higher than that of sub-bituminous coal (heat value 20.58 MJ/Kg). In other words, if the calorific value of 25MJ/Kg is calculated, the calorific value produced per 1 metric ton of algae is equivalent. The calorific value produced by 1.044 tons of bituminous coal, or the calorific value produced by 1.215 tons of sub-bituminous coal. If the microalgae are cultured in a biofilm culture mode, the harvested algae can be subjected to subsequent drying and roasting using waste heat to produce biocoal, which can replace some of the currently used coal, thereby reducing coal combustion. The amount of use.

At present, the research on microalgae as a source of biofuels mainly includes algae development/screening, microalgae culture technology, algae recovery and biomass extraction. In the development and screening of algae species, algae species with rapid growth, high biomass yield, high algal oil content and easy recovery of algae are still needed as the basis for the preparation of microalgae biomass fuel. By developing potential algae species to prepare biofuels or high-priced active substances, and at the same time slowing carbon dioxide emissions, the company achieves the goal of environmental protection and industrial profitability (Farrelly, DJ et al ., Carbon sequestration and the role) Of biological carbon mitigation: A review., Renewable and Sustainable Energy Reviews, 2013, 21: 712-727).

The invention collects soil samples containing microalgae in the Alishan mountainous area of Chiayi, Taiwan, and cultures and separates the microalgae in C medium, and selects a culture mode capable of culture, high oil content, high calorific value and high temperature resistance. The 74C strain was identified as a microalgae of the genus Micractinium . Based on the analysis of algae oil content, composition and algae calorific value, ash content and sulfur content, 74C strains were found to have potential as raw materials for biofuels and edible oils. In addition, the 74C strain can be attached to the surface of the carrier of different materials to form a biofilm; the algae oil can be produced by the biofilm culture mode, and only the algae on the surface of the carrier can be scraped off when the algae is harvested. High concentration of algae mud can be obtained, which simplifies the harvesting process during microalgae cultivation and reduces production costs.

Accordingly, it is an object of the present invention to provide an isolated M. genus isolate, wherein the M. genus isolate comprises the 18S rDNA sequence of the nucleotide sequence set forth in SEQ ID NO: 1 and SEQ ID NO: The ITS region sequence of the nucleotide sequence shown in 2.

Another object of the present invention is to provide a method of culturing the isolated M. genus isolate to obtain a culture product of Micromandala.

Another object of the present invention is to provide a culture product of Micromandala obtained by the above method, wherein the culture product of Micromania can be used as a source of raw fuel and edible oil.

Another object of the present invention is to provide a method for obtaining triglyceride from the above culture product of Micromonas.

Another object of the present invention is to provide a method of culturing the isolated Miscanthus isolate to immobilize carbon dioxide.

The invention is described in detail in the following sections. Other features, objects, and advantages of the invention are apparent from the embodiments of the invention and the appended claims.

Figure 1 is a microscopic examination of the 74C strain. Fig. 1A is a bright field observation, the diameter of the algae cells is about 3~6μm, and the microscopic magnification is 1,000X; Fig. 1B is stained by Nile Red, observed by a fluorescent microscope, and the yellow oil droplets are distributed inside the algae body, and the microscopic magnification is 1,000. X.

Figure 2 is an evolutionary tree analysis of the 18S rDNA and ITS region sequences of the 74C strain.

Figure 3 is an evolutionary tree analysis diagram of the ITS region sequence of the 74C strain.

Figure 4 shows the growth of 74C strains at different culture temperatures (30 ° C, 35 ° C, 37 ° C and 40 ° C).

Figure 5 shows the growth of 74C strains at different culture pH values (pH 4, pH 5.5, pH 6.5, pH 7.5 pH 8.5 and pH 9.5).

Figure 6 shows 74C strains at different culture salinities (0% (w/v), 1.0% (w/v), 1.5% (w/v), 2.0% (w/v), 2.5% (w/ v), 3.0% (w/v) and 4.0% (w/v) growth.

Fig. 7 shows the attachment and oil production of the attached carrier of the 74C strain grown on different materials. Group A is a carrier attached to various materials; Group B is a 74C strain attached to each carrier. Long case; group C is a microscope bright field view of 74C algae attached to each carrier, the microscopic magnification is 400X; group D is the fluorescent light of 74C algae attached to each carrier after Nile red staining Microscopic observation, the microscopic magnification is 400X.

The present invention can be understood from the various aspects of the invention, the embodiments and the description of the embodiments disclosed herein. Unless otherwise defined herein, terms (including technical and scientific terms) used in connection with the present invention shall have the meaning as understood by those of ordinary skill in the art. And, as will be appreciated, unless the definitions provided herein are otherwise specified, in the case of any potential ambiguity, the definition of terms should be consistent with such commonly used terms (as defined in the dictionary). It is to be understood that the terminology used herein is for the purpose of describing the particular embodiments

It must be noted that the singular forms "a", "an" and "the" are used in the <RTIgt; Therefore, unless the context requires otherwise, the singular terms shall include the plural, and the plural terms also include the singular.

The scope of the present invention is expressed by "a particular value" and / or another "about" specific value. When the range is expressed in the above manner, it includes a range from a particular value and/or to another particular value. Similarly, when a value can be approximated by the term "about", it will be understood that it is another aspect of a particular value. It can be further appreciated that when referring to other endpoints and other endpoints themselves, the endpoints of each range are meaningful. According to the present invention, "about" can mean ± 20%, preferably ± 10%, more preferably ± 5%.

In the present invention, the term "separated" or "isolated" means that the substance is removed from its original environment (or natural environment if it is naturally present). The term "isolated" or "isolated" does not necessarily mean that the material is purified.

An object of the present invention is to provide a strain of Micromandala, wherein the strain of Micromania comprises the 18S rDNA sequence of the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: The sequence of the ITS region of the nucleotide sequence shown in 2. In a preferred embodiment of the present invention, the microorganism is a strain deposited in the Food Industry Development Research Institute of the Corporation and registered as BCRC 980044, or is deposited with the Food Industry Development Research Institute. The strain with the accession number BCRC 980044 has a variant strain that has substantially identical identification characteristics.

