KR101244469B1 - Method and Device for Producing Bio-Diesel and Fermentation Material by Culturing Microalgae - Google Patents

Abstract

The present invention relates to a method for producing biodiesel by fractionating lipids from microalgae by contacting a cultured microalgae with a lipid extracting solvent and cultivating the fractionated microalgae or producing various fermented products such as ethanol, To a method and an apparatus capable of simultaneously producing biodiesel and a fermentation product and reducing carbon dioxide. The method for producing biodiesel and fermented product by culturing microalgae according to the present invention comprises the steps of: culturing microalgae; The lipid of the microalgae culture medium is dissolved in the lipid extraction solvent by bringing the lipid extraction solvent into contact with the culture solution by mixing the microalgae culture solution with a lipid extraction solvent which does not affect survival of the microalgae, A step of fractionating a lipid-soluble solution and a microalgae dissolved in a lipid extraction solvent; Regenerating a part of the fractionated microalgae; Fermenting the remainder of the fractionated microalgae with one of ethanol, butanol and organic acid; Separating the lipid from the fractionated lipid dissolution solution; A process for producing biodiesel from the separated lipid; And separating any one of ethanol, butanol and organic acid from the fermentation product in the fermentation process; .

Description

TECHNICAL FIELD The present invention relates to a method and apparatus for producing biodiesel and a fermented product by microalgae culture,

The present invention relates to a method and an apparatus for producing biodiesel and a fermented product by culturing microalgae.

Due to the rapid population growth and industrial development of the 20th century, the use of fossil fuels is rapidly increasing, and the energy shortage problem, which is balancing the supply and demand of energy, is gradually becoming a reality. As coal and oil resources are gradually depleted worldwide There is a growing interest in the development of alternative energy sources, among which research has recently attracted new interest in recycling natural resources and waste oil, which can be obtained in large quantities through cultivation, as an energy source.

The chemical industry, which is most directly affected by high oil prices and environmental regulation threats, is not a new direction as a 21st century chemical industry to reduce reliance on fossil raw materials and to improve energy efficiency and environmentally friendly industrial structure.

As one of the global trends in the chemical industry, the recent remarkable move is the growth of the biochemical industry using biomass resources such as plants, algae, and organic waste. Biomass technology using biomass is a technology to produce biofuels and bio-based chemicals through biotechnological conversion technology using biocatalysts such as enzymes and microorganisms and conversion technology using chemical and catalytic conversion .

Because biomass is the only carbon source that is neutral and renewable to carbon dioxide emissions, biochemical technology is emerging as an eco-friendly chemical technology that can reduce dependence on crude oil in the chemical industry based on fossil feedstocks and reduce carbon dioxide emissions.

Biochemical technologies using plant raw materials and industries formed from them have recently started to take a new axis in the chemical industry with many investments and industrialization centered on countries and companies such as the USA, Europe, Japan and Brazil.

Since 90% of the 200 billion tonnes of photosynthetic output produced on Earth each year is in the ocean, the biomass of aquatic plants is estimated to be similar in size to the total oil reserves. Such biomass can be converted into bio-energy and organic acids such as lactic acid by applying bio-refinery process applied with thermochemical and biochemical technologies, and bio-energy can be converted into bio-ethanol, bio-butanol and biodiesel Liquid fuels, gas fuels such as hydrogen and methane, bio-diesel that can replace diesel, and bio-ethanol that replaces gasoline.

Biodiesel is fatty acid methyl ester, a pollution-free fuel produced from vegetable oil. It has a purity of 95% or more. It has similar physical properties to diesel oil. Therefore, diesel automobile additives It is used as vehicle fuel by itself.

Biodiesel has the effect of responding to the oil price era and improving the environment by reducing air pollution and greenhouse gas emissions from existing fossil energy use. In addition, since biodiesel is produced from renewable biomass, there is no problem of depletion of energy resources, and carbon dioxide, which is a problem of warming due to fuel use, is recovered during the production of biomass, and thus the net emission of carbon dioxide is very small. In addition, since biodiesel has a high oxygen content (more than 10% oxygen), it has a high rate of complete combustion and can reduce particulate matters such as carcinogens. Especially, it has low toxicity and high biodegradability, .

On the other hand, biodiesel has the disadvantages of low stability of components, aging phenomena such as oxygen, water, heat and impurities during long-term storage, the possibility of long-term environmental impact due to fertilizers and pesticides used for raw material production, It is pointed out as a disadvantage that the economical efficiency can be lowered because the possibility of raw material supply is not smooth.

Ethanol can be chemically synthesized, but is mostly produced by biological methods. Biological methods utilize biomass as a substrate to produce bioethanol by fermenting microorganisms such as yeast (S. cerevisiae, S. uvarum, etc.) or bacteria. When starch is used as a raw material, amylase, which is an acid or a starch degrading enzyme, is used to convert starch to glucose first to produce ethanol.

The bioethanol obtained from the biomass is almost completely mixed with the existing internal combustion engine in the form of gasoline or gasohol, mixed fuel of ETBE (Ethyl Tertiary Buthyl Ether) or hydrated ethanol (95% ethanol + 5% water) Can be used, can keep the air / fuel ratio low, has a high latent heat and octane value, and has a low flame temperature.

Butanol fermentation is carried out by bacteria that are not yeast, and microorganisms producing Clostridium spp., Especially Clostridium acetobutylicum, are produced by converting carbohydrates into biobutanol under anaerobic conditions.

It is known that most of Clostridium microorganisms stop the main metabolic activity when the concentration of organic solvent including butanol exceeds 20 g / l. A known butanol resistant strain for this inhibition is Clostridium beijerinckii NCIMB 8052. In addition to the development of butanol resistant strains, attempts have been made to solve these problems through fermentation processes. In general, a process using a batch reactor is mainly used because it can prevent the contamination and is easy to operate.

Bio-butanol, like bioethanol, is a fuel substance that can replace gasoline. Especially, it has higher energy density because it has two more carbon atoms than ethanol fuel. Especially, it does not dissolve with water, so it can be used as it is without causing corrosion of various petrochemical infrastructures such as oil pipeline compared to ethanol. Has recently come to the fore. In addition, due to the low vapor pressure of butanol, it does not increase the RVP (Reid vapor pressure) of the fuel. As an octane enhancer, it enables the use of inexpensive by-products (eg, butane) give.

When hydrogen is used as an energy source, it can be produced from the constituents of water and organic materials. Therefore, there is a low risk of depletion of raw materials and a high density of energy per unit weight (three times that of gasoline).

The hydrogen production process can be largely chemical production and biological production. The hydrogen production technology that is currently being commercialized is mainly produced by the fossil fuel steam reforming reaction such as petroleum or natural gas, the crude oil refining process, the separation from the by- And electrolysis of water.

However, these methods mainly use natural gas or crude oil as raw materials and do not meet the purpose of development as alternative energy. Therefore, hydrogen production as an alternative energy must ultimately be made by environmentally friendly technology using water or organic waste resources as a clean technology using microorganisms and the like.

Hydrogen production using microorganisms is a process in which green algae directly produces hydrogen from water through photosynthesis, hydrogen production, hydrogen production in a biogenic environment using biomass as a substrate, photosynthesis of Clostridium and Enterobacter and E. coli And the process of producing hydrogen and organic acids in the process of fermenting biomass under the conditions of cancer involved.

Anaerobic fermentation converts biomass to hydrogen and organic acids in anaerobic environments by absolute and tubular anaerobic microorganisms. This process does not rely on light, but the hydrogen production rate (1.6 to 26.9 LH ₂ · L-1 · day-1) is faster than the rate of photosynthesis (0.04 to 0.21 LH ₂ · L -1 · day -1) There are advantages. Disadvantages are that the conversion efficiency from organic materials is low, but the hydrogen production rate is high, which is considered to be the closest to commercialization. Biological hydrogen production is a technology that produces energy and can also be an environmental treatment technology. Although theoretically 2 to 4 mol of hydrogen is generated from 1 mol of glucose through anaerobic fermentation in microbial culture, the production rate of organic acids produced during fermentation, pH changes, kinds of harmful substances in the medium, kinds of carbon sources, types of nitrogen sources, It affects production.

