JPH10290698A - Production of ethanol using microalga - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply and efficiently producing ethanol from microalgae without requiring a step for crushing cell walls of the microalgae in order to extract starch in the microalgae, a step for purifying the extracted starch and a step for gelatinizing the starch by heating. SOLUTION: This method for producing ethanol using microalgae comprises (A) a step for culturing the microalgae having the ability to hydrolyze starch accumulated in cells into glucose and extracellulary secrete the resultant glucose in a dark and anaerobic atmosphere under bright conditions, concentrating the prepared culture solution and providing a slurry, (B) a step for keeping the obtained slurry in the dark and anaerobic atmosphere, hydrolyzing the starch in the cells of the microalgae into the glucose and extracellularly secreting the glucose and (C) a step for feeding an alcoholic fermentation microorganism into the slurry containing the extracellularly secreted glucose, fermenting the glucose and obtaining the ethanol.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing ethanol from starch accumulated in microalgae.
The obtained ethanol can be used as a fuel or a chemical raw material.

[0002]

2. Description of the Related Art Conventionally, ethanol has been produced by using fossil resources such as coal and petroleum as raw materials, chemically synthesizing via ethylene, and heating raw starch using biomass such as sugar cane sugar and corn starch as raw materials. Alternatively, it is produced by a method of fermentation by a microorganism such as yeast via hydrolysis by acid or saccharification by mold.

Some microalgae such as chlorella, Donariella and Chlamydomonas, which are biomass raw materials, have a dry weight of 5%.
Those containing a large amount of starch of about 0% are known.
A method for producing ethanol from starch extracted from these microalgae is described in, for example, Japanese Patent Publication No. 54-11397.
And Japanese Patent Publication No. 55-11316.

[0004]

When ethanol is produced using starch in microalga as a raw material, starch in cells of microalga is once extracted, separated and recovered. However, since the cell wall is often strong, mechanical crushing using a large power such as ultrasonic crushing or chemical crushing with a cell wall degrading enzyme such as expensive cellulase is required. In the process of starch extraction and purification, operations such as treatment with a large amount of organic solvent and centrifugation are required. Furthermore, since the extracted starch is in a raw state, it is necessary to heat-treat the starch using a large amount of energy to gelatinize the starch before saccharification by a saccharifying enzyme. An object of the present invention is to provide a method for easily and efficiently producing ethanol from microalgae without requiring such a step of crushing a cell wall.

[0005]

The process for producing ethanol using microalgae according to the present invention comprises the steps of: (A) decomposing starch accumulated in cells into glucose in a dark and anaerobic atmosphere; Culturing and concentrating the microalgae having the ability to secrete extracellularly under light conditions to obtain a slurry; and (B) maintaining the slurry in a dark and anaerobic atmosphere, Breaks starch into glucose,
And a step of secreting the glucose extracellularly;
(C) supplying an alcohol-fermenting microorganism to the slurry containing glucose secreted extracellularly to ferment glucose and obtain ethanol.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The microalgae used in the present invention is a microalga having the ability to degrade starch accumulated in cells into glucose and to secrete the glucose out of the cells under a dark and anaerobic atmosphere. is there. Examples of such microalgae include CHI-2 strain. CHI-2
The strain is a green algae isolated from the seawater of Chichijima and grows rapidly by photoautotrophic culture. Also,
The CHI-2 strain accumulates a large amount of starch in cells,
By maintaining in a dark and anaerobic atmosphere, the starch in the cells is converted to glucose and secreted out of the cells. CH
The characteristics of the I-2 strain are shown below.

(1) Separation source: Chichijima Ginny Beach seawater,
(2) Shape: elliptical, (3) Size: 10 to 15 μm,
(4) Flagella: two cells of cell length, (5) Motility: yes, (6) Photosynthetic pigment: chlorophyll a, chlorophyll b, carotenoid (detected by thin layer chromatography), (7) Proliferation: table Using a seawater medium shown in Table 1 (a medium to which the components shown in Table 1 were added per liter of seawater), a 15,000 lux fluorescent lamp was irradiated, and 0.5% by volume of carbon dioxide was distributed at 300 ml / min. And maintained at a temperature of 25 ° C., growing at a rate of about 0.6 g—dry weight / liter / day. (8) Starch content: 4% in dry algal cells
0% by weight, (9) Glucose producing ability: room temperature (20 to 35
C) and secretes extracellular glucose under dark anaerobic conditions.