The term "variant strain" means any micromandala strain that covers all cytogenetic components that have been altered by chemical mutation induction, spontaneous mutation, genetic engineering, transformation or transfection, such that they affect their physical or biochemical properties. . However, the variant strain should have all taxonomic identification features deposited in the Institute of Food Industry Development and deposited under the registration number BCRC 980044.

In the present invention, the term "similarity" means the degree of similarity between two nucleic acid sequences. The difference between the two nucleic acid sequences may occur at the 5' or 3' end position of the reference nucleotide sequence, or may be individually dispersed among the nucleotides in the reference sequence, or interspersed in one or more adjacent sequences within the reference sequence Anywhere between the end positions of the group. Whether any particular nucleic acid molecule has at least 95%, 96%, 97%, 98%, 99%, or 100% similarity to a reference nucleotide sequence refers to the use of standard algorithms well known in the art in two molecules The comparisons made between them can be determined conventionally using publicly available computer programs such as the BLASTN algorithm.

It is an object of the present invention to provide a method of preparing a culture product of Micromania. In an embodiment of the present invention, the method comprises inoculating the Miscanthus isolate of the present invention in a liquid medium, culturing under irradiation and aeration to obtain the culture product. In a preferred embodiment of the present invention, the method comprises culturing the microorganism of the present invention with one or more vectors in a liquid medium, and culturing under illumination and aeration to obtain the culture product. Wherein the Micromandala isolate and the one or more vector lines are separately added to the liquid medium, or the Micromandala isolate is first inoculated on the one or more carriers, and then the inoculated vector is placed. In the liquid medium.

In the present invention, the term "culture product" means a product rich in microalgae cells obtained by culturing microalgae in a medium. In the present invention, the microalgae cells in the culture product may not necessarily be separated from the culture medium, and the culture product may be in a liquid state, a solid state or a viscous state.

In the present invention, the "medium" used for culturing the Miscanthus isolate may be any aqueous medium which allows the growth, propagation and production of triglycerides and/or fatty acids of the Miscanthus isolate, such as C medium [per 100mL contains 15mg Ca(NO ₃ ) ₂ ̇4H ₂ O, 10mg KNO ₃ , 5mg β-glycerophosphate disodium ̇5H ₂ O, 4mg MgSO ₄ ̇7H ₂ O, 0.01μg vitamin B12, 0.01μg biotin (Biotin 1 μg of thiamine HCl, 0.3 mL of PIV trace element solution (containing 100 mg Na ₂ EDTA ̇ 2H ₂ O, 19.6 mg FeCl ₃ ̇ 6H ₂ O, 3.6 mg MnCl ₂ ̇ 4H ₂ O, 1.04 mg ZnCl ₂ per 100 mL, 0.4 μg CoCl ₂ ̇6H ₂ O, 0.25 μg Na ₂ MoO ₄ ̇2H ₂ O and water), 50 mg Tris (hydroxymethyl) aminomethane and water], BG-11 medium [containing 1,500 mg NaNO ₃ , 40 mg K ₂ HPO per 100 mL ₄ , 75mg MgSO ₄ . 7H ₂ O, 27.18 mg CaCl ₂ , 6 mg citric acid, 6 mg ammonium ferric citrate, 1 mg Na ₂ . Mg. EDTA. 2H ₂ O, 20 mg Na ₂ CO ₃ , 2.86 mg HBO ₃ , 1.181 mg MnCl ₂ ̇ 4H ₂ O, 0.222 mg ZnSO ₄ ̇7H ₂ O, 0.39 mg Na ₂ MoO ₄ ̇ 2H ₂ O, 0.0718 mg CuSO ₄ ̇ 5H ₂ O, 0.049 mg Co(NO ₃ ) ₂ . 6H ₂ O and water], and MA medium [10 mg Ca(NO ₃ ) ₂ ̇ 4H ₂ O, 10 mg KNO ₃ , 5 mg NaNO ₃ , 4 mg Na ₂ SO ₄ , 5 mg MgCl ₂ ̇6H ₂ O, 10 mg per 100 mL Β -glycerol phosphate disodium ̇5H ₂ O, 0.5 mg Na ₂ EDTA ̇ 2H ₂ O, 0.05 mg FeCl ₃ ̇6H ₂ O, 0.5 mg MnCl ₂ ̇4H ₂ O, 0.05 mg ZnCl ₂ , 0.5 mg CoCl ₂ ̇6H ₂ O, 0.08 mg Na ₂ MoO ₄ ̇ 2H ₂ O, 2 mg H ₃ BO ₃ and 50 mg Bicine].

The condition for cultivating the Miscanthus isolate in the present invention means that the pH of the medium, the salinity, the culture temperature, the illumination, the aeration condition, and the culture time, etc., allow the growth of the Miscanthus isolate, Reproduction and manufacture of triglycerides and/or fatty acids. Those skilled in the art can adjust the composition and culture conditions of the medium based on prior knowledge.

In an embodiment of the present invention, the pH of the medium for culturing Micromandala may be from about pH 2.5 to about pH 11.0 (eg, about pH 2.5, about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH 8.5, about pH 9.0, about pH 9.5, about pH 10.0, about pH 10.5 or about pH 11.0), preferably from about pH 4.0 to about pH 9.5.

In an embodiment of the present invention, the micro-mandala culture temperature may be from about 15 ° C to about 60 ° C (eg, about 15 ° C, about 20 ° C, about 25 ° C, about 30 ° C, about 35 ° C, about 40 ° C, About 45 ° C, about 50 ° C, about 55 ° C or about 60 ° C), preferably about 20 ° C to about 40 ° C; and the amount of light can be from about 100 lux to about 4,000 lux, preferably about 2,000 lux of brightness continuous illumination .

In the embodiment of the present invention, the salinity of the culture medium of the culture of Micromania can be adjusted as needed. As used herein, "salinity" means the salt content dissolved in the medium. The culture medium may have a salinity of from 0% (w/w) to about 6.0% (w/w) (e.g., 0% (w/w), about 0.5% (w/w), about 1.0% (w/). w), about 1.5% (w/w), about 2.0% (w/w), about 2.5% (w/w), about 3.0% (w/w), about 3.5% (w/w), about 4.0 % (w/w), about 4.5% (w/w), about 5.0% (w/w), about 5.5% (w/w) or about 6.0% (w/w), preferably 0% ( w/w) to about 4.0% (w/w).