Natural resources such as hemicellulose and lignin in addition to cellulose, as in the case of wood, require a process for removing them, which may be a problem in economy. Various efforts to solve these problems and efforts to find new natural resources Interestingly, attention is focused on microalgae recently.

In the biochemical industry, basic compounds that can be widely used or used in the manufacture of biochemicals can be classified as platform chemicals. Although economics are key, essentially all chemicals can be produced using biomass biochemical conversion technology.

After examining the technical complexity of the marketability and synthetic methods of these substances and derivatives in future alternative chemical candidates, the basic compounds that can be produced from monosaccharide raw materials through biological or chemical modifications are called the 12 best-value-added compounds, Biomass-derived alternative chemical raw materials are organic acids and alcohol compounds. Currently, the number of alternative chemical raw materials produced using biotechnology is still limited.

In addition to the 12 compounds, lactic acid and butanol, both ethanol and 2-hydroxypropionic acid, can also be used as important platform chemicals. Ethanol and butanol are not only used as biofuels but also have utility as raw materials for chemical products. Even lactic acid, one of the representative fermented products, can be used as raw material for chemical products if it can secure price competitiveness with propylene, which is a representative basic chemical product of petrochemicals.

Lactic acid is one of the most important compounds of biomass-derived compounds, although it is not included in the 12 basic compounds of US DOE. Lactic acid can be a raw material for various compounds such as lactide, lactate, acrylic acid, pyruvic acid, and 1,2-propanediol by esterification, dehydration, oxidation and hydrogenation. In particular, Lactide is a very important compound as a monomer of PLA (polylactic acid), which is a representative biomass-derived bioplastics. PLA is known to reduce carbon dioxide emissions by 44% and energy consumption by 36% compared to fossil based PET (polyethylene terephthalate). Biodegradable polymer PLA is in operation after the construction of a 140,000 ton production plant using lactic acid obtained from corn starch in 2002. PLA products are used in packaging and textile fields under the name of NatureWorks PLA and Ingeo fiber. In particular, the world's six major automakers have established sustainable plastic strategies for all automotive plastics by 2020, Mega trends are expected to boost PLA demand. However, due to the difficult and cost-competitive nature of PLA manufacturing process, it is still known that there are no commercial products other than PLA products of NatureWorks, and supply shortage compared to demand. In 2010, the expected market for lactic acid and PLA is expected to exceed W3tr, and production capacity expansion is expected to continue.

Microalgae, which make up the majority of aquatic plants, have been cultivated for a long time for pharmaceutical and health food production, but have not been cultivated for large commercial purposes.

Microalgae can be divided into cellulite, which generally contains a lot of fiber, and an inner extract filled with a variety of saccharides, though it varies depending on the species. The lipids contained in some species are very similar to vegetable oils and are well suited to making them biofuels. Microalgae contain 20-65% lipid, 20-40% 10-40% carbohydrate, 30-70% protein, and some lipid content is 80% of the dry matter for the biomass.

Since the microalgae fibers are mainly cellulose and have a relatively constant diameter than the plant-based cellulose fibers, the influence on the change in the physical properties of the composite material due to the size imbalance in one fiber, which is pointed out as a disadvantage of the vegetable cellulose, have.

In general, microalgae are less than 30 microns in size and can be automatically cultivated in a closed production system such as a culture facility due to the characteristics of algae. For maximum production, a culture system capable of producing appropriate culture conditions is very important.

Birds have the characteristics that they do not compete with other crops in land or space (1-3% of terrestrial plant land is enough), the growth rate is very fast (2 times in 24 hours) to be. The production efficiency of biofuels produced by microalgae is even higher than that of agricultural crops. For example, in the same land area, microalgae productivity is 40 times higher than fluid and 100 times higher than soybean.

Microalgae is a very promising biomass because it can be cultivated in large quantities in fresh water or in nursing water, so that fertile land and related water are not needed or can be minimized. In addition, microalgae can be propagated in large quantities in bio-reactors, which can maximize productivity by artificially controlling conditions ideal for temperature and breeding.

In power plants, carbon dioxide and nitrogen dioxide, which are pollutants, become nutrients necessary for microalgae breeding. Therefore, if carbon dioxide and nitrogen dioxide emitted from power plants are used for algal culture, contamination by carbon dioxide and nitrogen dioxide can be prevented as well.

The general laboratory method of producing biodiesel, bioethanol, biobutanol and organic acids from microalgae is the centrifugation, which is a process that inhibits the growth of microalgae for the purification of biodiesel, bioethanol and organic acids after microalgae culture , Filtration and drying process, and then extracted with a solvent having high selectivity for lipids and used as biodiesel or fermented using appropriate enzymes and microorganisms to produce bioethanol or an organic acid (for example, lactic acid).

However, this lipid production method has difficulty in reusing biomass or re-use of biomass because of leaching and disappearance of carbohydrates and intracellular proteins, which are useful biomass fuels, due to destruction of microalgae during dehydration and extraction, And complicated process steps leading to high operating costs.

It is an object of the present invention to provide a method and an apparatus for culturing microalgae to produce useful fermentation products such as ethanol, butanol, and organic acids together with biodiesel.

It is an object of the present invention to provide a method for producing biodiesel by effectively applying a lipid extracting solvent to a cultured microalgae to effectively fractionate the lipid and further cultivating the fractionated microalgae or cultivating various kinds of useful fermented products including ethanol, And to provide a method and an apparatus for producing biodiesel and various fermentation products at the same time.

It is an object of the present invention to provide an improved method for continuous cultivation of microalgae and a contact between a microalgae culture medium and a lipid extraction solvent to obtain lipids and microalgae at a high recovery rate unlike existing complex and costly production methods And the production cost is low and biodiesel is produced from the fractionated lipids and the fractionated microalgae are used for cultivation or production of useful fermentation products such as ethanol, butanol and organic acid, And to provide a method and apparatus for simultaneously producing diesel and fermentation products and reducing carbon dioxide.

The present invention provides a method for producing biodiesel and a fermented product by culturing microalgae.

The method for producing biodiesel and fermented product by culturing microalgae according to the present invention comprises the steps of: culturing microalgae; The lipid of the microalgae culture medium is dissolved in the lipid extraction solvent by bringing the lipid extraction solvent into contact with the culture solution by mixing the microalgae culture solution with a lipid extraction solvent which does not affect survival of the microalgae, A step of fractionating a lipid-soluble solution and a microalgae dissolved in a lipid extraction solvent; Regenerating a part of the fractionated microalgae; Fermenting the remainder of the fractionated microalgae with one of ethanol, butanol and organic acid; Separating the lipid from the fractionated lipid dissolution solution; A process for producing biodiesel from the separated lipid; And separating any one of ethanol, butanol and organic acid from the fermentation product in the fermentation process; .

Alternatively, the method for producing biodiesel and fermented products by culturing the microalgae according to the present invention comprises the steps of: culturing microalgae; Mixing the cultured microalgae culture liquid with a lipid extraction solvent that does not affect survival of the microalgae, and contacting the lipid extraction solvent with the culture solution to dissolve the lipid of the culture solution in the lipid extraction solvent, A step of fractionating a lipid-soluble solution and a microalgae dissolved in a lipid extraction solvent; Regenerating a part of the fractionated microalgae; Fermenting the remainder of the fractionated microalgae with one of ethanol, butanol and organic acid; And separating any one of ethanol, butanol and organic acid from the fermentation product in the fermentation process; .

Preferably, in the method of the present invention, at least one of the steps of vibrating and pulverizing the microalgae culture broth and stirring the mixture when the microalgae culture broth and the lipid extraction solvent are mixed is further performed, Thereby increasing the contact between the algal culture fluid and the lipid extraction solvent.
Preferably, the lipid extraction solvent is a hydrocarbon solvent.

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Preferably, the method according to the present invention can also produce hydrogen by producing hydrogen from the ethanol fermentation process.

According to the present invention, there is provided an apparatus for producing biodiesel and a fermented product by culturing microalgae.

