JPH11196885A - Marine micro-alga producing ethanol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a marine micro-alga that can produce ethanol and to provide a process that can efficiently produce ethanol by using the micro-alga in no need of a large amount of water and even in a dry region having reduced amount of rain fall. SOLUTION: This is a micro-alga in Chlamydomonas that grows in an aqueous solution containing salts of sea water concentrations to accumulate starch in its cells and produce ethanol from the starch in the cells by keeping the cells in a dark and anaerobic atmosphere. The ethanol is by culturing a micro- alga, Chlamydomonas sp. MT-JE-SH-1 in an aqueous solution containing the salts of the seawater concentrations to accumulate starch in the cells of the micro-algae. Then, the slurry including the cell bodies of the micro-algae cultured is held in the dark place and anaerobic atmosphere, while the pH of the culture mixture is kept at 6.0-9.0, thereby forming ethanol.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel microalga used for producing ethanol useful as a fuel or a chemical raw material, and a method for producing ethanol using the microalga.

[0002]

2. Description of the Related Art Conventionally, ethanol is produced by a method of chemically synthesizing fossil resources such as coal and petroleum via ethylene, or a method of fermenting and producing biomass such as sugar cane sugar or corn starch as a raw material. ing. When the raw material is starch, the starch is saccharified to monosaccharides by the action of acid hydrolysis or mold, and further converted to ethanol by the action of an alcohol-fermenting microorganism such as yeast.

[0003] Among biomass raw materials, microalgae represented by chlorella, Donariella, Chlamydomonas and the like are known to have extremely high productivity among photosynthetic organisms. About 20 ~ of weight
50% by weight), and there is a method for producing ethanol by the above-described method using these microalga starches as raw materials. (Literature: Japanese Patent Publication No. 54-11397,
Japanese Patent Publication No. 55-11316

[0004] The present inventors have also noticed that some of the starch-containing microalgae perform a reaction of converting intracellular starch into ethanol, and cultivate such starch-containing microalgae. It has been found that ethanol can be produced by producing ethanol while maintaining the pH of a concentrated slurry in a dark and anaerobic atmosphere while maintaining the pH near neutrality, and filed a patent application as Japanese Patent Application No. 5-239845.

[0005] In the conventional method of producing ethanol by using ethanol fermentation microorganisms from microalgae starch as a raw material, it is necessary to once extract, separate and recover the starch in the microbial cells as a raw material. Many were strong, requiring a lot of power for mechanical disruption and requiring expensive cell wall degrading enzymes. In addition, extraction and purification of starch require a large amount of organic solvent and power for centrifugation.

[0006] Further, since the extracted starch is in a raw state, it is necessary to heat-treat the starch before saccharification by the saccharifying enzyme to gelatinize the starch. In this step, a large amount of energy for heating is required. That was also a problem. Usually, this heating energy is supposed to account for 20 to 30% of the energy of the whole ethanol production process.

[0007] On the other hand, the present inventors have found that after cultivating a starch-containing microalgae, ethanol is produced while maintaining the pH of the slurry obtained by concentrating the microalgae at around neutral, and in a dark and anaerobic atmosphere. The method proposed in Japanese Patent Application No. 5-239845 does not need to extract and separate the starch in the microalgae cells, does not require heating for gelatinization of raw starch, further does not require saccharification, and has a fine ethanol fermentation. Because the algae themselves do it,
The process is greatly simplified as compared with the conventional method, and the energy for manufacturing can be reduced.

[0008]

The above-mentioned method proposed by the present inventors still has the following problems.
Larger microalga culture scales require more water for cultivation, but in areas where the amount of sunshine is favorable for microalga cultivation, where there is little rainfall, securing fresh water is a bottleneck. There are many. Therefore, microalgae that can be cultured in such an area without requiring a large amount of freshwater are desired.
Even in such areas with little rainfall, the seawater can be used almost inexhaustibly in the coastal areas, so the present inventors proliferate vigorously in seawater, contain a large amount of starch, and have excellent ethanol conversion performance of intracellular starch. We thought that microalgae could solve the above problems, but there was no report on such microalgae so far. An object of the present invention is to provide such a microalga and to provide a method for producing ethanol using the microalga.