In the present invention, the term "ventilating" means continuously introducing air containing carbon dioxide into a liquid medium, and the aeration amount may be from about 0.05 vvm to about 1 vvm, preferably from about 0.1 vvm to about 0.5 vvm, most preferably about 0.1vvm. The concentration of carbon dioxide in the air may range from about 0.04% (v/v) to about 20% (v/v), preferably from about 0.1% (v/v) to about 15% (v/v), more preferably. It is from about 5% (v/v) to about 10% (v/v).

In the present invention, the term "carrier" means any substrate which can be attached to and/or immobilized on the surface of the microorganism, including but not limited to a mesh material (for example, fiber filter paper, cotton cloth). , burlap, filter cloth, wood pulp cloth, non-woven fabric, denim, nylon cloth, microfiber woven fabric and canvas), foam (for example, polyvinyl alcohol (PVA) foam) or any combination thereof; In any shape, including but not limited to flakes, spheres, rings, spirals Body, cube, cuboid and polygon.

In the present invention, the term "biofilm" means a membranous colony formed by aggregation of the cells of the Micromania of the present invention.

In the method for preparing a culture product of Micromandala according to the present invention, the step of isolating the culture product may be included as needed, and the separation step may be, for example, centrifugation, filtration, aggregation coagulation, and/or scraping of the biofilm from the carrier. Know the method steps.

The present invention also provides a culture product obtained by the above method. The culture product of the present invention is rich in triglycerides and/or fatty acids, particularly triglycerides, and can be used as a source of raw fuel and edible oil. Studies have shown that the amount of oil produced by microalgae under attachment growth accounts for only 10% (w/w) of the dry weight of algae, which is much lower than the amount of oil produced by suspension growth (Gross, M. & Wen, Z ., yearlong evaluation of performance and durability of a pilot-scale Revolving Algal Biofilm (RAB) cultivation system, Bioresour.Technol, 2014,171:... 50-58; and Sebestyén, P et al, Biomass and lipid production by microalgae In a new biofilm based photobioreactor, presented at Young Biotechnologist National Conference, 2014, Szeged). However, the oil content of the dried algae of the microorganism of the present invention is 52.08% (w/w), which is much higher than the oil content of the algal dry of the common algae strain under the attachment culture.

As used herein, "triglyceride" means an ester compound having one glycerol molecule and three fatty acid molecules, wherein the three fatty acid molecules may have identical, partially identical or completely different carbon numbers and unsaturated bonds. .

The "fatty acid" herein means a carboxylic acid compound having 8 to 30 carbon atoms and 0 to 6 unsaturated bonds, preferably a carboxylic acid compound having 12 to 20 carbon atoms and 0 to 5 unsaturated bonds. More preferably, it is a carboxylic acid compound having 16 to 18 carbon atoms and 0 to 3 unsaturated bonds.

The triglyceride and the fatty acid can be obtained by any extraction and separation method well known in the art, for example, Folch, J. et al ., A simple method for the isolation and purification of total lipids from animal tissue. , J. Bio . Chem ., 1957, 23: 497-509), Balasubramanian S. et al ., Oil extraction from Scenedesmus obliquus using a continuous microwave system-design, optimization, and quality characterization., Bioresour. Technol ., 2011, 102: 3396-3403) and Sajilata et al . (Sajilata MG et al ., Supercritical CO ₂ extraction of γ-linolenic acid (GLA) from Spirulina platensis ARM 740 using response surface methodology., J.Food Eng . , 2008, 84: 321-326). Briefly, the method may comprise crushing Microman's genus cells in a manner such as grinding or ultrasonication, and extracting triglycerides and/or fatty acids in Microman's cells by a suitable solvent. Triglycerides and/or fatty acids are then obtained by techniques such as HPLC and/or ion exchange resins.

The culture product of the microorganism of the present invention can be chemically converted (such as hydrogenation, transesterification, hydrothermal carbonisation, fermentation or pyrolysis, etc.) or extracted into a solid, liquid or gaseous biomass fuel, including (but not limited to) biodiesel, biomethanol, bioethanol, biobutanol, biomethane, biohydrogen or biomass coal.

The above biomass fuel can be obtained by any method known in the art, such as Tran DT et al ., Effect of solvents and oil content on direct transesterification of wet oil-bearing microalgal biomass of Chlorella vulgaris ESP- 31 for biodiesel synthesis using immobilized lipase as the biocatalyst, Bioresour.Technol, 2013,135:.. 213-221), Cheng HH et al (Cheng HH, et al, Biological butanol production from microalgae-based biodiesel residues by Clostridium acetobutylicum. ., Bioresour.Technol., 2015, 184: 379-385), Sambusiti C., et al ., Algae as promising feedstocks for fermentative biohydrogen production according to a biorefinery approach: A comprehensive review., Renew .Sust.Energ.Rev ., 2015, 44: 20-36) and Heilmann SM et al ., Hydrothermal carbonization of microalgae., Biomass . Bioenerg ., 2010 , 34 (6): 875-882 and Heilmann SM et al ., Hydrothermal carbonization of microalgae II. Fatty acid, char, and algal nutrient products., Appl.Energ ., 201 1,88(10):3286-3290).

All publications, patents and patent documents mentioned herein are hereby incorporated by reference in their entirety.

The following examples are provided to assist those skilled in the art in practicing the invention. </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Changes are still within the scope of the invention.

Example Materials and Methods

1.C medium

Ca(NO ₃ ) ₂ ̇4H ₂ O 15 mg, KNO ₃ 10 mg, β-glycerophosphate disodium H5H ₂ O 5 mg, MgSO ₄ ̇7H ₂ O 4 mg, vitamin B12 0.01 μg, biotin (Biotin) 0.01 μg, 1 μg of Thiamine HCl, 0.3 mL of a PIV trace metal solution and 50 mg of Tris were mixed, and the volume was hydrated to 100 mL, and the pH was adjusted to 7.5, followed by autoclaving. If it is 1.5% (w/v) acacia solid medium, it needs to be sterilized by adding 15g of vegetable gum.