An apparatus for producing biodiesel and fermented products by culturing microalgae according to the present invention comprises: a culture tank for culturing microalgae; A solvent tank for storing a lipid extraction solvent which does not affect the survival of the microalgae; A mixing tank for mixing the microalgae culture liquid from the culture tank and the lipid extraction solvent from the solvent tank; A mixture from the mixing tank is divided into a lipid dissolving solution in which the lipid of the microalgae culture liquid is dissolved in the lipid extraction solvent and a fractionation tank in which the microalgae culture liquid is fractionated by the microalgae; A fermentation tank for fermenting a part of the microalgae fractionated from the fractionation tank with ethanol; A circulation line for circulating the fraction of the microalgae fractionated from the fractionation tank to the culture tank; A distillation tank for separating lipids from the fractionated lipolysis solution from the fractionation tank and separating ethanol from the fermentation product of the fermentation tank; And a diesel production tank for producing biodiesel from the lipids separated from the distillation tank; .

Alternatively, the apparatus of the present invention may further comprise: a culture tank for culturing microalgae; A solvent tank for storing a lipid extraction solvent which does not affect the survival of the microalgae; A mixing tank for mixing the microalgae culture liquid from the culture tank and the lipid extraction solvent from the solvent tank; A mixture from the mixing tank is divided into a lipid dissolving solution in which the lipid of the microalgae culture liquid is dissolved in the lipid extraction solvent and a fractionation tank in which the microalgae culture liquid is fractionated by the microalgae; A fermentation tank for fermenting part of the fractionated microalgae from the fractionation tank with butanol; A circulation line for circulating the fraction of the microalgae fractionated from the fractionation tank to the culture tank; A distillation tank for separating lipids from the fractionated lipid dissolution solution from the fractionation tank; A diesel production tank for producing biodiesel from lipids separated from the distillation tank; And a butanol production tank for separating butanol from the fermentation product of the fermentation tank.

Alternatively, the apparatus of the present invention may further comprise: a culture tank for culturing microalgae; A solvent tank for storing a lipid extraction solvent which does not affect the survival of the microalgae; A mixing tank for mixing the microalgae culture liquid from the culture tank and the lipid extraction solvent from the solvent tank; A mixture from the mixing tank is divided into a lipid dissolving solution in which the lipid of the microalgae culture liquid is dissolved in the lipid extraction solvent and a fractionation tank in which the microalgae culture liquid is fractionated by the microalgae; A fermentation tank for fermenting a part of the microalgae fractionated from the fractionation tank with an organic acid; A circulation line for circulating the fraction of the microalgae fractionated from the fractionation tank to the culture tank; A distillation tank for separating lipids from the fractionated lipid dissolution solution from the fractionation tank; A diesel production tank for producing biodiesel from lipids separated from the distillation tank; A sedimentation tank for precipitating an organic acid from a fermentation product of the fermentation tank to separate an organic acid; .

Preferably, the apparatus of the present invention further comprises: a first peristaltic pump for supplying the culture solution of the microalgae of the culture tank and the lipid extraction solvent of the solvent tank at a constant flow rate to the mixing tank; And a second peristaltic pump in which a portion of the microalgae fractionated in the fractional tank is transferred to the fermentation tank and the remainder of the microalgae fractionated from the fractional tank is transferred to the culture tank through the circulation line.

Preferably, the apparatus of the present invention is characterized in that the lipid dissolution solution fractionated in the fractionation tank and the fermentation product of the fermentation tank are transferred to the distillation tank, or the lipid dissolving solution fractionated in the fractionation tank is transferred to the distillation tank, And a third peristaltic pump for transferring the fermentation product of the fermentation product to the distillation tank and transferring the fermentation product of the fermentation tank to the distillation tank.

Preferably, the mixing tank further comprises at least one of an apparatus for vibrating and pulverizing the microalgae culture liquid and a stirring device for stirring the mixture so as to increase the contact between the microalgae culture medium and the lipid extraction solvent .

Preferably, the mixing tank and the fraction tank may be combined into one reaction tank.
In the present invention, 'a lipid extracting solvent which does not affect the survival of microalgae' means that, even when lipids are extracted (dissolved) from microalgae by contact with microalgae, the amount of microalgae Refers to a solvent that does not substantially affect survival and cell activity, and the term "lipid extraction solvent" used in describing the present invention is used as such.

The method and apparatus for producing biodiesel and fermented products by culturing microalgae according to the present invention are characterized in that the microalgae are cultivated to separate lipid and microalgae to collect biodiesel and fermented products (ethanol, butanol, organic acid) It is possible to produce biodiesel and fermentation products at the same time by using microalgae and to maximize their productivity. Therefore, the use of carbon dioxide GHG mitigation and development of alternative energy sources are expected to accelerate.

The method and apparatus according to the present invention improve the extraction (production) capacity of lipids through effective contact between the microalgae culture medium and the lipid extraction solvent and improve the yield of biomass and abundant lipid energy that can be harvested from microalgae .

The method and apparatus according to the present invention are advantageous in that since the microalgae culture liquid is circulated between the culture tank and the fraction tank by using peristaltic pumps unlike the prior art methods requiring a dehydration process, The recovery rate of the biomass can be obtained, and various useful fermentation products such as ethanol and organic acid can be obtained from the biomass which is not greatly damaged.

The method and apparatus according to the present invention produce biodiesel and fermented products without destroying the cells of microalgae, thereby preventing the loss of other components in the cell and improving the usability of microalgae extracted with lipids have.

1 is a block diagram of an exemplary apparatus to which the method of producing biodiesel and fermentation products according to the present invention is applied;
2 is a graph showing the effect of a lipid extraction solvent on the survival of microalgae,
FIG. 3 is a graph showing the effect of a solvent (alkane) for extracting lipids and vibration pulverization on lipid extraction from microalgae,
FIG. 4 is a graph of GC-TOF-MS analysis results of biodiesel produced by the present invention. FIG.

Hereinafter, a method and an apparatus for producing biodiesel and fermented products by culturing microalgae according to the present invention will be described in detail. The following examples are illustrative of the present invention but are not intended to limit the scope of the present invention.

First, a biodiesel and fermentation product production apparatus according to the present invention will be described with reference to FIG. 1, a biodiesel and fermentation product production apparatus 1 according to the present invention includes a culture tank 10, a solvent tank 20, a mixing tank 30, a fractionation tank 40, a fermentation tank 50, a circulation line 60, a distillation tank 70, a diesel production tank 80, a butanol production tank 90, and a settling tank 100.

The culture tank 10 is a device for culturing microalgae. The culture tank 10 can be widely used as a system suitable for culturing microorganisms including microalgae. The incubation tank 10 may be equipped with a pond, an artificial incubator, bioreactors, plastic bags, tubes, fermentors, shake flasks, pneumatic lift columns lift columns), and there is no limit to the type of microorganism that can ferment.

The solvent tank 20 is a device for storing and supplying a lipid extraction solvent for extracting (dissolving) lipids from a microalgae culture medium, without affecting the survival of microalgae.

The mixing tank 30 is an apparatus for uniformly mixing the microalgae culture liquid from the culture tank 10 and the lipid extraction solvent from the solvent tank 20. The mixing tank 30 preferably mixes the microalgae culture liquid and the lipid extraction solvent at a ratio of 5: 1.

Preferably, the vibration pulverizing device 31 is mounted on the mixing chamber 30 to vibrate and pulverize the microalgae culture liquid. The vibration crushing apparatus 31 treats the culture liquid with high energy resonance or acoustical radiation of ultrasonic waves. Vibrational pulverization pulverizes molecular aggregates to separate or permeate the molecular aggregates. Vibrating milling makes the microalgae culture into small particles, which allows the microalgae to be exposed (contacted) with more lipid extraction solvent to improve lipid extraction efficiency. A stirring device 32 for stirring the mixture of the microalgae culture liquid and the lipid extraction solvent may be provided in place of the vibration shredding device 31 or in place of the vibration shredding device 31. The stirring device 32 may also be a microalgae culture solution And lipid extraction solvent to improve lipid extraction efficiency.