[0009]

Means for Solving the Problems As means for solving the above problems, the present invention provides (1) a method of growing at a salt concentration of seawater, accumulating starch in the cells, and maintaining the cells in a dark and anaerobic atmosphere; Chlamydomonas sp. MT-JE-SH-, a microalga belonging to the genus Chlamydomonas, which produces ethanol from starch in soil
1 and (2) Chla, a microalga belonging to the genus Chlamydomonas
After culturing mydomonas sp. MT-JE-SH-1 at the salt concentration of seawater to accumulate starch in the cells, the slurry containing the cultured alga bodies was adjusted to pH 6.0 to 9.0. It is intended to provide a method for producing ethanol, characterized in that ethanol is produced while keeping a dark and anaerobic atmosphere while keeping ethanol.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors have collected a large amount of seawater, searched for microalgae that vigorously proliferate in seawater, contain a large amount of starch, and have excellent ethanol conversion performance of intracellular starch.
As a result of the separation and examination, the present inventors succeeded in separating microalgae having excellent performance capable of solving the above-mentioned problem from the Red Sea seawater, and have reached the present invention. This strain converts intracellular starch into ethanol and releases this ethanol out of the cell.Therefore, there is no need to break down the cells and extract and separate the starch as in the conventional method. Since saccharification is unnecessary, the production energy can be reduced by a simple process.

The microalga of the present invention was identified as Chlamydomonas based on the following photosynthetic pigment components and morphological characteristics, and the present inventors designated this algal strain as Chlamydomonas sp. MT-JE.
-SH-1 strain. In addition, the strain was submitted to the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology for deposit.
Was rejected for the reason. It should be noted that many types of microalgae classified into the genus Chlamydomonas are known as being freshwater, and several types are also reported in seawater.

The Chlamydomonas sp. MT-JE-SH-1 strain of the present invention was separated from seawater by the following procedure. The obtained Red Sea seawater (about 5000 ml) is centrifuged (3000x
g, 10 min), discard most of the supernatant, suspend the remaining precipitate and a little seawater, and apply 1 ml of the suspension to a modified ES medium having the composition shown in Table 1 and using genlan gum as a support. did. Next, this gellan gum medium was added to 1500 Lu
The culture was allowed to stand still for about one month in an environment of x and 25 ° C., whereby a green-yellow spread colony-like microalgae could be obtained. However, this microalgal colony was determined by microscopic observation to be a mixture of multiple types of microalgae. Next, from this mixed microalgae, an elliptical shape was obtained using a capillary pipette under a microscope. One microalgae was lifted up, transplanted to a microtiter plate containing 100 μl of the modified ES medium, and these microalgae were statically cultured in an environment of 1500 Lux and 25 ° C. for about 2 months. Five strains grew to tens of thousands as a single species of microalgae.

[0013]

[Table 1]

Next, these were transferred to an L-shaped tube containing 10 ml of the modified ES medium, and were cultured for about 3 weeks at 3500 Lux, 25
When shaking culture was performed at 60 ° C. and 60 ° C., three of the strains grew in a floating state without adhering to the L-shaped tube until the OD _750, whose cell concentration was simply indicated by turbidity, became 3 or more. This 10 ml was further transferred to a 100 ml Erlenmeyer flask containing 60 ml of the modified ES medium, and was heated at 25 ° C. for 60 r.
By performing shaking culture at pm for 2 weeks, a seed strain used for mass culture of microalgae could be obtained. Next, 15,000 L of these three strains was placed in a 1300 ml flat large culture bottle.
The cells were cultured in an atmosphere of ux, 25 ° C. and 0.5% CO ₂ (air), and the growth rate, starch content, and ethanol production performance were comparatively evaluated. As a result, among them, one strain having the best performance was obtained, and this strain was obtained from Chlamydomonas sp.
It was named MT-JE-SH-1 strain.

The bacteriological characteristics of Chlamydomonas sp. MT-JE-SH-1 strain are shown below. The micrographs of the vegetative cells of the present algal strain (preferred growth environment, cells under vigorous growth under abundant nutrient conditions) and immobile cells (cells under poor growth environment, nutrient depletion conditions) are shown in FIG. FIG. 2 shows a transmission electron micrograph of a thin section of a vegetative cell. FIG. 3 is an explanatory diagram schematically showing the micrograph of FIG. Shape: oval Size: Vegetative cells 7 to 12 μm Immobilized cells 10 to 16 μm Flagella: Two vegetative cells that are about 1.5 times the cell length. Motility: Yes (only in the state of vegetative cells) Photosynthetic pigment: Contains chlorophyll a, chlorophyll b, and carotenoid (detected by thin layer chromatography) Cell structure: Has a cell wall on the outer periphery of the cell. Has eye spots. It has a cup-shaped chloroplast. It has a round pyrenoid at the base of the chloroplast. It has a large number of starch granules inside the chloroplast. Salt tolerance: The salt concentration suitable for growth in culture under photoautotrophic conditions is 2 to 5% by weight of NaCl, and the salt concentration near seawater (about 3.5% by weight) seems to be optimal.