The PIV trace metal solution was prepared by sequentially adding Na ₂ EDTA ̇2H ₂ O 100 mg, FeCl ₃ ̇6H ₂ O 19.6 mg, MnCl ₂ ̇4H ₂ O 3.6 mg, ZnCl ₂ 1.04 mg, CoCl ₂ ̇6H ₂ O 0.4 μg. After autoclaving with Na ₂ MoO ₄ ̇2H ₂ O 0.25 μg, the volume was then hydrated to 100 mL.

2. Algae collection, separation and culture

Approximately 10 g of the soil sample from the Alishan mountain area of Chiayi, Taiwan, was placed in a 50 mL centrifuge tube, and about 30 mL of C medium was added and cultured at 25 ° C. During the culture, the growth of the algae was observed by a microscope, and then an appropriate amount of the culture medium containing the algae was taken out, transferred to a plate medium, and cultured at 25 ° C. After the algae grows, a single algae species is taken and spread in the plate medium, and the above steps need to be repeated until the sieve is applied to the single algae body. For plate culture, a single algal was applied to a C medium plate and cultured at 25 ° C. In a large number of cultures, a freshly cultured single algae body is scraped from the plate medium and added to the liquid C medium (about 800 mL) to make the absorbance of the culture solution (OD ₆₈₂ nm) about 0.1 to 0.15, and the aeration culture is carried out at 25 ° C. The culture period is about 30 days.

3. Oil staining analysis

20 μL of the cultured algae was mixed with 1 μL of Nile Red (0.1 mg/mL in dimethyl sulfoxide) for oil droplet staining, and after staining, it was allowed to stand at room temperature for 10 minutes, and then observed with a fluorescence microscope ( Chen, W. et al ., A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae ., J. Microbio. Methods, 2009, 77:41-47 and Huang, GH, et al ., Rapid screening method for Lipid production in alga based on Nile red fluorescence., Biomass . Bioenerg ., 2009 , 33 : 1386-1392).

4. Molecular identification of algae species

Scraped from the plate medium amount of freshly cultured algae, which was collected in a microcentrifuge tube 2mL acquires genome (genomic) DNA in accordance ZYMO RESEARCH ZR Fungal / Bacterial DNA MiniPrep ^TM kit manual operation, and to a NanoDrop ( ND-1000 spectrophotometer) detects DNA concentration.

The genomic DNA of the algae was used as a PCR template, and the 18S rRNA and ITS regions (including the internal transcribed spacer 1, the 5.8S ribosomal RNA, the internal transcribed spacer 2 and the front end of the 28S ribosomal RNA) A related primer set (http://biology.duke.edu/fungi/mycolab/primers.htm) to amplify its gene fragment. The PCR reaction solution was as follows: an appropriate amount of genome DNA was used as PCR template, 10mM dNTP 3μL, 10X PCR buffer 4μL, 10mM 5 'end of the primer and the 3' end of each primer and Taq enzyme 0.5μL 5U. The PCR reaction conditions were 96 ° C for 5 minutes; (96 ° C, 30 seconds; 50 ° C, 20 seconds; 72 ° C, 3 minutes) for a total of 40 cycles; 72 ° C, 10 minutes; and finally maintained at 4 ° C. 5 μL of the product was taken for electrophoresis.

The PCR product was purified and sequenced with appropriate primers (http://biology.duke.edu/fungi/mycolab/primers.htm), and the sequencing results were made with Vector NTI Suite 9 software (VNTI) and NCBI/Blastn (http Sequence recombination and sequence similarity alignment analysis were performed at ://www.ncbi.nlm.nih.gov/BLAST/). In addition, the results obtained by sequencing were compared with the algae and algae which were obtained by NCBI/Blastn and the algae and algae which were close to several algal species centers. The evolution tree was analyzed and compared with MEGA 6.0. Then use the Maximum Likelihood to plot the evolution tree in GTR+G+I, with Bootstrap 100 times.

5. Analysis of culture characteristics of algae

5.1 Culture temperature (heat resistant temperature)

The 74C algae were isolated and purified at 25 ° C, and then subjected to long-term subculture at 20 ° C. In order to test the growth tolerance of 74C strains at different culture temperatures, appropriate algae were inoculated on multiple C medium plates and cultured at 30 ° C, 35 ° C, 37 ° C and 40 ° C, respectively. Then, the growth of the algal bodies at different temperatures was observed on the 14th day and the 21st day of the culture, respectively.

5.2 Culture pH

The liquid C medium with pH 4~pH9.5 was prepared, and the appropriate amount of algae was inoculated into liquid C medium with different pH values. The light culture was carried out at 25 °C, and the algae were observed on the first and the 14th day of culture. Growth at different pH values shape.

5.3 Culture salinity (salt tolerance)

Prepare a liquid C medium containing 0% (w/v) ~ 4% (w / v) NaCl, and take an appropriate amount of algae inoculated in liquid medium of each salinity, and illuminate at 25 ° C, respectively. The growth of algal bodies at different salinities was observed on day 1 and day 14.

5.4. Algae attachment culture

The growth and attachment of the 74C strain on the carrier of 8 different materials was observed. The carrier tested comprises: (1) fiber filter paper; (2) cotton cloth a; (3) filter cloth; (4) wood pulp cloth; (5) cotton cloth b; (6) non-woven fabric; (7) denim; 8) Floral cloth. The different vectors and 74C strains were placed in a 250 mL Erlenmeyer flask containing 50 mL of C medium, and shake-cultured at 75 rpm for 21 days. The carrier was taken out to observe the attachment of the algae and attached to the carrier. The algae were stained with Nile red, allowed to stand at room temperature for 10 minutes, and observed under a fluorescent microscope.