The fractionation tank 40 is a device for fractionating a mixture from the mixing tank 30 into a lipid dissolution solution (a solution in which the lipid of a microalgae culture liquid is dissolved in a lipid extraction solvent) and microalgae.

1, the mixing tank 30 and the fractionation tank 40 are constructed separately from each other. However, as shown by a dotted line, the mixing tank 30 and the fractionation tank 40 May be integrated into one reaction tank 110 in which their functions are integrated.

The fermentation tank 50 is a device for fermenting a part of the fractionated microalgae from the fractionation tank 40 with ethanol, butanol or organic acid (lactic acid).

The fermenter (50) is an ethanol fermenter when the apparatus (1) of the present invention is constructed for producing ethanol, a butanol fermenter when it is constructed for producing butanol, and an organic acid fermenter when it is constructed for producing organic acids.

The circulation line 60 circulates the remaining microalgae that have not been introduced into the fermentation tank 50 from the fractionated microalgae from the fractionation tank 40 to the culture tank 10 again.

The distillation tank 70 is a device for separating and separating lipid from the fractionated lipid dissolution solution from the fractionation tank 40 and distilling and separating ethanol from the fermentation product from the fermentation tank 50.

The diesel production tank 80 is a device for producing biodiesel from lipids separated from the distillation tank 70.

The butanol producing tank 90 is a device for separating and separating butanol from the butanol fermentation product from the fermentation tank 50.

The settling tank 100 separates and separates organic acids (lactic acid) from the fermentation product of organic acid (lactic acid) from the fermentation tank 50.

In the apparatus 1 according to the present invention, the microalgae culture liquid of the culture tank 10 and the lipid extraction solvent of the solvent tank 20 are preferably supplied to the mixing tank 30 at a constant flow rate through the first peristaltic pump 2 Lt; / RTI >

The line from the culture tank 10 and the line from the solvent tank 20 are connected to the first peristaltic pump 2 and the line from the first peristaltic pump 2 is connected to the mixing tank 30 And the line from the mixing tank 30 is connected to the fractionation tank 40.

In the fractionation tank 40, the mixture from the mixing tank 30 is fractionated into a lipid dissolving solution (layer), an intermediate water (layer), and a lower layer microalgae (layer) Is partially transferred to the fermentation tank 50 through the second peristaltic pump 3 and fermented by an organic acid such as ethanol, butanol or lactic acid by the fermenting microorganism, and the remaining microalgae are fractionated through the circulation line 60 And then recycled to the culture tank 10 to be cultured so that lipids are accumulated again.

To this end, the line from the fractionation tank 40 is connected to the second peristaltic pump 3, the line from the second peristaltic pump 3 is connected to the fermentation tank 50, 2 is connected between the peristaltic pump (3) and the culture tank (10).

Preferably, the lipid-dissolving solution (layer) fractionated in the fractionation tank 40 is transferred to the distillation tank 70 by the third peristaltic pump 4 to separate the lipids, and the fermentation product of the fermentation tank 50 3 peristaltic pump 4 to the distillation tank 70 to separate the ethanol.

To this end, the line from the lipid solution (layer) of the fractionation tank 40 is connected to the third peristaltic pump 4, the line from the fermentation tank 50 is also connected to the third peristaltic pump 4, 3 The line from peristaltic pump 4 is connected to distillation tank 70.

Alternatively, the lipid-dissolving solution (layer) fractionated in the fractionation tank 40 is transferred to the distillation tank 70 by the third peristaltic pump 4 to separate the lipids, and the fermentation product of the fermentation tank 50 3 peristaltic pump 4 to be fed to the butanol producing tank 90 to produce butanol or to the settling tank 100 to produce lactic acid.

The line from the lipid dissolving solution (layer) of the fractionation tank 40 and the line from the distillation tank 70 are connected to the third peristaltic pump 4 and the line from the fermentation tank 50 and the line And the line from the sump 90 or the settling tank 100 is also connected to the third peristaltic pump 4.

The line from the distillation tank 70 is connected to the diesel production tank 80 to transfer the separated lipids from the distillation tank 70 to the diesel production tank 80.

Hereinafter, a method for producing biodiesel and a fermentation product according to the present invention will be described with reference to FIGS. 1 to 4. FIG.

1. Microalgae culture

Examples of the microalgae cultured in the culture tank 10 include diatom strain, chlorpicela, cyanobacteria, Xanthophyceaei, yellow cochlea, chlorella, cryptecoidium, Schizocytrium, nanochlorobushi, woolenia, Cyclodextrins, cyclodextrins, cyclodextrins, cyclodextrins, cyclodextrins, cyclodextrins, cyclodextrins, cyclodextrins, and the like.

Chlorella protothecoides can be used in the present invention. C. protothecoides can be cultured at a cell density 10 times higher than most microalgae, which is very suitable for securing biomass. C. protothecoides can harvest biomass at yields up to 35 gfw / L under ideal conditions under heterotrophic conditions and can store 55% of the biomass as lipid.

C. protothecoides can grow sub-nutritionally to glucose or corn sweetener hydrolyzate (CSH). Heterotrophic growth can increase lipid content and reduce direct dependence on solar energy. The energy density of biodiesel produced from C. protothecoides is substantially equivalent to that of petroleum-based diesel. The cold filter plugging temperature of biodiesel produced from C. protothecoides is lower than that of diesel fuel. Chorella is easy to engineer by molecular biology and is capable of culturing in large scale photobiorectors with enhanced CO ₂ .

2. Solvent and ultrasonic irradiation used for lipid extraction from microalgae

The main cost involved in biodiesel production is the process of harvesting biodiesel from a large volume of culture. In the conventional biodiesel production method including harvesting microbial algae from a culture medium, drying and then destroying the microbial algae to extract lipids and replenishing the culture liquid, the cost of such a process is 40-60% of the total cost. In addition, since the conventional method is difficult to harvest biomolecules other than lipids after the biomass is destroyed after extracting lipids, the present invention is a non-destructive and low-cost technique to mitigate and overcome such conventional problems.

Microalgae is a major component of lipids, carbohydrates and proteins, has a high potential for lipid production, and other biomolecules can also be used as a major source of bioenergy. When cultured in heterotrophic condition, 15-55% of the microalgae are lipid. If lipids can be harvested without destroying microalgae, non-lipid biomass (carbohydrates and proteins), 45-85% of microalgae biomass, can be harvested easily and used as a raw material for fermentation of ethanol or lactic acid, Can be used.

For this reason, in the method of producing biodiesel according to the present invention, only a part of the microalgae after the lipid is extracted is used for fermentation of ethanol, butanol or organic acid (lactic acid), and the remaining microalgae are circulated and regenerated.
As a lipid extraction solvent which does not affect the viability of the microalgae, a hydrocarbon solvent can be used.

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The lipid extraction solvent may include at least one solvent in the C4-C16 hydrocarbon solvent, and preferably at least one solvent in the C10-C16 hydrocarbon solvent.

Exemplary lipid extraction solvents that can be applied to the present invention include 1,12-dodecanedioic acid diethyl ether, n-hexane, n-heptane, n-heptane, n-octane, n-dodecane, dodecyl acetate, decane, dihexyl ether, isopar ), 1-dodecanol, 1-octanol, butyoxyethoxyehteane, 3-octanone, cyclic paraffins, varsol, isoparaffin isoparaffins, branched alkanes, oleyl alcohols, dihecylether, 2-dodecane, and the like.

Ultrasonic irradiation of the microorganism without cell damage by the vibration shredding apparatus 31 is dose-dependent at low frequencies. As the frequency increases, microorganisms survive even at long exposure times. A study was conducted to determine the frequency range and the intensity over different exposure times in a frequency domain (20 kHz to 1 MHz) with no influence on cell activity and to optimize the extraction of lipids, And the exposure time affect the extraction efficiency.

It can be used to optimize the extraction of lipids without damaging microalgae with ideal intensity over different exposure times in the appropriate frequency range (20 kHz to 60 kHz). In other words, frequency, intensity and exposure time affect lipid extraction efficiency. Because the cell size, cell shape, cell wall composition, and physiological conditions have a complex effect on the interaction of cells and ultrasound, 20 kHz and 1 MHz, 20-100 kHz, 20-60 kHz, 30-50 kHz, or 40 kHz, among the various frequencies. With an appropriate combination of lipid extraction solvent and vibration milling, almost 100% lipid (10% of total cellular fatty acid) extraction efficiency can be achieved.