The production of ethanol using the microalgae of the present invention is described in Japanese Patent Application No. 5-239845 previously proposed by the present inventors.
It can be carried out according to the process described in the specification. An example of this process is shown in FIG. 4 and a specific manufacturing method will be described based on this. In FIG. 4, the microalga culturing means 1 comprises:
This is a means for culturing the Chlamydomonas sp. MT-JE-SH-1 strain of the present invention independently of light. This includes a water depth of about 10-30c
A flow channel type culture tank having an upper surface of about m can be used, and the culture can be performed by receiving sunlight while giving inorganic nutrients such as nitrogen and phosphorus based on seawater.
Here, the seawater is supplied to the culture tank after foreign substances such as predators of microalgae contained therein are removed by filtration or the like. The cultivation is performed at around normal temperature, for example, at 25 to 35 ° C.

Next, the concentration of the microalgae in the culture solution
When about 0.1 to 3.0 g (as dry solid content) per 000 ml of the culture is stopped, the microalgae concentration means 2
To concentrate. Specifically, in the microalga enrichment means 2, when the microalgae is highly sedimentable, the solid content is temporarily settled by 1 to 5% by weight / volume% (10% per 1000 ml).
５０50 g), and then further subjected to centrifugation, a belt filter or the like to further solid matter of 10 to 20% by weight / volume (10%).
100-200 g per 100 ml). In this process, by concentrating to the highest possible solid content within the range of fluidity, handling as a slurry and slow stirring in the subsequent stage are possible, and the ethanol concentration can be increased,
Ethanol concentration in the latter stage is advantageous.

The slurry of the microalga thus obtained is led to a holding means 3 capable of holding a dark and anaerobic atmosphere to produce ethanol. The holding means 3 is provided with a slow stirring means such as a slurry pump or a stirrer.
It consists of a semi-sealed container with H monitor. Here, the dark atmosphere means a state where light is blocked to such an extent that photosynthesis is not performed. The anaerobic atmosphere refers to a state in which O ₂ is consumed by respiration of microalgae in an air atmosphere, and the O ₂ concentration becomes zero.

The slurry is introduced into the holding means 3 and is kept in a dark and anaerobic atmosphere while gently stirring to produce ethanol by the fermentation action of the intracellular starch performed by the microalga itself. During this time, the pH of the slurry is monitored by a pH monitor, and an alkali solution or an acid solution is added from a pH adjusting means 4 provided with a means for supplying an alkali solution such as NaOH and an acid solution such as HCl to adjust the pH of the slurry. 6.0 to 9.0, preferably 6.5 to 8.0.
To keep in the range. The pH adjusting means 4 operates in conjunction with the pH monitor of the holding means 3 and continuously adjusts the pH in the tank.
H can also be adjusted. The residence time of the microalgae in the holding means 3 varies depending on the type of the microalgae, the holding conditions, and the like, but is generally in the range of 5 to 50 hours.
The temperature in this step is around normal temperature, for example, 25 to 35 ° C.

The ethanol concentration in the slurry is 5 to 50 g
At about / 1000 ml, the mixture is led to the ethanol separation / concentration means 5 to separate and concentrate ethanol. The ethanol separation / concentration means 5 uses a concentration method using an ethanol or water separation membrane or a supercritical extraction method using a solvent such as propane in addition to distillation, thereby concentrating the ethanol to a desired concentration up to absolute ethanol. be able to.
The residue obtained after separating ethanol can be dried and then disposed of by incineration or the like, or can be fermented and used as compost.

[0021]

The contents of the present invention will be described more specifically with reference to the following examples. Example 1 Chlamydomonas sp. MT-JE-SH-1 strain was cultured using a mixture of nutrients having the composition shown in Table 2 in filtered seawater as a medium. 10 liters of this medium and the Chlamyd
omonas sp. MT-JE-SH-1 strain 4 g (as a dry solid content) was placed in a flat transparent container, and about 1500 was taken with a white fluorescent lamp.
The cells are cultured at 25 ° C. for 5 days while continuously irradiating with 0 Lux and aerating with air (0.5% CO ₂ added) to obtain a culture solution containing 24 g (as a dry solid content) of algal cells in 10 liters. Was. The microalgae after the completion of the culture showed a starch content of 25% by weight per dry weight.