6. Algal analysis

6.1 analysis of algae oil content

After collecting the algae, it is freeze-dried into algal flour, and the quantitative algae powder is weighed to extract the oil. The method of extracting fats and oils is described in the method of Folch et al . (Folch, J. et al ., A simple method for the isolation and purification of total lipids from animal tissue., J. Bio . Chem ., 1957, 23: 497-509). The modification is carried out by placing 30 mg of freeze-dried algal flour (A value) into a 2 mL microcentrifuge tube, adding about 2.0 mL of chloroform/methanol (v:v=2:1) and a suitable amount of large glass beads to The percussive cell disrupter (Retsch® MM400) was shaken for approximately 5 minutes and repeated twice. After centrifugation at 10,000 rpm for 5 minutes, the supernatant was removed and added to a disposable 15 mL centrifuge tube, and then about 2.0 mL of chloroform/methanol (v:v=2:1) was added to a 2 mL microcentrifuge tube. The sonication and centrifugation were performed, and the supernatant was taken out and added to another disposable 15 mL centrifuge tube, and the above extraction and centrifugation steps were repeated until the extract was colorless. An equal volume of 145 mM NaCl solution was added to a 15 mL centrifuge tube containing the extract, mixed well in a test tube rotary mixer, and centrifuged at 4,500 rpm for 10 minutes through a centrifuge tube. The lower layer of liquid was taken with a glass pipette into a weighed glass bottle (B value). The liquid in the glass bottle was air-dried overnight and weighed (C value), and the percentage (D value) of the oil content of the dried algae was calculated. Algae dry oil content calculation formula:

6.2 Fatty acid map analysis

A proper amount of dry algae was scraped and placed in a glass test tube, and 1 mL of solution I (NaOH 45 g, methanol 150 mL, and ddH ₂ O 150 mL) was added to shake the algae. Heat at 100 ° C for 5 minutes, then shake all the algae and continue heating for 25 minutes. 2 mL of Solution II (6N HCl 325 mL and methanol 200 mL) was added, and the mixture was heated at 80 ° C for 10 minutes, and then rapidly cooled. Further, 1.25 mL of Solution III (200 mL of hexane and 200 mL of tertiary butyl methyl ether) was added, and the mixture was slowly mixed for 10 minutes, and the lower layer liquid was sucked by a glass pipette tip and discarded. The upper liquid was added to 3 mL of solution IV (NaOH 10.8 g and ddH ₂ O 900 mL), and after mixing for 5 minutes, the supernatant liquid was taken up and the upper layer liquid was analyzed for its fatty acid content by GC/MS (HP 5973 GC/MS System). GC/MS analysis method reference to the method of Valencia, I. et al ., 2007 (Valencia, I. et al ., Development of dry fermented sausages rich in docosahexaenoic acid with oil from the microalgae Schizochytrium sp.: Influence on nutritional properties, sensorial quality And oxidation stability., Food Chem ., 2007, 104: 1087-1096). The GC/Mass analysis conditions were: capillary column: SP-2560, 75m x 0.18mm ID, 0.14μm; inlet temperature: 250°C; ion source temperature: FID 250°C; column oven temperature: initial temperature 140°C, After 5 minutes, the temperature was raised to 240 ° C at a heating rate of 4 ° C / min for 2 minutes; carrier gas: helium; column flow: 40 cm / sec @ 175 ° C; sample to be analyzed injection volume: 1 μL; split ratio : 1 / 100; fatty acid standard: 37-Component FAME Mix (Cat. 18919-1 AMP, Sigma-Aldrich).

6.3 Analysis of oil composition

The 1:1 extracted algal oil sample was analyzed for its oil composition by HPLC. HPLC analysis conditions: Separation column was Silica gel (4.6 mm id × 250 mm, particle size 5 μm) manufactured by Merck, Germany; solvent A was hexane; solvent B was hexane / ethyl acetate / isopropanol (80:10:10 (v/v)); solvent A/B was 98:2 (v/v) at 0 minutes, linearly increased to solvent A/B of 50:50 (v/v) at 8 minutes, Linear increase to 8.5 minutes at solvent A/B of 2:98 (v/v), maintaining the same gradient for 15 minutes, linear reduction to 20:00 for solvent A/B of 98:2 (v/v); flow rate: 1.2 mL/ Min; Evaporative Light Scattering Detector (ELSD) conditions: gas flow rate 2.6L / min; evaporator temperature is 40 ° C (Zhan Guojing et al, glycerol and vegetable oil using lipolytic enzymes to produce esterification reaction 1 , 3-biguanide glycerol. Taiwan Agricultural Chemistry and Food Science , 2010, 45: 19-25).

7. Analysis of biomass yield and oil production efficiency of algae-attached culture

After the 74C strain was cultured in a large amount in a suspension culture, the concentration of the algae solution was adjusted to an absorbance (OD ₆₈₂ nm ) of 0.5, and then 30 mL of the algae solution was taken, and the algae body was fixed to dryness by air filtration and finished. On the surface of a 0.45 μm nitrocellulose/ethyl fiber membrane, 2 parts of immobilized algae were prepared. One blank group (including algae which had not been attached for growth) was directly subjected to freeze-drying, and the dry weight (W _P ) of the filter paper was subtracted, and the dry weight (W _AI ) of the algae before the culture was used. Take aseptically treated water-absorbing carrier (such as sponge, thick filter paper, etc.), place it on the bottom of the sterile plate, add appropriate amount of liquid C medium to keep it moist, and then place the filter paper containing immobilized algae on the water-absorbing carrier. On the top, the plate was placed in a zipper bag to prevent evaporation of water, and the light was cultured at 30 ° C for 24 hours. After 14 days of culture, the filter paper containing the immobilized algal body was taken out from the plate, and the algae body was dried by freeze-drying, weighed, and the dry weight of the filter paper was subtracted as the dry weight of the algae (W _AF ) after the cultivation. After the algae body was weighed, the algae body was scraped off from the surface of the carrier to analyze the oil content, and the oil content (OC _A ) of the attached algae body was recorded. The calculation formula for calculating the biomass yield and oil content of algae is: P _AB = (W _{AF -} W _AI ) / A / TP _AO = OC _A × P _AB

P _AB : biomass yield of algae in attached culture (g/m ² /day)

P _AO : yield of algae oil in attached culture (g/m ² /day)

W _AF : dry weight of algae attached to culture (g)

W _AI : dry weight of algae attached to culture (g)

A: Area of algae attachment culture (m ² )

T: days of algae attachment culture (day)

OC _A : oil content (%) (w/w) of the cultured algae

8. Biomass yield, oil production efficiency analysis and carbon sequestration efficiency analysis of algae suspension culture