When the microalgae culture solution and the lipid extract solution mixture are stirred in place of the vibrational milling, the lipid extraction efficiency can be improved by improving the contact between the microalgae culture medium and the lipid extraction solvent. It is, of course, also possible to carry out vibration pulverization and stirring together.

3. Biodiesel Production

Production methods of biodiesel are divided into direct use, supercritical fluid method, and transesterification method. Direct use is a direct mixture of light oil and vegetable oil. As time goes by, the viscosity of the diesel increases and hinders the operation. The supercritical fluid method has a fast reaction rate, and all fatty acids and glycerin are pyrolyzed and no by-products are generated.

The transesterification method, which is a commonly used method, is a method of decomposing oil into fatty acid ester and glycerin by mixing triglyceride and alcohol in the presence of a catalyst, wherein the separated fatty acid ester becomes biodiesel. The transesterification process is divided into a chemical process using a chemical catalyst and an enzyme process using a biocatalyst.

The chemical method is used in most processes because the catalyst cost is low and high conversion rate is obtained in a short time. In general, acid catalysts (HCl, H ₂ SO _4, etc.) or base catalysts (NaOH, KOH, etc.) are used. Acid catalysts are suitable for waste oils with high free fatty acids. However, they have a disadvantage of high energy consumption due to corrosion, Have. Base catalysts are used in many commercial processes because of their faster reaction rates than acid catalysts. Enzymatic method is a method in which lipase is used as a catalyst to make esterification. As compared with the chemical method, the use amount of alcohol is small and the separation and purification process of glycerin can be omitted. In addition, all the fatty acids contained in the oil can be esterified and the purity of the product is high. In order to overcome the disadvantages of this enzyme method, the graft chain is formed by using the radiation graft polymerization method in the porous hollow fiber membrane, and the activity of the enzyme is maintained by introducing the hydrophilic anion exchanger.

The types of diesel reactors to be applied to the diesel production tank 80 include batches, continuous type, and the like, and there are general catalytic reactors, cyclic reactors, and tubular reactors depending on the type. In the production of bio - diesel using plant lipids, the optimization and integration of the process according to the acidity, viscosity, and unsaturated fatty acid content of plant lipids needs to be developed through future research. Preferably, in embodiments of the present invention, biodiesel is produced from fractionated lipids by chemical transesterification.

4. Production of ethanol

Generally, the raw materials of ethanol include saccharides (sugar cane, sugar beet), starchy maize (corn, potato, sweet potato, etc.), woody (wood, rice straw, waste paper and the like) In the case of starchy and woody plants, ethanol can be produced through a fermentation process using an appropriate pretreatment process and saccharification process.

In the present invention, microalgae fractionated by using anaerobic cellulose-degrading bacteria Clostridium phytofermentans cells (American Type Culture Collection 700394 ^T ) is directly fermented (sample preprocessing, glycation process and ethanol fermentation process ) Can produce ethanol and hydrogen.

Anaerobic cellulose degrading bacteria are isolated from a variety of habitats (eg, soil, sediment, wetland, mammalian gut, etc.) (Madden, et al ., (1982) Int J Syst Bacteriol 32, 87-91; Madden; Murray et al . (1986) Syst Appl Microbiol 8, 181-184; He et al ., (1991) Int J Syst Bacteriol 41, 306-309; Monserrate et al ., (2001) Int J Syst Evol Microbiol 51, 123-132. ).

Clostridium phytofermentans cells (American Type Culture Collection 700394 ^T ), a representative anaerobic cellulose degrading bacterium, are isolated from the wet slit at the bottom of an intermittent riverbed in the woodland near the Qawbin Reservoir in Massachusetts, USA .

In general, Clostridium piotopermantans cells are long, thin, and straight mobility rod cells that form round terminal spores (0.9-1.5 μm in diameter). Additional features of Clostridium phytopertentans cells can be found in Warnick et al., Int. J. Systematic and Evol. Microbiology, 52, 1155-1160 (2002).

Clostridium phytopermants can ferment a wide spectrum of substances with fuel with high efficiency. The fuels can advantageously be made using waste, such as lactose, wastepaper, leaves, grass cuts, and / or sawdust (Korean Patent Laid-Open No. 10-2008-0091257).

Clostridium phytopermantans may be used alone or in combination with yeast or fungi (e.g., Saccharomyces cerevisiae, Pichia stipitis, Trichoderma species, Aspergillus species) or other bacteria (e.g., Zymommonas moblis, Klebsiella oxytoca, Escherichia coli, clostridium acetobu Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium papyrosolvens, Clostridium cellulolyticum, Clostridium josui, Clostridium termitidis, Clostridium cellulosi, Clostridium celerecrescens, Clostridium < RTI ID = 0.0 > Host lithium popul retina (Clostridium populeti), Clostridium cellulose Robo lance (Clostridium cellulovorans)) may be used in combination with one or more other micro-organisms and the like.

For example, the cellulose decomposition clostridium (strain C7) has a 2.5-fold higher ethanol yield than the clostridial alone culture when co-cultured with Zymomonas mobilis in a medium containing cellulose as a growth substrate Leschine and Canale-Parola, Current Microbiology, 11: 129-136, 1984).

The microbial mixture may be provided as a solid mixture (in a freeze-dried mixture) or as a liquid dispersion of microorganisms, and may be grown by co-culturing with clostridium piotopermantans, or before or after the addition of clostridium phytopermantan By adding another microorganism, the microorganisms may be added sequentially to the culture medium.

5. Production of butanol

Biological pretreatment and saccharification of cellulosic biomass can produce biobutanol with high process efficiency and yield by saccharifying cellulosic biomass by pure biological treatment, without chemical treatment such as acid or base treatment or high pressure / high temperature physical treatment have.

The biobutanol production process can be classified into a (glycation) pretreatment process, a saccharification process, a fermentation process, and a purification process.

The butanol fermentation in the present invention can improve the yield of saccharides such as glucose obtained through the saccharification process by simultaneously performing the saccharification process and the fermentation process in the fermentation tank 50 and optimizing each process. In the present invention, the cellulosic biomass is a non-woody biomass derived from fossil crops such as microalgae.

Generally, the saccharification process can be divided into a saccharification process and a biological saccharification process. In the case of saccharification, dilute acid or concentrated acid can be used to convert cellulose and hemicellulose structure into a sugar form.

The most commonly used enzymatic saccharification among biological saccharides is that the cellulase adsorbs on the reaction surface of the cellulose and decomposes it to generate cellobiose. Cellobiose is a colorless crystal having the formula C ₁₂ H ₂₂ O ₁₁ and is hydrolyzed by β-glucosidase to produce glucose molecules.

In the present invention, enzyme saccharification can be performed by decomposing the biomass of the culture solution to produce glucose by applying the beta-glucosidase immobilized on the carrier to a fractional and transferred microalgae culture.

Preferably, the glycation process in the present invention can be performed by using Clostridium thermocellum instead of a biological enzyme method to sacrifice hemicellulose and cellulose of microalgae (Biotechnology Letters Vol 7 No 7 509-514 (1985)) .

Butanol fermentation can be carried out with the anaerobic microorganisms Clostridium acetobutylicum, Clostridium saccharoperbutylacetonicum, or Clostridium beijernckii.

In the present invention, butanol fermentation can be performed using butostanol using Clostridium acetobutylicum, but not limited thereto.

As a technique for separating the produced butanol from the culture solution, pervaporation, extraction, distillation, gas stripping, or adsorption may be used. The separation of butanol in the present invention can be carried out using a hydrophobic ionic liquid, but is not limited thereto.

6. Production of organic acids (lactic acid)

Lactic acid (2-hydroxypropanoic acid) is produced by microbial fermentation or chemical synthesis. Lactobacillus fermenting bacteria such as Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella can be used for anaerobic fermentation by using a single strain ( Lactobacillus brevis subsp. Brevis ) .