[0008]

[Table 1]

The present invention will be described below by dividing it into first to third steps. First step : First, after culturing the microalgae under light conditions, the microalgae is concentrated to obtain a slurry. Culture of microalgae under light conditions is performed photoautotrophically, if necessary, using a culture solution consisting of seawater to which various salts such as nitrogen phosphorus have been added.
"Bright conditions" refers to a state in which sufficient light has been irradiated to allow microalgae to grow photoautotrophically. Light conditions can usually be met by exposure to sunlight.

[0010] The concentration of the culture solution after culturing the microalgae under light conditions utilizes the extremely high sedimentation of the microalgae, and once the solid content is about 3 to 5% by weight (dry weight) by natural sedimentation. And then further concentrated by centrifugation or a belt filter until the solid content becomes 15 to 30% by weight. Concentration so as to have a high solid content as much as possible within the range having fluidity facilitates handling when transported as a slurry, etc., and allows efficient subsequent concentration of ethanol.

Second step : Next, the slurry obtained in the first step is kept in a dark and anaerobic atmosphere to decompose the starch in the cells of the microalgae in the slurry into glucose, and to remove the extracellular glucose. To be secreted. The holding period of the slurry containing the microalgae in a dark and anaerobic atmosphere is generally 2 to 3 times.
About a day, glucose is secreted extracellularly by the microalga itself without adding the operation of crushing the microalgae,
Its concentration is up to about 20,000 mg / L.

Third step : Ethanol can be obtained by fermenting glucose by supplying an alcohol-fermenting microorganism to a slurry containing extracellularly secreted glucose. Glucose in the slurry is fermented by alcohol-fermenting microorganisms and almost completely becomes ethanol. Here, the alcohol-fermenting microorganism means a microorganism capable of alcohol-fermenting glucose, and specifically includes yeast, Zymomonas, and the like.

[0013] production plant view 1 ethanol shows an example of a plant employing the ethanol production process of the present invention. The microalga culturing means 1 includes a culture tank having an open upper surface of a flowing water channel having a water depth of about 10 to 30 cm and a culture solution in the culture tank in order to culture the microalgae photoautotrophically. While giving inorganic nutrients to the culture solution in the culture tank,
Microalgae can be cultured by irradiating sunlight. Here, the seawater serving as the base of the culture solution is supplied to the culture solution after being filtered by a filter to remove living organisms that feed on the microalgae and the like.

The culture solution containing microalgae cultured in the microalgae culture means 1 is supplied to the microalgae microalgae concentration means 2 and concentrated to a predetermined concentration by operations such as spontaneous sedimentation and centrifugation. After the concentration, the slurry containing the microalgae from which the supernatant has been removed is supplied to a means 3 for maintaining a dark and anaerobic atmosphere (hereinafter also referred to as "a means for maintaining darkness or the like").
The holding means 3 is a sealed or semi-sealed container provided with a slow stirring means such as a slurry pump or a stirrer. The microalgae supplied to the dark or the like holding means 3 breaks down the starch in the cells into glucose by its own metabolic action and secretes glucose out of the cells.

Then, the alcohol-fermenting microorganism supply means 4
When a small amount of alcohol-fermenting microorganisms supplied from the above is inoculated in a slurry in the holding means 3 such as darkness, glucose becomes
Almost completely converted to ethanol by alcoholic fermentation. Here, the alcohol fermentation microorganism supply means 4 comprises:
One or more alcohol-fermenting microorganisms are cultured in pure culture or in a state close to pure culture, and the culture is maintained in a dark or similar holding means 3.
It is a means that can supply the slurry held in the inside for a certain period. As the alcohol-fermenting microorganism supply means 4, a shake flask, a jar fermenter, or the like used for ordinary culture of microorganisms can be used. Further, the means 4 may include a centrifugal separator or the like for converting the culture solution containing the cultured alcohol-fermenting microorganism into a concentrated slurry.

The slurry in which ethanol has been produced by the alcohol-fermenting microorganism is supplied to an ethanol separation / concentration means 5. The ethanol separation / concentration unit 5 is a unit for separating and concentrating ethanol from a slurry containing ethanol, which contains microalgae and alcohol-fermenting microorganisms. In addition to distillation, a concentration method using ethanol or water separation membrane,
By a method such as supercritical extraction using a solvent such as propane, the concentration can be increased to an arbitrary concentration up to the production of anhydrous ethanol.