Place an appropriate amount of algae in a 1 L serum bottle containing 1 L of C medium, adjust the absorbance (OD ₆₈₂ nm ) of the culture solution to about 0.5, then take 50 mL of the solution, collect the algae by suction filtration, and add the algae. Freeze, dry and weigh (as the starting point for algae growth W _AI ). After that, it was placed on a carbon dioxide carbon sequestration screening platform and monitored with a 1 L volume of algae solution. The carbon sequestration platform continuously feeds 5% (v/v) carbon dioxide into the algae liquid of the potential algae strain with aeration of 0.1vvm, and continuously monitors the influent and outflow carbon dioxide concentration by using the carbon dioxide automatic monitoring system (concentration unit is %, 1% = 10,000 ppm), convert the carbon dioxide concentration unit to mg/m ³ by calculation of the gas concentration conversion formula, and calculate the daily fixed carbon dioxide milligrams (Ya-jun H., Brief Discussion on Conversion Coefficient between the Concentration Units Ppm and mg/m ³ of Nitrogen Oxides (NO _X )., Sichuan Environment, 2010, 29(1): 24-46). After incubation at 30 ° C, take 50 mL of algae solution (four replicates, take a total of 200 mL of liquid), collect the algae by suction filtration, and freeze, dry and weigh the algae (as the end point of algae growth W _AF ) . In addition, after the algae body was weighed, the algae body was scraped off from the surface of the carrier to analyze the oil content of the algae, and the oil content (OC _A ) of the algae was recorded.

Calculation formula for carbon dioxide gas concentration conversion: concentration (mg/m ³ ) = concentration (ppm) × (molecular weight / 22.4) × [273 / (273 + t)]

Molecular weight (carbon dioxide): 44.01

t: measured temperature (30 ° C)

Formula for calculating carbon sequestration efficiency: R=(C _in -C _out )×V×Q×T

R: fixed milligrams per day (mg)

V: microalgae culture volume (m ³ )

Q: gas permeation (vvm, volume per volume per minute)

T: daily ventilation time (minutes)

C _in : carbon dioxide influent concentration (mg/m ³ )

C _out : carbon dioxide outflow concentration (mg/m ³ )

The formula for calculating the biomass yield and oil production efficiency of algae is: P _AB =(W _AF -W _AI )/A/TP _AO =OC _A ×P _AB

P _AB : algal biomass yield in suspension culture (mg/L/day)

P _AO : oil yield of algae in suspension culture (mg/L/day)

W _AF : dry weight (mg) after suspension culture of algae

W _AI : dry weight (mg) before suspension culture of algae

A: algal fluid volume (L)

T: days of algae suspension culture (day)

OC _A : Algae dry oil content in suspension culture (%) (w/w)

9. Algae calorific value determination

After the 74C strain was cultured in a large amount, the algal bodies were collected and lyophilized and stored. 1 g of freeze-dried algae was placed in an oven and dried at 105 ° C for 2 hours. After drying the dried algae in a desiccator and cooling to room temperature, the weight of the dried algae after drying (W _HD ) was recorded on the scale. . The dried algae body is placed in a thermal bomb meter and placed in a water bath with an adiabatic incendiary bomb jacket. After ignition and combustion, the combustion heat released by the sample will be absorbed by the peripheral water bath. The temperature of the water bath rises, multiplied by the sum of water and the thermal charge of the incendiary bomb, multiplied by the specific heat of the water, and divided by W _HD , the calorific value of the sample can be obtained.

10. Algae ash determination

After the 74C strain was cultured in a large amount, the algal bodies were collected and lyophilized and stored. 1 g of the freeze-dried algae body was placed in a crucible, and dried in an oven at 105 ° C for 2 hours. The dried algae body was placed in a desiccator and cooled to room temperature, and the weight of the dried body after drying was recorded by Libra ( W _AD ). The dried algae sample and the crucible are removed and placed in a high temperature furnace at 800±50°C for 3 hours, the furnace temperature is lowered to 300° C., the crucible and the sample are transferred to a desiccator and cooled to room temperature. The weight of the residue (W _ASH ), the W _{ASH is} divided by W _AD to obtain the algal ash content.

11. Determination of sulfur content in algae

The 74C algae body is burned in a high-temperature pure oxygen environment, and the mixed gas containing carbon dioxide, water, nitride and sulfur dioxide produced after combustion is sent to the copper reduction tube by helium gas, and the nitride is reduced to nitrogen gas, other gases. According to the gas adsorption characteristics, they are respectively adsorbed by the fillers in different adsorption tubes. Nitrogen is directly sent from the helium gas to the Thermal conductivity detector (TCD) to detect its content. Different adsorption tubes desorb carbon dioxide, water and sulfur dioxide separately at different desorption temperatures, and then the components are respectively introduced into a heat conduction detector to detect the content of individual components to obtain the composition percentage of sulfur.

Example 1. Identification of algal strains

The 74C strain was isolated and purified from soil samples from the Alishan Mountains in Chiayi, Taiwan. The morphology was observed by microscope (1,000X), and it was found that the 74C strain was present in a non-clustered single algae body. There was no bristles outside the cell wall, and the cells were round. According to the number of days of algal culture, the cell diameter was about 3~6μm (Fig. 1A). After staining with Nile Red, a large number of distinct and yellow oil droplets were observed inside the algae by a fluorescence microscope, indicating that oil droplets could accumulate in the algae (Fig. 1B).

The 18S rDNA and ITS regions of the 74C strain were subjected to DNA sequencing to obtain an 18S rDNA sequence (SEQ ID NO: 1) of length 1,744 bp and an ITS sequence (SEQ ID NO: 2) of 674 bp in length. After comparing the 18S rDNA and ITS sequences with NCBI's nr database, it was found to have the highest similarity to the sequence of the following five strains: (1) Micractinium sp. (Accession No. JX889639.1), the similarity is 99%; (2) Micractinium sp. CCAP 248/2 (Accession No. FR865695.1), similarity is 95%; (3) Micractinium pusillum (Accession No. FM205866.1), similarity is 95%; (4 Micractinium sp. CCAP 248/7 (Accession No. FM205835.1) with a similarity of 95%; and (5) Micractinium pusillum (Accession No. FM205872.1) with a similarity of 95%.