Lactic acid is widely used in food additives and other industrial applications, accounting for around 50,000 tonnes per year. Lactic acid is an intermediate for biodegradable polymers, environmentally friendly solvents, plant growth regulators, and specialty chemicals, and is unlimited for use. Synthetic lactic acid produced from petroleum is low in production cost but is not suitable for making biodegradable polymers such as PLA (polylactate) because D (-) and L (+) type exist together. PLA is a substitute for polyethylene, polystyrene, and polypropylene, which are degradable plastics derived from the petrochemical industry. It is expected to increase the demand for biodegradable plastics as well as being biodegradable.

In order to produce biodegradable polymers such as PLA, it is more effective to process only L (+) lactic acid biosynthesis by biotechnological fermentation rather than DL-lactic acid production by petrochemical synthesis process. However, since the lactic acid produced by the fermentation process is present together with various impurities in the fermentation broth, a recovery process for removing them is required. In addition, since the separation and purification process of the lactic acid production process accounts for more than 50% of the total production cost, an efficient recovery process from the fermentation liquid is essential for the economical production of lactic acid. Examples of separation and purification methods include solvent extraction, electrodialysis, ion exchange resin, nanofiltration, and reverse osmosis.

In the present invention, microorganisms are removed by centrifugation in a fermentation liquid containing impurities such as lactate and protein produced through the organic acid fermentation process, and then lime (Ca (OH) ₂ ) is added in the precipitation tank 100, (Ca (LA) ₂ ) and then recovering the precipitate.

Hereinafter, an example of a method for producing biodiesel and a fermentation product according to the present invention will be described with reference to specific examples.

Example 1. Culture of microalgae

Chlorella protothecoides were used in the present invention while being maintained on a proteose agar slant.

KH ₂ PO ₄ (0.7 g), K ₂ HPO ₄ (0.3 g), MgSO ₄ .7H ₂ O (0.3 g) and FeSO ₄ .7H ₂ O (3 mg) were added to the basic medium. Urea (1 g), Arnon's A Solution (1 ml), thiamine hvdrochloride (10 μg), pH 6.3. The cultures were carried out in 5% CO ₂ , 20 °, 15,000 lux fluorescent lamps.

The composition of the Arnon's A5 solution was H ₂ BOS ₃ (2.9 g), MnCl ₂ .4H ₂ O (1.8 g), ZnSO ₄ .7H ₂ O (0.22 g), CuSO ₄ .5H ₂ O ₃ (0.018 g).

The heterotrophic culture of Chlorella protothecoides is carried out by adding 0.01% urea and 1.0% glucose instead of 0.1% urea in the basal medium.

Example 2. Effect of lipid extraction solvent on microalgae

C. protothecoides culture medium was treated with a 5: 1 ratio of at least one lipid extracting solvent of C10 to C16 alkanes for 5 minutes. Then, 1 ml of the fractionated C. protothecoides solution was diluted 1 / 100,000 and diluted with 1.5% agar plates . The results are shown in FIG. 2. The lipid extraction solvent had no effect on cell survival. The results are shown in FIG.

Example 3 Effect of Lipid Extraction Solvent and Ultrasound on the Lipid Extraction of Microalgae

The growth rate of C. protothecoides was treated with the culture medium in the log section, hexane and decane solvent (lipid extraction solvent) at a ratio of 5: 1 for 5 minutes, and then shaken at 40 kHz for 2 seconds in the water bath.

Lipid extracted with lipid extraction solvent was saponified, and free fatty acid was measured by LC-MS analysis using C17 as a standard. The results are shown in Fig. 3, in which 10% of the total cellular fatty acids were extracted by 5 minutes of lipid extraction solvent mix and an additional 2 seconds of vibration milling. Short vibration milling increased the lipid extraction by 75%.

Example 4. Microalgae culture - fraction of lipid extraction solvent mixture

C. protothecoides were cultured in a 5 L culture tank (10) at a stirring speed of 150 rpm and an illuminance of 15,000 lux until the log interval. The cultured microalgae stock solution or the concentrated culture liquid was used as a raw material. Centrifugation or filtration was used for microalgae concentration. The microalgae culture solution in the culture tank 10 and the decane solvent (lipid extraction solvent) in the solvent tank 20 were transferred to the mixing tank 30 at a ratio of 5: 1, mixed and left for 5 minutes And then subjected to vibration crushing at 40 kHz for 2 seconds by the post-vibration crushing device 31.

The mixture of the microalgae culture medium and the lipid extraction solvent was transferred to the fractionation tank 40 and fractionated for 60 minutes to confirm that the lipid-soluble solution layer, the water layer and the microalgae biomass layer were separated.

Thereafter, the lipid-soluble solution layer was transferred to the distillation tank 70, a portion of the microalgae layer was transferred to the fermentation tank 50, and the remainder was transferred to the culture tank 10 through the circulation line 60.

Example 5. Isolation of microalgae lipids from a fractionated algae lipid solubilization solution layer

Buchi 210/215 rotovapor (Buchi, Switzerland) equipped with round bottom flask as distillation tank 70 was used. The separated lipid-soluble solution layer was transferred to the distillation tank 70 and placed in a round-bottomed flask. The cold raw water was introduced into the condenser and the oil bath of the distillation flask was set at 174 ° C.

When the distillation started, a decane passed through the apparatus and flowed into the condenser and collected into the receiving flask in liquid form. When the distillation was terminated, the volume of decane recovered in the receiving flask and the microalgae lipids left in the distillation flask were determined by LC-MS analysis with C17 as the standard and volume. The recovered decane solvent (lipid extraction solvent) was transferred to the solvent tank 20 for reuse.

Example 6. Production of biodiesel from microalgae lipids

The microalgae lipids separated by distillation were transferred to a diesel production tank (80: stirrer). The methanol and the caustic soda were stirred to prepare methoxide, and the prepared methoxide was added to the agitator and stirred to react the microalgae lipid with the methoxide to form the biodiesel, glycerin and soap solid components. These products were supplied to a centrifuge and centrifuged. The biodiesel was placed at the upper part due to the difference in specific gravity of each product, and the upper part and lower part of the lower part were separated so that heavy glycerin and soap components were positioned.

The thus-separated glycerin and soap components were discharged into separate glycerin storage tanks. Water in the upper portion of the biodiesel and twice the amount of biodiesel was introduced into a stirrer and stirred, thereby being contained in biodiesel The glycerin, the soap component and the methanol component were dissolved in water, and the thus stirred solution was separated into a centrifugal separator to separate the components such as glycerin dissolved in the water. And then discharged to a glycerin storage tank through a separate discharge line. Finally, a distillation unit, which is a heater unit, was operated to distill 1 to 2% of water remaining in the biodiesel, Respectively.

The GC-TOF-MS (GC-6890N, Agilent Technologies, USA) analysis of the harvested biodiesel is shown in FIG.

Example 7. Ethanol fermentation in fractionated microalgae

1, the fermenter 50 is constructed using a microbial fermenter (INNO 200603, Innobio, Korea). Clostridium phytopermans were grown in a culture tube containing GS-2 medium containing the indicated amounts of fractionated microalgae, respectively.

The GS-2 medium contained yeast extract 6.0, urea 2.1, K ₂ HPO ₄ 2.9, KH ₂ PO ₄ 1.5, MOPS 10.0, triosodium citrate dihydrate 3.0, and cysteine hydrochloride 2.0 (each expressed in g / L) . The initial pH of the culture medium was 7.5, and the initial Clostridium phytopermementus concentration was 0.8-1.1 × 10 ⁷ cells / mL and cultured under an atmosphere of N ₂ at 30 ° C.

The ethanol concentration was determined upon completion of fermentation of the fractionated microalgae. Clostridium phytopermantans produced hydrogen simultaneously with ethanol fermentation. The ethanol concentration was analyzed using HPLC (Breeze HPLC system, Waters Co., USA) equipped with RI detector. The column was Aminex HPX-87H (3007.8 mm, Bio-rad). When the initial fractionated microalgae concentration was 10 g / L, the concentration of ethanol was 0.23-0.26% (v / v). When the initial fractionated microalgae concentration was 20 g / L, the concentration of ethanol was 0.42-0.54% (v / v). When the initial fractionated microalgae concentration was 40 g / L, the concentration of ethanol was 0.92 ~ 1.20% (v / v). The ethanol distillation was carried out using a distillation tank (70).