[0017]

The following examples illustrate some specific examples of the method of the present invention. Example 1 An eight-fold amount of the seawater medium shown in Table 1 was mixed in 1 liter of sterile seawater, and used as a culture solution under light conditions. 50 liters of this culture solution and a culture seed of the CHI-2 strain (20 g as a dry algal body) were placed in a flat transparent container, and continuously irradiated with white fluorescent light at about 15,000 lux for 0.5 liter.
The cells were cultured at 25 ° C. for 6 days while air to which volume% carbon dioxide was added was aerated. After culturing, 130 g in 50 liters
A culture solution containing (dried alga bodies) was obtained. The microalga after culture contained 40% starch per dry weight.

Next, this solution was concentrated by a centrifugal sedimentation method, and 130 g of CHI-2 strain alga was placed in 500 ml of the solution.
(Dry weight) was obtained. This slurry was transferred to a 1,000 ml Erlenmeyer flask, and nitrogen gas was injected into the slurry for a short time to remove oxygen in the Erlenmeyer flask. The flask was then sealed, and slowly shaken at 30 ° C. under dark conditions (1).
(00 strokes / min) to produce glucose. The amount of produced glucose was measured using a glucose CII-Test Wako kit (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, after 1 day, a maximum of 15,000 mg was contained in the microalga slurry.
/ Liter of glucose was confirmed to have formed.

Y-6-malt extract medium (pH 6 medium containing 2% malt extract, 2% glucose and 0.1% peptone) at 25 ° C. for 4 days at 100 strokes / min
Yeast having alcohol fermentation ability ( Zygosaccharomyces roux) pre-cultured under shaking conditions for minutes
ii ) The slurry of IAM12879) was added to the above-mentioned microalga slurry containing glucose at a volume ratio of 50/1, and the mixture was subjected to 60 strokes / day at 25 ° C. for 4 days under an anaerobic atmosphere.
After shaking culture for 1 minute, 6,600 mg / liter of ethanol was produced.

Example 2 A microalga slurry obtained by collecting and concentrating microalgae cultured in the same procedure as in Example 1 was mixed with yeast ( Zygosacc) having alcohol fermentation ability and precultured in the same procedure as in Example 1.
haromyces rouxii IAM1287
9) mixing the slurry, and under a dark and anaerobic atmosphere,
The cells were cultured at 25 ° C. for 4 days with shaking at 100 strokes / minute, and glucose production and ethanol fermentation were performed in parallel. As a result, 6,700 mg / liter of ethanol was produced.

From the results of Examples 1 and 2, it can be seen that glucose can be almost completely converted to ethanol by allowing alcohol-fermenting microorganisms to act on glucose secreted from microalgal cells.

[0022]

According to the method of the present invention, a microalga having starch accumulated therein is cultured in a culture medium under light conditions, and then the microalga slurry obtained by concentrating the culture medium is subjected to dark and anaerobic atmosphere. Can be produced simply by holding the microorganism in a culture medium, and ethanol can be easily produced by adding an alcohol-fermenting microorganism to a culture solution containing glucose.

Therefore, in comparison with the conventional method for producing ethanol from microalgae, the present invention provides a step of crushing the cell wall of the microalgae for extracting the starch in the microalgae, a step of purifying the extracted starch, a step of purifying the starch. Ethanol can be simply and efficiently produced without the need for a gelatinization step by heating.

Further, the green alga CHI-2 strain used in the present invention can grow well at the salt concentration of seawater, so that a large amount of available seawater can be used for culture under light conditions. Enables large-scale production of ethanol. On the other hand, the yeast and microalgae remaining after the step of obtaining ethanol can be used as feed for cultured fish, feed for rotifer, a feed organism, feed for livestock, and the like. As described above, there is also a side effect that diversified use of biomass is realized.

[Brief description of the drawings]

FIG. 1 is a diagram showing an example of a plant using the ethanol production method of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Microalga culture means 2 Microalga concentration means 3 Dark and anaerobic atmosphere holding means 4 Alcohol fermentation microorganism supply means 5 Ethanol separation and concentration means

Claims (1)

[Claims] (A) In a dark and anaerobic atmosphere, microalgae capable of decomposing starch accumulated in cells into glucose and secreting the glucose out of cells are cultured under light conditions. Concentrating to obtain a slurry; and (B) maintaining the slurry in a dark and anaerobic atmosphere, decomposing starch in the cells of the microalgae into glucose, and secreting the glucose out of the cell. And (C) a step of supplying an alcohol-fermenting microorganism to the slurry containing extracellularly secreted glucose to ferment glucose to obtain ethanol, and a method for producing ethanol using microalgae.