After comparing the ITS sequence of the 74C strain (SEQ ID NO: 2) with the NCBI nr database, it was found to have the highest similarity to the sequence of the following four strains: (1) Chlorella sp. (Accession No) .JQ315187.1), similarity is 100%; (2) Micractinium sp. (Accession No. JX889639.1), similarity is 99%; (3) Micractinium sp. CCAP 211/92 (Accession No. FM205863.1 ), the similarity is 99%; and (4) Micractinium sp. (Accession No. JQ710681.1), the similarity is 98%.

In addition, the 18S rDNA of the 74C strain and the DNA sequence (full length) of the ITS region were analyzed by NCBI/Blastn, and the obtained algal strains were analyzed with an evolutionary tree of several closely related algae and algae. The results show that 74C is assigned to the Micractinium sp. group (Fig. 2). In the evolution tree alignment analysis of the ITS sequence, it was shown that the 74C strain was also attributed to the Micractinium sp. group (Fig. 3). The above sequence alignment analysis showed that the 74C strain may belong to the genus Micractinium .

Based on the results of morphological and molecular identification, the 74C strain should be identified as Micractinium sp., and it is likely to be a new species, so it was named Micractinium sp.74C.

Example 2, culture conditions of 74C strain

(1) Culture temperature (heat resistant temperature) test

The results in Figure 4 show that the 74C strain was continuously and significantly grown on the C medium plate at a culture temperature of 30 °C to 40 °C. Accordingly, the 74C strain can be grown in a temperature range of from about 20 ° C to about 40 ° C and can be thermally resistant to growth in a temperature range of from about 30 ° C to about 40 ° C.

(2) Medium pH test

The results in Figure 5 show the sustainable and significant growth of the 74C strain in liquid C medium at pH 4 to pH 9.5. Accordingly, the 74C strain can be grown in an environment of from about pH 4 to about pH 9.5.

(3) Medium salt test

The results in Fig. 6 show that the 74C strain has a sustained and significant growth phenomenon in liquid C medium having a salinity of 0% (w/v) to 4.0% (w/v). Thus, the 74C strain can be grown from 0% (w/v) to at least about 4.0% (w/v) salinity.

Example 3: Attachment culture of 74C strain

The 74C strains were separately cultured with the following carriers of 8 different materials: (1) fiber filter paper, (2) cotton cloth a, (3) filter cloth, (4) wood pulp cloth, (5) cotton cloth b, (6) Non-woven fabrics, (7) denim and (8) floral fabrics were tested for adhesion. The results in Figure 7 show that the 74C strain can be attached. It is grown on (1) fiber filter paper, (5) cotton cloth b, (6) non-woven fabric, (7) denim and (8) floral fabric, etc., among which (1) fiber filter paper and (8) floral cloth The best results are attached. In addition, the 74C algae attached to each carrier was stained with Nile red, and observed by a fluorescent microscope. It was found that there was a large amount of oil droplets distributed in the algae (Fig. 7), so the 74C strain had great potential for oil production.

Example 4: Analysis of oil content and composition of 74C strain

The 74C strain was cultured in suspension and attached, and the algae was collected, freeze-dried into algal flour, the quantitative algae powder was weighed and the oil was extracted, and the oil content, oil composition and fatty acid composition of the algae were analyzed. . The results showed that the oil content of 74C algae in suspension culture was 47.7% (w/w), and the oil content of 74C algae in the attached culture was 52.08% (w/w). Compared with the general algae strain, the algae dry oil content in the attachment culture is reduced to less than 10% compared with the suspension culture (Gross, M. & Wen, Z., Yearlong evaluation of performance and durability of a pilot-scale Revolving Algal Biofilm (RAB) cultivation system, Bioresour . Technol ., 2014 , 171:50-58), the oil content of 74C algae in the attached culture is higher than that in the suspension culture, and this characteristic shows that the 74C strain is more Suitable for cultivating by attachment.

The results of oil composition in Table 1 showed that there was no significant difference in the composition of oil and fat in 74C strains under suspension culture and attachment culture, mainly based on triglyceride (TAG), with a content of 99.81~99.89% (w/w). ), the rest contains a small amount of 1,3-bisguanidinoglyceride (1,3-DAG, 0.08~0.17% (w/w) of total oil content) and 1,2-bis-decyl glyceride (1 , 2-DAG, accounting for 0.01~0.02% (w/w) of the total oil content, this component is suitable as a source of biofuel.

The fatty acid composition results in Table 2 showed that the fatty acid composition of 74C algae in suspension culture and attachment culture was mainly C16 and C18 fatty acids, and the composition ratio of each fatty acid was similar, and the C16, C18:1 and C18:2 fatty acids were higher. In addition, after calculation, the unsaturation (DU) of the oil contained in 74C algae is 109~116, which is in line with the European standard for biodiesel (DU value must be less than 137) (Ramos, MJ, et al ., Influence . of fatty acid composition of raw materials on biodiesel properties Bioresour.Technol, 2009,100:. 261-268). Accordingly, the 74C strain is suitable as a source of refining biomass fuel, whether it is cultured in suspension or attached. In addition, according to the composition of the fatty acid in the oil produced by the 74C strain and the content of the triglyceride, it is also suitable as a source of edible oil.

Example 5: Biomass yield and oil production efficiency of 74C strains attached to culture

The 74C strain was affixed with fiber filter paper, cultured at 30 ° C for 24 hours, and the algae were collected to calculate the algal biomass yield and oil production efficiency on the surface of the carrier. The results showed that the culture was attached to the laboratory scale. The yield of 74C algae biomass is up to 6.7g/m ² /day, and the oil production efficiency can reach 1.44g/m ² /day. The oil production efficiency is higher than that of the chlorella strain cultivated by the circulating microalgae biofilm growth system (RAB) (oil production efficiency is 0.27 g/m ² /day).