These results indicate that higher concentrations of fractionated microalgae do not inhibit the action of clostridial peatotermentans, as the concentration of ethanol increases with increasing initial concentration of fractionated microalgae. Finally, it can be seen that these cellulosic feedstocks are fermented with ethanol without chemical pretreatment of the microalgae feedstock from which clostridium phytopor mentans is fractionated and without the addition of cellulases or other enzymes.

Example 8. Production of ethanol

The fermentation product produced by the ethanol fermentation was transferred to the distillation tank 70 to distill ethanol. At this time, the oil bath is operated so that the evaporation of ethanol is carried out, and the temperature is heated to above the evaporation temperature of ethanol. When the temperature is higher than the evaporation temperature of water, the water is evaporated and the concentration of ethanol may be lowered by mixing with ethanol. Therefore, the vaporization temperature is formed between 78.3 and 85 ° C and heated for a certain period of time. In an ethanol storage tank.

Example 9. Butanol fermentation in fractionated microalgae

1, the fermenter 50 is constructed using a microbial fermenter (INNO 200603, Innobio, Korea).

Clostridium thermocellum was anaerobically cultured in DSM medium at 60 ℃, anaerobic condition, 150 rpm. The fractionated microalgae were transferred to a 5 L fermenter (50), inoculated with the C. thermocellum culture (5%, v / v), sacrificed by incubation at 60 ° C under anaerobic conditions and 150 rpm for 3 days [Biotechnology Letters Vol 7 No 7 509-514 (1985)]. After inoculation, nitrogen gas was injected into the culture solution to maintain anaerobic conditions.

For butanol fermentation, Clostridium acetobutylicum, a spore suspension, was heated at 80 ° C for 10 min. Then, anaerobically cultured in a medium at a temperature of 37 ° C.

The fermenter (50) was inoculated with the C. acetobutylicum culture (5%, v / v), and anaerobic butanol fermentation was carried out at 37 ° C under anaerobic conditions with stirring at 180 rpm. During the fermentation period, samples were taken periodically to analyze microbial growth and butanol concentration.

The used DSM medium contained 1.3 g of (NH ₄ ) ₂ SO ₄ per liter, 2.6 g of MgCl ₂ .6H ₂ O, 1.43 g of KH ₂ PO ₄ , 7.2 g of K ₂ HPO ₄ .3H ₂ O, 0.13 g of CaCl 2 ₂ · 6H ₂ O, 1.1 mg of FeSO ₄ · 7H ₂ O, 6.0g of sodium β-glycerophosphate, yeast extract of 4.5g (yeast extract), 10g of a filter paper, a ball mill, the cellulose or BIOS cell, 0.25 g of reduced Gt; glutathione < / RTI > and 1 mg of resazurin. The pH was adjusted from 5.0 to 8.0 with 1 M HCl or 1 M NaOH.

The C. acetobutylicum culture medium contained 0.75 g KH ₂ PO ₄ , 0.75 g K ₂ HPO ₄ , 0.4 g MgSO ₄揃 H ₂ O, 0.01 g MnSO ₄揃 H ₂ O, 0.01 g FeSO ₄揃 7H ₂ O, 0.5 g of cysteine, 5 g of yeast extract, 2 g of asparagine.H ₂ O, 2 g of (NH ₄ ) ₂ SO ₄ . The acetone, butanol and ethanol produced by the microorganisms after the continuous process were quantitatively analyzed using gas chromatography (Agilent technology 6890N Network GC system) equipped with a FID (Flame Ionization Detector), and the column was HP-INNOWAX (30 cm x 250 Mu m x 0.25 mu m, Agilent technology) was used. The temperature of the sample injecting portion and the detecting portion was set to 250 DEG C, and the oven was set to rise to 170 DEG C at a rate of 10 DEG C / minute at 50 DEG C. [ The fermentation broth contained 13 g / L of butanol, 8 g / L of acetone and 0.5 g / L of ethanol.

Example 10. Production of butanol

The culture solution containing the butanol, acetone and ethanol was transferred to a butanol producing tank 90 to obtain an ionic liquid BMIM-TFSI [1-butyl-3-methylimidazolium bis (trifluoromethylsulfonyl) Butanol was prepared by using 1-butyl-3-methyl imidazolium bis (trifluoromethylsulfonyl) imide and BMIM-PF6 [1-butyl-3-methyl imidazolium hexafluorophosphate] Respectively. The fermentation broth in the fermentation tank 50 is transferred to the butanol production tank 90 and mixed with the same amount of BMIM-TFSI (Sigma Aldrich, USA) in the fermentation broth and BMIM-PF6 (Sigma Aldrich, USA) The mixed solution was vortexed to isolate butanol. In this case, it is also possible to use a solution prepared by using a general ionic liquid production method. As a result, 60 ± 1% of butanol was isolated when BMIM-TFSI was used, and 64 ± 1% of butanol was isolated when BMIM-PF6 was used.

Example 11. Organic acid fermentation in fractionated microalgae

1, the fermenter 50 is constructed using a microbial fermenter (INNO 200603, Innobio, Korea). Lactobacillus brevis subsp. brevis were cultured in PYG medium (medium composition: 20.0 g of peptone, 5.0 g of glucose, 10.0 g of yeast, 0.08 g of NaCl, 0.5 g of cysteine hydrochloride, 0.008 g of calcium chloride, 0.008 g of MgSO ₄ , 0.04 g of K ₂ HPO ₄ , KH ₂ PO ₄ 0.04 g, sodium bicarbonate 0.4 g, pH 7.1-7.3), harvested by centrifugation, and washed with 0.085% saline. This was inoculated into the fermentation tank 50 containing the transferred microalgae. The initial pH of the medium was 7.2, and the initial lactic acid fermentation strain was 0.8-1.1 × 10 ⁸ cells / mL and cultured with N ₂ gas at 30 ° C. The concentrations of malate, lactate, acetate, citrate and butyrate were determined at the completion of fermentation of the microalgae.

Concentrations of malic acid, lactic acid, acetic acid and citric acid were measured by HPLC (HP placard, Japan) with a Platinum EPS C18 organic acid analytical column (250 mm × 4.6 mm, 5 μm) and adjusted to pH _2.0 with 0.05 M KH ₂ PO ₄ . The nitric acid was analyzed by gas chromatography (HP placard, Japan) using CP 58 Wax (FFAP) (30 cm x 0.25 mm ID, 0.25 m) column.

The results showed that the concentrations of malic acid, lactic acid, acetic acid, citric acid and lactic acid were 870.30 + 13.15, 746.16 + 8.91, 4,233.23 + 76.06, 318.04 + 47.75 and 1.99 + 1.99 mM, respectively.

These results show that these cellulosic feedstocks are fermented with lactic acid without the chemical pretreatment of microalgae feedstock in which the lactic acid fermentor strain is fractionated and without the addition of cellulases or other enzymes.

Example 12. Production of lactic acid

After the fermentation liquid from the fermentation tank 50 is transferred to the settling tank 100, Ca (OH) ₂ is added to the fermentation liquid to adjust the pH to 10 and then heated to increase the solubility of calcium lactate (Ca-lactate) And the protein was allowed to solidify. It was filtered at high temperature and recovered with calcium lactate and cooled to precipitate calcium lactate. After dissolving calcium lactate at high temperature, CaSO ₄ was precipitated by treatment with sulfuric acid, and lactic acid was recovered.

Example 13. Growth of fractionated microalgae

After the microalgae lipid extraction, microalgae were regrown. In Example 3, 10% (v / v) of the microalgae culture inhibited by the lipid extraction solvent by lipid extraction was transferred to the culture tank and cultivated. As a result, the non-growth rate was 0.028 h -1. In comparison with the non-growth rate of about 0.033 h-1 in the culture tank, it was found that there is no problem in cultivating the strain after the simultaneous extraction.