[0022]

[Table 2]

Next, 10 liters of this culture solution was concentrated by a centrifugal sedimentation method, and 100 ml of Chlamydomonas s.
p. An algal cell slurry solution containing 24 g (as a dry solid content) of algal cells of the MT-JE-SH-1 strain was transferred to a semi-closed container having a volume of 200 ml, and nitrogen gas was injected into the slurry liquid for a short time. After removing the oxygen in the container, it was shaken (30 reciprocations / min) at 30 ° C. under dark conditions to produce ethanol. During this time, 0.1N NaOH or 0.1N HCl was appropriately added to maintain the pH of the slurry in the range of 6.5 to 8.0. The produced ethanol was analyzed by gas chromatography, and the change with time in the ethanol concentration was examined. As a result, as shown in the graph of FIG. 5, a maximum of 18 g / 1000 ml of ethanol was obtained after about 2 days. In the graph of FIG. 5, the horizontal axis represents the retention time (hr) under anaerobic dark conditions, and the vertical axis represents the ethanol concentration (g / 1000 ml).
Represents

[0024]

EFFECT OF THE INVENTION The microalga Chlamydomonas sp. MT-
By using the JE-SH-1 strain, cultivation is performed using seawater obtained almost infinitely, and the algae slurry obtained by concentrating the culture solution is kept in a dark and anaerobic atmosphere while maintaining the pH near neutrality. To produce ethanol. This eliminates the bottleneck of securing fresh water in the cultivation of ethanol-producing microalgae, and it is difficult to secure water despite the abundance of sunlight and the advantage of photosynthetic production. Ethanol production becomes possible even in areas where it was not possible, making it possible to use the ethanol production process by microalgae on a large scale. In addition, since the present invention can be produced using seawater, fertilized land and freshwater do not compete with food production by agriculture, and degraded land such as deserts and seawater that have not been used so far are used. Ethanol, which is used as energy and chemical raw materials, can be produced in large quantities. Moreover, the present invention has the effect of two birds per stone in that it is effective as a measure for reducing carbon dioxide in the atmosphere on a global scale because the carbon dioxide is fixed and effectively used by photosynthesis by sunlight.

[Brief description of the drawings]

FIG. 1 Microalgae of the present invention Clamydomonas sp. MT-JE-SH-1
It is a microscope picture (2,500 times magnification) instead of a drawing which shows the shape of the vegetative cell and the immobile cell of a strain.

FIG. 2 Microalgae of the present invention Clamydomonas sp. MT-JE-SH-1
It is a transmission electron microscope photograph (magnification 14,000 times) which replaces the drawing and shows the intracellular structure of the vegetative cells of the strain.

FIG. 3 is an explanatory view schematically showing the electron micrograph of FIG. 2;

FIG. 4 is a microalga Clamydomonas sp. MT-JE-SH-1 of the present invention.
It is a flow figure showing the outline of the ethanol production process by a strain.

FIG. 5: Microalgae of the present invention Clamydomonas sp. MT-JE-SH-1
It is a graph which shows the time-dependent change of the density | concentration of ethanol produced | generated in the microalga slurry in the Example of ethanol production using a strain.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:89) (72) Inventor Shin Hirayama 1-8-1 Koura, Kanazawa-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Ryohei Ueda 1-8-1 Koura, Kanazawa-ku, Yokohama-shi, Kanagawa Prefecture Mitsubishi Heavy Industries, Ltd.Basic Technology Research Laboratory (72) Inventor Seiji Kagawa 2-5-1 Marunouchi, Chiyoda-ku, Tokyo Sanishi Heavy Industries, Ltd. Inside

Claims (2)

[Claims] 1. A microalga Chlamydomonas sp. MT- belonging to the genus Chlamydomonas which grows at a salt concentration of seawater, accumulates starch in cells, and produces ethanol from intracellular starch by keeping the cells in a dark and anaerobic atmosphere. JE-SH-1. 2. A microalga Chla belonging to the genus Chlamydomonas.
After culturing mydomonas sp. MT-JE-SH-1 at the salt concentration of seawater to accumulate starch in the cells, the slurry containing the cultured alga bodies was adjusted to pH 6.0 to 9.0. A method for producing ethanol, comprising maintaining ethanol in a dark and anaerobic atmosphere while maintaining it.