Example 6. Biomass yield, oil yield and carbon sequestration efficiency of suspension culture of 74C strain

The carbon sequestration efficiency of the 74C strain was tested using a carbon dioxide carbon sequestration efficiency evaluation platform, and 5% (v/v) carbon dioxide was continuously supplied to the algae solution at a ventilation rate of 0.1 vvm, and the inflow was continuously monitored using an automatic carbon dioxide monitoring system. The concentration of carbon dioxide in the outflow (concentration in %, 1% = 10,000 ppm), and the number of milligrams of fixed carbon dioxide per day is calculated. After suspension culture at 30 ° C, the collected algal body analysis showed that the 74C algal biomass yield, oil yield and carbon sequestration efficiency were 100.4 mg / L / day, 45.7 mg / L / day and 508 mg / L / day, respectively. .

Example 7: Analysis of biochar characteristics of 74C strain

After the 74C strain was cultured in large amounts, the algal bodies were collected, freeze-dried to algal flour, and the quantitative algal flour was weighed and the calorific value, ash and sulfur were measured. The results in Table 3 show that the algae powder has a calorific value of 25.41 MJ/Kg, and the ash and sulfur contents are 2.07% (w/w) and 0.362% (w/w), respectively. In addition, compared with the coal-fired bituminous coal and sub-bituminous coal used in the current power plants, the ash and sulfur content of the 74C strain are only 13.8% (w/w) and 32.9% (w/w) of the coal-fired coal procurement standards. . Compared with biomass fuels such as weeds, bean stems or straw (Wang Yuqi and Feng Dingshu, research on incineration of agricultural wastes, Journal of Agricultural Machinery, 1993, 2:1-11), 74C strains have high calorific value. It also has a lower ash content. The above results prove that the 74C strain can also be used as a source of high-quality biomass fuel (such as raw coal), and can significantly reduce the generation of pollutants to reduce environmental pollution.

in conclusion

The present invention firstly found a micromania sp. isolate 74C which was initially identified as Micractinium sp. The strain can be grown in an environment having a temperature of from about 20 ° C to about 40 ° C, from about pH 4 to about pH 9 and from 0% (w/v) to at least about 4% (w/v), with good adhesion. Features can be attached to carriers grown on different materials. In addition, the oil content of the dried algae of the 74C strain in suspension culture was 47.7% (w/w), and the oil content of the algae in the biofilm formed by the attachment culture was 52.08% (w/w), which instead Higher than the oil content of the algae in suspension culture. Under the attachment culture, the biomass yield and oil production efficiency of 74C algae were 6.7g/m ² /day and 1.44g/m ² /day, respectively, while the biomass yield of 74C algae in suspension culture The oil yield and carbon sequestration efficiency were 100.4 mg/L/day, 45.7 mg/L/day and 508 mg/L/day, respectively. In addition, regardless of whether it is cultured by suspension culture or attachment culture, the content of triglyceride in 74C is more than 99% (w/w) of the total content of oil and fat, and the fatty acid composition is mainly C16 and C18 fatty acids, and fatty acids are not. The saturation is 109~116. Further analysis showed that the 74C algae had biochar characteristics, and its calorific value was 25.41 MJ/Kg, and the ash and sulfur contents were 2.07% (w/v) and 0.362% (w/v), respectively. The above results show that the 74C strain ( Micractinium sp.) has the characteristics of forming a biofilm and can be cultured in a mode of cultivation; it has a high-quality algae oil composition and can be used as a source of raw diesel oil and edible oil; The ability to fix carbon can reduce the role of carbon dioxide in the environment to reduce environmental pollution; and because of its low ash and low sulfur content, it can be used as a source of biofuels and can significantly reduce pollutant generation to reduce environmental pollution. .

【Biomaterial Storage】

Domestic deposit information

Food Industry Development Institute, January 24, 2017, BCRC 980044.

Foreign deposit information

China, China Type Culture Collection, December 12, 2016, CCTCC M 2016743.

<110> Food Industry Development Research Institute

<120> MICRACTINIUM SP. and its use

<130> 294

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1744

<212> DNA

<213> Micractinium sp.

<220>

<221> 18S rDNA

<222> (1)..(1744)

<400> 1

<210> 2

<211> 728

<212> DNA

<213> Micractinium sp.

<220>

<221> ITS

<222> (1)..(728)

<400> 2

Claims (18)

An isolated strain of Micractinium sp., which is a strain of algae deposited under the No. BCRC 980044 deposited in the Institute of Food Industry Development. A method for preparing a culture product of Micractinium sp., which comprises inoculating a strain of Micromonas sp. according to claim 1 in a liquid medium, and culturing under irradiation and aeration to obtain the culture product. The method of claim 2, wherein the M. genus isolate is co-cultured with the one or more vectors in the liquid medium. The method of claim 3, wherein the micromania isolate and the one or more vector lines are separately added to the liquid medium, or the micromania isolate is first inoculated on the one or more carriers. The inoculated vector is then placed in the liquid medium. The method of claim 3 or 4, wherein the carrier is a mesh material, a foam, or any combination thereof. The method of claim 5, wherein the mesh material is fiber filter paper, cotton cloth, burlap cloth, filter cloth, wood pulp cloth, non-woven fabric, denim cloth, nylon cloth, microfiber woven cloth or canvas. The method of claim 5, wherein the foam is a polyvinyl alcohol (PVA) foam. The method of any one of claims 2 to 4, wherein the pH of the liquid medium is from about pH 3.5 to about pH 10. The method of any one of claims 2 to 4, wherein the culture is carried out at a temperature of from about 25 ° C to about 45 ° C. The method of any one of claims 2 to 4, wherein the liquid medium has a salinity of from 0% (w/w) to about 4.5% (w/w). The method of any one of claims 2 to 4, further comprising the step of isolating the culture product. The method of claim 2, which can be used to fix carbon dioxide. A culture product of the genus Micractinium sp., which can be obtained by the method of any one of claims 2 to 11. The micromandarin culture product of claim 13, which comprises triglyceride and a fatty acid. A method of producing a triglyceride and/or a fatty acid, which comprises isolating a triglyceride and/or a fatty acid from the culture product of Micromonas sp. A method of preparing a biomass fuel comprising using a culture product of Micromonas sp. as claimed in claim 13 or 14 as a raw material. The method of claim 16, wherein the biomass fuel is biodiesel. The method of claim 16, wherein the raw fuel is raw coal.