1: Biodiesel production apparatus of the present invention 2: First peristaltic pump
3: second peristaltic pump 4: third peristaltic pump
10: culture tank 20: solvent tank
30: mixing tank 31: vibration crushing device
32: stirrer 40: fractionation tank
50: Fermenter 60: Circulation line
70: Distillation tank 80: Diesel production tank
90: Butanol production tank 100: Settling tank
110: Reactor

Claims (13)

The process of culturing microalgae;
The lipid of the microalgae culture medium is dissolved in the lipid extraction solvent by bringing the lipid extraction solvent into contact with the culture solution by mixing the microalgae culture solution with a lipid extraction solvent which does not affect survival of the microalgae, A lipid dissolving solution dissolved in a lipid extracting solvent and fractionating the microalgae;
Regenerating a part of the fractionated microalgae;
Fermenting the remainder of the fractionated microalgae with one of ethanol, butanol and organic acid;
Separating the lipid from the fractionated lipid dissolution solution;
A process for producing biodiesel from the separated lipid; And
Separating any one of ethanol, butanol and organic acid from the fermentation product in the fermentation process;
≪ / RTI > The method of claim 1, wherein the biodiesel and fermented product are produced by microalgae culture. The process of culturing microalgae;
Mixing the cultured microalgae culture liquid with a lipid extraction solvent that does not affect survival of the microalgae, and contacting the lipid extraction solvent with the culture solution to dissolve the lipid of the culture solution in the lipid extraction solvent, A lipid dissolving solution dissolved in a lipid extracting solvent and fractionating the microalgae;
Regenerating a part of the fractionated microalgae;
Fermenting the remainder of the fractionated microalgae with one of ethanol, butanol and organic acid; And
Separating any one of ethanol, butanol and organic acid from the fermentation product in the fermentation process;
≪ / RTI > wherein the fermentation product is cultivated in a microalgae culture. The method according to claim 2, further comprising at least one of vibrating and pulverizing the microalgae culture broth and stirring the mixture when mixing the microalgae culture broth with the lipid extraction solvent, Wherein the contact of the lipid extraction solvent is increased. delete [Claim 3] The method according to claim 2, wherein hydrogen is also produced in the fermentation process of ethanol to produce hydrogen together with the microalgae. A culture tank (10) for culturing microalgae;
A solvent tank (20) for storing a lipid extraction solvent which does not affect the survival of the microalgae;
A mixing tank (30) for mixing the microalgae culture liquid from the culture tank (10) and the lipid extraction solvent from the solvent tank (20);
A fractionation tank (40) for fractionating the mixture from the mixing tank (30) into a lipid dissolution solution in which the lipid of the microalgae culture liquid is dissolved in the lipid extraction solvent and the microalgae;
A fermentation tank (50) for fermenting a part of the microalgae fractionated from the fractionation tank (40) with ethanol;
A circulation line (60) for circulating the fraction of the microalgae fractionated from the fractionation tank (40) to the culture tank (10);
A distillation tank (70) for separating lipids from the fractionated lipid solution from the fractionation tank (40) or for separating ethanol from the fermentation product of the fermentation tank (50); And
A diesel production tank 80 for producing biodiesel from lipids separated from the distillation tank 70;
Wherein the biodiesel and fermentation product are produced by microalgae culture. The method according to claim 6,
A first peristaltic pump (2) for feeding the microalgae culture solution of the culture tank (10) and the lipid extraction solvent of the solvent tank (20) to the mixing tank (30) at a constant flow rate;
A part of the microalgae fractionated in the fractionation tank 40 is transferred to the fermentation tank 50 and the remainder of the microalgae fractionated from the fractionation tank 40 is recovered through the circulation line 60 A second peristaltic pump (3) for transferring to the bath (10); And
A third peristaltic pump (4) for transferring the lipid-soluble solution fractionated in the fractionation tank (40) and the fermentation product of the fermentation tank (50) to the distillation tank (70);
Wherein the biodiesel and fermentation product are produced by microalgae culture. A culture tank (10) for culturing microalgae;
A solvent tank (20) for storing a lipid extraction solvent which does not affect the survival of the microalgae;
A mixing tank (30) for mixing the microalgae culture liquid from the culture tank (10) and the lipid extraction solvent from the solvent tank (20);
A fractionation tank (40) for fractionating the mixture from the mixing tank (30) into a lipid dissolution solution in which the lipid of the microalgae culture liquid is dissolved in the lipid extraction solvent and the microalgae;
A fermentation tank (50) for fermenting part of the microalgae fractionated from the fractionation tank (40) with butanol;
A circulation line (60) for circulating the fraction of the microalgae fractionated from the fractionation tank (40) to the culture tank (10);
A distillation tank 70 for separating lipid from the fractionated lipid dissolution solution from the fractionation tank 40;
A diesel production tank 80 for producing biodiesel from lipids separated from the distillation tank 70; And
A butanol production tank 90 for separating butanol from the fermentation product of the fermentation tank 50;
Wherein the biodiesel and fermentation product are produced by microalgae culture. 9. The method of claim 8,
A first peristaltic pump (2) for feeding the microalgae culture solution of the culture tank (10) and the lipid extraction solvent of the solvent tank (20) to the mixing tank (30) at a constant flow rate;
A part of the microalgae fractionated in the fractionation tank 40 is transferred to the fermentation tank 50 and the remainder of the microalgae fractionated from the fractionation tank 40 is recovered through the circulation line 60 A second peristaltic pump (3) for transferring to the bath (10); And
A third peristaltic pump 4 for transferring the lipid-lysing solution fractionated in the fractionation tank 40 to the distillation tank 70 and transferring the fermentation product of the fermentation tank 50 to the butanol production tank 90; ;
Wherein the biodiesel and fermentation product are produced by microalgae culture. A culture tank (10) for culturing microalgae;
A solvent tank (20) for storing a lipid extraction solvent which does not affect the survival of the microalgae;
A mixing tank (30) for mixing the microalgae culture liquid from the culture tank (10) and the lipid extraction solvent from the solvent tank (20);
A fractionation tank (40) for fractionating the mixture from the mixing tank (30) into a lipid dissolution solution in which the lipid of the microalgae culture liquid is dissolved in the lipid extraction solvent and the microalgae;
A fermenter (50) for fermenting a part of the microalgae fractionated from the fractionation tank (40) with an organic acid;
A circulation line (60) for circulating the fraction of the microalgae fractionated from the fractionation tank (40) to the culture tank (10);
A distillation tank 70 for separating lipid from the fractionated lipid dissolution solution from the fractionation tank 40;
A diesel production tank 80 for producing biodiesel from lipids separated from the distillation tank 70; And
A sedimentation tank 100 for precipitating an organic acid from a fermentation product of the fermentation tank 50 to separate an organic acid;
Wherein the biodiesel and fermentation product are produced by microalgae culture. 11. The method of claim 10,
A first peristaltic pump (2) for feeding the microalgae culture solution of the culture tank (10) and the lipid extraction solvent of the solvent tank (20) to the mixing tank (30) at a constant flow rate;
A part of the microalgae fractionated in the fractionation tank 40 is transferred to the fermentation tank 50 and the remainder of the microalgae fractionated from the fractionation tank 40 is recovered through the circulation line 60 A second peristaltic pump (3) for transferring to the bath (10); And
A third peristaltic pump 4 for transferring the lipid-soluble solution fractionated in the fractionation tank 40 to the distillation tank 70 and transferring the fermentation product of the fermentation tank 50 to the sedimentation tank 100;
Wherein the biodiesel and fermentation product are produced by microalgae culture. 12. The method according to any one of claims 6 to 11,
The mixing tank 30 is provided with a vibrating crushing device 31 for vibrating and pulverizing the microalgae culture liquid so as to increase the contact between the microalgae culture medium and the lipid extraction solvent and a stirring device 32 for stirring the mixture The apparatus for producing biodiesel and fermentation products by microalgae culture, further comprising at least one device. 12. The method according to any one of claims 6 to 11,
Wherein the mixing tank (30) and the fractionation tank (40) are integrated into one reaction tank (110), wherein the biodiesel and the fermentation product are produced by the microalgae culture.

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